Gene Expression in Recombinant Microorganisms (Biotechnology and Bioprocessing Series)

GENE EXPRESSION IN RECOMBINANT MICROORGANISMS

BIOPROCESS TECHNOIX)GY Series mitor

W. Courtney McGregor Xoma Corporation Berkeley, California

1. Membrane Separations in Biotechnology, edited by W. Courtney McGregor 2. Commercial Production of Monoclonal Antibodies: A Guide for ScaleUp, edited by Sally S. Seaver 3. Handbook on Anaerobic Fermentations, edited by Larry E. Erickson and Daniel Yee-Chak Fung 4. Fermentation Process Developmentof Industrial Organisms, edited by Justin 0. Neway 5. Yeast: Biotechnology and Biocatalysis, edited by Hubert Verachtert and Rend De Mot 6. Sensors in Bioprocess Control, edited by John V. Twork and Alexander M. Yacyn ych 7. Fundamentals of Protein Biotechnology, edited by Stanley Stein 0. Yeast Strain Selection, edited by Chandra J. Panchal 9. Separation Processes in Biotechnology, edited by Juan A. Asenjo 10. Large-scale Mammalian Cell Culture Technology, edited by Anthony S. Lubiniecki 11. Extractive Bioconversions, edited by Bo Mattiasson and Olle Holst 12. Purification and Analysis of Recombinant Proteins,edited by Ramnath Seetharam andSatish K. Sharma 13. Drug Biotechnology Regulation: Scientific Basis and Practices, edited by Yuan- yuan H. Chiuand John L. Gueriguian 14. Protein Immobilization: Fundamentals and Applications, edited by Richard F. Taylor 15. BiosensorPrinciples and Applications, edited by Loi'c J. Blum and Pierre R. Coulet 16. Industrial Application of Immobilized Biocatalysts, edited by Atsuo Tanaka, Tetsuya Tosa, and Takeshi Kobayashi 17. Insect Cell Culture Engineering, edited by Mattheus F. A. Goosen, Andrew J. Daugulis, and Peter Faulkner 18. Protein Purification Process Engineering, edited by Roger G. Harrison 19. Recombinant Microbes for Industrial and Agricultural Applications, edited by Yoshikatsu Murooka and Tada yuki lmanaka 20. Cell Adhesion: Fundamentals and Biotechnological Applications, edited by Martin A. Hjortso and Joseph W. Roos

edited by Juan A. Asenjo and Josd C. Merchuk 22. GeneExpressioninRecombinantMicroorganisms, edited by Alan Smith

21. Bioreactor System Design,

ADDITIONAL VOLUMES IN PREPARATION

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GENE EXPRESSION IN RECOMBINANT MICROORGANISMS editedby

Alan Smith

CIBA-GEIGY AG Basel, Switzerland

MarcelDekker, Inc.

NewYork.Basel

HongKong

Library of Congress Cataloging-in-Publication

Data

Gene expression in recombinant microorganisms le d i t e d by Alan Smith. p.cm. - (Bioprocesstechnology ; 22) Includes bibliographical references and index. ISBN 0-8247-9543-1 1. Microbialgenetics.2.Recombinantmicroorganisms.3.Gene expression.4.Geneticengineering-Methodology.I.Smith,Alan. II. Series. QH434.G44 1994 660’.65-d~20

94-35435

CP The publisher offers discounts on this book when ordered in bulk quantities. For more information, write to Special Sales/Professional Marketing at the address below. This book is printed on acid-free paper. Copyright 6 1995 by Marcel Dekker, Inc. All Rights Reserved. Neither this book nor anypart may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, microfilming, and recording, orby any information storage and retrieval system, without permission in writing from the publisher. Marcel Dekker, Inc. 270 Madison Avenue, New York, NewYork10016 Current printing (last digit): l 0 9 8 7 6 5 4 3 2 1 PRINTED IN THE UNITED STATES OF AMERICA

Series Introduction

Bioprocess technology encompasses all ofthe basic and applied sciences as well as the engineering requiredto fully exploit living systems and bring their productsto the marketplace. The technology that develops is eventually expressed in various methodologies and types of equipment and instruments builtup along a bioprocess stream. Typically in commercial production, the streambegins at the bioreactor, which can bea classical fermentor, acell culture perfusion system, or an enzyme bioreactor. Then comes separation of the product from the living systems and/or their components followed by an appropriate number of purification steps. The stream ends with bioproduct finishing, formulation, and packaging. A given bioprocess stream may have some tributaries or outlets and may be overlaid with a variety of monitoring devices and control systems. As with any stream, it will both shape and be shaped with time. Documenting the evolutionary shaping of bioprocess technology is the purpose of this series. Now that several products from recombinant DNA and cell fusion techniques are on the market, the new era of bioprocess technology is well established and validated. Books of this series represent developments ili

iv

Series Introduction

in various segments of bioprocessing that have paralleled progress in life the sciences. For obvious proprietary reasons, some developments in industry, although validated, maybe published only later,if at all. Therefore, our continuing series willfollow the growthof this fieldas it is available from both academia and industry. W. Courtney McGregor

Preface

The last decade has witnessed a remarkable growth inthe development of recombinant DNA technology. Applications include the use of genetically engineered microorganisms for the productionof biopharmaceuticals such as insulinand interferon; the early diagnosis of disease; gene therapy for inherited diseases; and the creation of transgenic animals for food production, for production of therapeutics, and for use in screening for new biologically active substances. Agricultural applications include the development of new plant strains, of plants resistant to pests and pesticides, and the deliberate release of engineered organisms for crop protection. The technology has also been exploitedto further our understanding of basic metabolism and its regulation. This in turn has led to new approaches inthe production of substances derived from multigene pathways such as antibiotics. Slowly, but inevitably, the once impenetrable barriers between species have been broken down. This rapidly developing field has also been accompanied abypublic debate about the ethics of genemanipulation and about environmental and safety aspects. It is not the intention of this bookto address thewhole field of gene technology, but rather to focus on one of the least contentious areas-gene V

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Preface

expression in recombinant microorganisms. Nevertheless, the chapter on regulatory and safety aspects will confront the environmental question and attempt to put into perspective the risksand benefits of this exciting technology now at our disposal. Scientists with international reputations have been recruited to compile up-to-date reviews on gene expression in recombinant microorganisms. Each chapter provides a specialist’s account of gene expression in one of the important classes of microorganism used to express both homologous and heterologous genes. Each chapter describes the state-of-theart for the expression system, provides examples of the expression of homologous and heterologous genes, documents the industrial and commercial exploitation of the system, and speculates on future prospects and developments. More precisely, the features discussed include host strain characteristics, selectable markers, protease-deficient strains, transformation systems, plasmid vectors, integrating vectors, copy number, stability, constitutiveand regulated promoters, activators, secretion signals, posttranslational modification, glycosylation, S -S bridges, and protein folding. Where appropriate, the authors describe aspects of metabolic regulation and fermentation process technology. The individual chapters are “validated”by extensive referenceto the published literature. The chapter on regulatory and safety aspects reviews current legislation in the United States, England, Europe, and elsewhere. The measures taken for large-scale operation are also addressed, including the evolution of good industrial large-scale practice (GILSP), as regulatory authorities seek to achieve a balance between safety requirements and regulatory excess. The gram-negative bacterium Escherichia colihas been thefront-runner in the commercial application of rDNA technology and there are highly developed fermentation processes operating on a large scale for wellestablished products. Joan Stader’s chapter describes how this leading position is based on the expanding knowledge about the basic biology of this organism. The availability of a range of regulated promoters provides numerous options for efficient gene expression. In particular, the system basedon the T7-RNA polymerase aispowerful tool for high-level expression of heterologous genes. However, this organism brings with it a number of problems. Among themare the difficulties of protein secretion across the dual membrane system of the gram-negative bacterium, the contamination of products with endotoxins,and the reluctance with which correctly folded proteins containing disulfide bridges are produced. The synthesis of fusion proteins is one way to overcome some of these

Preface

VI1

problems and can be combined with specific procedures for protein purification using affinity chromatography. The bacilli have a number of features that make these gram-positive bacteria potentially attractive hostsfor gene expression. There isa long history of successful industrial exploitation and the capacity to secrete large amountsof homologous proteins. In hischapter, Matti Sarvas describes the expression vectors based on host a-amylases and proteases. Most work has been done using Bacillus subtilis, but B. brevis also has promise as a host for the expression of heterologous genes. The major problems that have been confrontedare the degradation of secreted proteins by the host’s own exoproteases andthe apparently narrow rangeof heterologous proteinsthat can be secreted in reasonable yield. There appears to be an incompatibility with theBacillus secretory machinery in many instances.A case-by-case judgment is clearly called for since sometimes this host may indeed be the system of choice. Others have turned to the eukaryotes for solutions to problems encountered with prokaryotic host/vector systems. Thus the yeast Saccharomyces cerevisiae has been successfully exploited for the production of certain heterologous proteins. Hinnen and co-workers describe the expression system based on the indigenous 2-pm plasmid. Gene expression can be constitutive or inducible and there are examplesof the efficient secretion of proteins into the culture medium. The anticlotting protein hirudin is an example ofa heterologous proteinthat is well expressedand secreted. The host strain can also be tailored using molecular genetic procedures to eliminate proteasesthat would otherwise degrade the foreign protein. Experience has shown, however,that some proteinsare poorly secreted by yeast and glycosylation can present additional problems. The example of insulin-like growthfactor shows that there is still much to be learned about posttranslational events leadingto a correctly folded, biologically active product. The methylotrophic yeasts, represented by the genera Pichia and Hansenula, present an interesting alternative. Gerd Gellissenand colleagues describe the exploitation of the strongly inducible promoters associated with growth of these organisms on methanol. Here the use of a cheap substrate and the possibility of growing cultures to high cell densitiesare attractive features to the fermentation technologist. The constructs are stable by virtue of integration into the host genome, and in the case of Hansenula the foreign gene can be present as multiple copies. As with Saccharomyces, there are examplesof both intracellular and extracellular products.

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Other candidates among the eukaryotes are the filamentous fungi. Again, there is a wealth of experience in large-scale fermentationof these organisms, and the genus Aspergirlus could be a viable alternative to the other well-established host/vector systemsfor the production of heterologous proteins.Jaap Visser and his colleagues document thefungal expression system, based on the database of knowledge established for Aspergillus nidulans and Neurospora crassa and now extended to the industrially more interesting strains such as A . niger. The filamentous fungi are particularly interestingfor their abilityto secrete large amounts of homologous proteins. Practical illustrations of the potential of this class of organism are provided by the production of pectinases for use in the food and beverage industryand of glucose oxidase, which hasapplications in the production of gluconic acid, in construction of biosensors, and in diagnostics. The interest in recombinant DNA technology is not, however, restricted to the production of heterologous proteins. The interest in secondary metabolites, such as antibiotics, has a much longer history. The genus Streptomyces is a prolific producerof low-molecular-weight compounds of commercial interest. Here also, recombinant DNA technology has been exploited to improve strains, to create new hybrid molecules, and indeed to clone complete.biosynthetic pathways for certain compounds. The availability of vectors that replicate in more than one species has opened up the way to express individual genes or clusters of genes in the organism of choice. In Chapter 6, Richard Baltz has compiledan extensive review of gene expression and regulation in Streptomyces, which illustrates the excellent progress in understanding these organisms and also points to the work that still remains to be done. our intention to provide a state-of-the-art In compiling this book it was documentation of gene expression in the most important of classes recombinant microorganisms,to provide comprehensive reference to the literature, and to illustrate progress with concrete examples. We hope that this book will be of special interest to all those working in the fieldof gene expression, whether they are studying the regulation of primary or secondary metabolism, expressing homologous genes or heterologous gene products for the biopharmaceutical industry.It is also our intention that this book should be of general interest to those in the scientific community as a whole seeking a comprehensive description of the scientific and commercial application of microbial recombinant DNA technology. Alan Smith

Contents

Series Introduction Preface Contributors

1. Gene Expression in RecombinantEscherichia coli Joan Stader 2. Gene Expression in RecombinantBacillus Matti Sarvas Recombinant 3.Expression Gene Yeast in Albert Hinnen, Frank Baton, Bhabotosh Chaudhuri, Jutta Heim, Thomas Hottiger, Bernd Meyhack, and Gabriele Pohlig

4. Gene Expression in Methylotrophic Yeasts Gerd Gellissen, Cornelis P. Hollenberg, and Zbigniew A . Janowicz

iii V

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1 53

121

1%

ix

X

Contents

5. GeneExpressioninFilamentousFungi:Expression of Pectinases and Glucose Oxidase in Aspergillus niger Jaap Visser, Henk-Jan Bussink, and Cor Witteveen

241

6. Gene Expression Recombinant in

309

Streptomyces

Richard H. Baltz 7. RecombinantMicroorganisms:Safetyand Regulatory Aspects Anthony J. Taylor

383

Index

407

Contributors

Richard H. Baltz Lilly Research Laboratories, a Division of Eli Lilly and Company, Indianapolis, Indiana HenkJan Busink WageningenAgriculturalUniversity,Wageningen, The Netherlands Frank Buxton CIBA-GEIGY AG, Basel, Switzerland Bhabotosh Chaudhuri CIBA-GEIGYAG,Basel,Switzerland Gerd Gellissen Rhein Biotech GmbH, Dusseldorf, Germany

CIBA-GEIGYAG,Basel,Switzerland AlbertHinnen* CIBA-GEIGYAG,Basel,Switzerland

JuttaHeim

Cornelis P.Hollenberg Heinrich-Heine University, Diisseldorf, Germany

Thomas Hottiger CIBA-GEIGYAG,Basel,Switzerland Zbigniew A. Janowicz Rhein Biotech GmbH, Dusseldorf, Germany *Present qffiliution: Hans-Knlill-Institut fur Naturstoff-Forschung, Jena, Germany. XI

Contrlbutors

xll

BerndMeyhackCIBA-GEIGY

AG, Basel,Switzerland

Gabriele Pohlig CIBA-GEIGY AG, Basel,Switzerland Matti Sarvas National Public Health Institute, Helsinki, Finland Joan Stader University of Missouri -Kansas City, Kansas City, Missouri Anthony J. Taylor* Health and Safety Executive, London, England Jaap Visser WageningenAgriculturalUniversity,Wageningen,The Netherlands

Cor Witteveen Wageningen Agricultural University, Wageningen, The Netherlands

*Present qffiliation: Department of Health, London, England.

GENE EXPRESSION IN RECOMBINANT MICROORGANISMS

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l Gene Expression in Recombinant Escherichia coli Joan Stader University of Missouri-Kansas City, Kansas City, Missouri

1 INTRODUCTION Twenty years ago, the successful expression of heterologous proteins in Escherichia coli was largelya matter of luck. Atthat time, the focus was primarily on promoter strengthand how well the bacterium could tolerate the foreign gene or protein product. In many casesthe DNA sequence was not known, and genes were localized by restriction mapping based on a limited number of available enzymes. If gene expression failed to materialize, few options for troubleshooting were available because our understanding of recombinant gene expression was not sufficiently advanced. Much progress has been made since that time; and although some proteins are difficult to express in E. coli, a persistent researcher who takes a systematic approach is likely to succeed. It follows that the more we understand about the host organism in general, the better our chances of making it produce recombinant proteins. This is why E. coli is sucha good host-no organism is better understood in termsof genetics, physiology, or biochemistry. It is this wealth of information that has led to our present high level of sophistication in 1

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E. coli recombinant gene expression, and consequently, it is now possible to express genes from almost any organism inE. coli. The primary aimof this chapteris to summarize the molecular genetic principles that are germane to successful recombinant gene expression in E. coli and to present options for designing such a project. Many of the variables that affect gene expression itself are understood well enough that they can eitherbe controlled or counteracted. For instance, the diminishing effect of low plasmid copy number could be balanced by optimal promoter strengthand translation initiation signals. In some cases, a high plasmid copy number in combination with a weak promoter may yield the same results. It is likely that maximization of all aspectsof expression isunattainable because eventually one step will become limiting. Thus optimization may be achieved through a combination of choices that can be changedor substituted as the situation dictates. Our present ability to be flexiblein our approach to heterologous gene expression in E. coli means that the solution to the problem is still empirical but possible in almost all cases. In this chapter I do not recommand any specificapproach to gene expression because each gene presents unique challenges. However, since these challengesare not known at the outset, some general recommendations can be made with respectto the main issues of plasmid copy number, regulation of gene expression, promoter strength, and translation signals. It makes senseto take the simplestapproach at the beginningand make modifications along the way to optimize expressionof a particular gene. For example, use of a high-copy-number vector is advisable because highDNA yields makeit easier to perform in vitro DNA manipulations. Tight gene regulation is recommended because many genes are toxic when presenton a high copy plasmid. If the promoter is regulated by a repressor protein,it is important to have sufficientlevels of repressor available. The T7 phage promoter, which is currently very popular for gene expression, doesnot eliminate the necessity for repression but simply diverts the problem from direct repression of the gene of interest to repression of the T7 polymerase gene. Optimization oftranslation signals is dealt with most easilyby using vectors that allow the constructionof in-frame gene fusionsthat place the gene of interestat the 3’ end of the fusion. Of course, the gene fusionapproach would be unsuitable in cases where the hybrid proteinproduct is uselessfor the intended purpose.Beyond these general recommendations, other choices to be made depend on the project’s objective and required downstream operations, such as fermentation, protein purification, cleavage of fusion proteins, and re-

Expression Gene

Recombinant in fscherlchia coli

3

naturation. Finally, personal preference or researcher skills can be accommodated by alternative choices.A researcher who possesses greater skills in the area of DNA manipulation may be better off engineering an expression schemethat involves a more complicated cloning strategy to avoid protein purification problems later, while another researchermay be more comfortable with the reciprocal arrangement. In this chapter I discuss some of the pros and cons of the various approaches. Thus protein overexpression should be approached systematically, first addressing themajor issues of choosinga vector, then optimizing expression by making sequence changes or gene fusions,and finally, by experimenting with different host strains and growth conditions. The scaling up of expression for fermentation requires special considerations that need not be addressed in the early stages of development. These issues are not a primary focusof this chapterbut are addressed in instances where they seem relevant.

2 PLASMID VECTORS Most current examples of overexpression inE. coli utilize derivativesof pBR322. These plasmids carry a ColEI origin of replication and their copy numbers range from 20 to 700 per cell, depending on whether or not sequences requiredfor regulation of replication are present. There are good reasons for utilizing the pBR322 derivatives, such as a known DNA sequence, a proven track record, and knowledge of the details related to its mechanisms of replication, stability, mobilization,and amplification (1,2). Other repliconsthat differ from ColEI plasmids ainnumber of characteristics may alsobe useful for certain special situations (see Ref. 3 and citations therein);for example, derivatives of plasmid pSClOl are generally used when a low-copy-number vector is required. Most expression vectors carry a selectable marker and a strong promoter. Some vectors carry terminators, antiterminators, a repressor gene, translation signals, secretion signals, reporter genes, orthe potential for producing a hybrid proteinproduct. In Table1 are listed gene expression plasmid vectors in current use which are available commerciallyor can be obtained by request from the laboratories that developed them. Vectors designed primarilyfor general cloningand sequencing, such asthe pUC series, are also used for expression purposesbut are not listed. They can be used as an alternative to subcloning a gene into one of the expression vectors and customized through the of usecassettesthat carry someof the gene expression elements.

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Expression Gene

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Plasmid stabilityand copy number should be of concern an in expression system. Higher plasmid copy number will increase gene expression only if copy number is limiting the in first place. If the gene of interest is under the control of a repressor, an increase in copy number will be accompanied by an increased requirement for repressor molecules. If an antibiotic marker is present onthe plasmid, higher levels of the drugare required. In addition, high plasmid copy number places a greater demand on the physiologyof the host cell through its requirementfor factors involved in plasmid replication and maintenance. This situation may lead to slow growth of the host cells or outgrowth of cells that have a lower copy number.

2.1

Plasmid Stability

There are two types of plasmid stability: structural and segregational. Structural stability refers to the absence of insertions, deletions, and DNA rearrangements. Segregational stability refers to the retention of a plasmid in a population of growing cells. The stability of a plasmid should always be monitored by confirming the presence and size of the plasmid and assaying for production of the gene of interest prior to and during expression, especially during fermentation. Strains containing an overas frozen glycerol stocks and removed expression plasmid should be stored immediately priorto use -they shouldnot be usedif they have beenon a plate for several days. Depending onthe magnitude of the project, it is worthwhile to perform pilot experiments to determine growth conditions that will allow for optimal plasmid stability. In expression systems, structural stability is usually maintained through the avoidance of DNA sequences that contain intramolecular homologies and by utilizing areCA host. Nevertheless, insertion sequences present a potential problem, as these elements transpose randomly and independent of homologyor RecA function. Even though the frequency of transposition is low (about 1 in 108 cell divisions), insertional inactivation of a toxic gene confers a selective growth advantage when it arises in the population. There are no E. coli hosts lacking insertion sequences, but selection for rare insertional activation events can be avoided through the proper choice of transcription signals controlling expressionof the toxic gene (see below). Segregational stability is facilitated by par sequences present on some plasmid vectors that ensure plasmid inheritance to both daughter cells during cell division or by plasmid-encoded genes that confer a strong selective advantage in a given growth medium. Toxicity of resident genes can leadto segregational instability throughthe selective advantage con-

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Stader

ferred on plasmidless cells. Otherfactors, such as transcriptional activity of plasmid genes, growth conditions, selectable markers, and the genetic background of the host, strongly influence segregational stability. Any one of these factors could have a profound effect on protein yields. 2.1.1 SelectableMarkers In general, there are two ways to select for retention of overexpression plasmids in bacterialcultures: a suicidal mechanism or antibiotic resistance. In the suicidalapproach, the hostcell is missingan essential function that is supplied on the plasmid. For example, an amino acid auxotroph can be transformed with a plasmid that encodes a wild-type copy ofthe defective chromosomal gene. If the cells are grown on minimal medium lacking that amino acid,cells that do not inherit the plasmid will not survive. The advantagesof this approach are that it is inexpensiveand eliminates the possibility that the purified product will have antibiotic contamination. The disadvantages are that expression of the selected gene is not regulated as it normally is in the chromosome, and the daughtercells may obtain sufficient quantitiesof the gene product in the cytoplasm to survive for several generations in the absence of the plasmid. Antibiotic resistance is more commonly used because it allows for an extended host range and provides more flexibility in growth conditions; however, heterogeneous populations may arise when the antibiotic is inactivated or bound to the resistance geneproduct, lowering the effective concentrationsand allowing plasmidless cells to survive. The mode of action, mode of resistance, and working concentrations of the most commonly usedantibiotics are described in Ref. 3. 2.1.2 Transcription Levels and Plasmid Stability A decrease in the stability of plasmids canying nontoxic cloned genes under the control of strong promotersmay bedue to high-level transcription and replication interfering with one another. For instance, a comparison of high-level expression between a pBR322 derivative and a pUC derivative indicated that recombinant protein levels were much higher with the lower copy pBR322 derivative(4). The lower protein levels seen from the pUC derivativeseemed to be related to plasmid stability, and there was a strong correlation between high transcriptional activityand plasmid instability. 2. l.3 Plasmid Stability Under Various Growth Conditions Unfortunately, thereis no formula for increasing the stability of recombinant plasmids relative to growth conditions because every expression

Expression Gene

in Recomblnant Escherichia

coli

9

system is unique. In general, growth conditions that do not select for plasmid loss shouldbe used. Cells carrying plasmids require more energy than do cells that are plasmid-free(5); thus nutrient(6) or oxygen (7) starvation conditions should be avoided. It was reported recently that the presence of the bacteriophageX cos fragment made plasmid-containing cells as fit as plasmid-free cells when grown under starvation conditions (8). The reason for this phenomenon is not known, nor isit known if it will be generally applicable. Scaling-up operations from growth in shake flasksto continuous culture may result in a decrease in plasmid stability and(9) measures should be taken to monitor plasmid loss and devise schemes to avoid it. Sometimes this will involve altering the construct (7,9) or switching host strains. been reported (Q, in which the acquiThe use ofa "preadapted" strain has sition of one or more mutations during growth in a fermentor madeit more fit as a plasmid host, but the nature of these changes was not studied. Finally, systematic studies of plasmid vector stability have shown that the presence ofthe tet gene accelerates plasmid loss in the stationary phase (10) and there is a strong selection for mutations in tet (6,11), probably because the tet gene product is an inner membrane protein and E. coli has a limited tolerance for highly expressed inner membrane proteins.

2.2 Plasmid Inheritance and Copy Number Low-copy-number plasmids that are derivatives of pSClOl are usually present at around six to eight copies per cell (12). Inheritance of these plasmids is controlled by thepar locus, and they are stable even in the absence of a selectable marker (9,12,13). The par locus isa cis-acting site that is recognized by hostfactors to ensure proper partitioning, but it has no effect on copy number (12). Copy number in pSClOl is positively controlled by the plasmid-encoded replication protein, RepA (14), a protein whose expression is autoregulated (15). There is no partitioning function in pBR322 derivatives (ColEI replicons); thusthe maintenance of high-copy-number plasmids in a growing as described above. culture requiresthat they confer a selective advantage, Inheritance of multicopy plasmids is thought to be simplya matterof receiving a certain portion of plasmids residing in the cytoplasm,but this has never been proven. Copy number in ColEI plasmids is controlled by negative regulation of replication. Initiationof replication requires the binding of the plasmidencoded primer, RNAII, to the ori region (18,19). RNAII is first made of replication. Initiationof replication requires the binding of the plasmid-

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Stader

as a larger precursor which is processed by host RNaseH as an activation step prior to initiation of replication. Negative regulationof replication occurswhen another plasmid-encodedRNAmolecule,RNAI, hybridizes with RNAIIto prevent processing (20,21). Anadditional level of regulation comes from the plasmid-encoded rop (rorn)gene product, a repressor of transcription of RNAII (22). The levels of RNAIand Rop are regulated by plasmid copy number (gene dosage). Thus low number a of plasmid copies favors replication, and a high number of copies results in inhibitionof replication. For pBR322 derivatives, the normal number of copies present per cell is about 20 (2,3,23). This number can be increased five-to sevenfold through deletion of rop (U), and a further increase in copy number can be achieved through a point mutation immediately 5‘ to the coding region of RNAI (25). The pUC vectorsboth carry types of mutation, and the resulting copy number is about 600 (25).The effect on copy number of the latter mutation alone hasnot yet been reported. Several efforts have been madeto construct hybrid plasmids to obtain the high copy number of the pBR322 derivatives and the stable partitioning of the pSClOl (13,16,17). For instance, it wasfound that placing the pSClOl par locus on pBR322 increased plasmid stability 3- to 10-fold (16). Another example isthe “runaway replication” type of plasmid (13), in which a low-copy-number plasmid carrying the rep and par loci from synpSClOl is coresident with amutant ColEl replicon whose RNAII is thesized under XPLcontrol. These plasmids requirethe presence of the temperature-sensitive X repressor, cI857. At the nonpermissive temperature, plasmid replication occursand copy number will increase to about 200 copies per genomeafter 6 h. The temperature-sensitive replicon can also be used as a cassette, so that runaway replication canbe conferred on other plasmids.

2.3 ChromosomalExpressionVector A seriesof expression vectors has now been produced which are designed to reside in the chromosome (26). These vectors (MudMON) wereproduced for increased stability in the absenceof selection. The gene to be expressed is cloned into a plasmid vector that contains bacteriophage Mu sequences capable of transpositioninto the chromosome and can be maintained asa defective prophage.The gene of interest is placed under the control of the Mu middle promoter, which is efficiently repressed. In the presence of a temperature-sensitive repressor and a helper phage, replication of the vector and expression of the geneare both induced at

Expression Gene Recombinant in Escherichia

coli

11

42°C. Several heterologous proteins have been expressed with this system, and the levels seem to be dependent on the cloned gene itself. Because Mu has abroad host range, these vectors can alsoused be in other bacterial host strains, such as Serratia, Erwinia, Citrobacter, Pseudomonas, and Agrobacterium.

3 TRANSCRIPTIONSIGNALS With the exception of theT7 RNA polymerase-specific system, thepromoters utilized inE. coli expression vectorsare the u70 type because they are the most thoroughly understood. The bulk of routine gene expression in E. coli is handled by u70, and the promoters it recognizes are characteristically flexible in terms of levels of gene expression during changing growth conditions. The promoters are controlled at one or more levelsto respond to inducers and activators either specificto the operon in question or as part of global regulation systems. Many of the cis elements that participate in controllingu70 gene expression are a considerable distance away from the actual RNA polymerase binding site (27), and such remote control regions are usually not utilized in plasmid constructions. Therefore, regulation from a given promoter on a small, high-copy-number plasmid can be considerably different from that which takes place in a single copyon the chromosome. RNA polymerase holoenzyme recognizes a consensus DNA sequence centered at the - 35 and - 10 positions upstream of the start of transcription (+ 1). A close correspondence to the consensus sequence (Tathe rate-limiting ble 2) is required for efficient RNA polymerase binding, step in transcription initiation. Depending onthe operon, there are any number of cis- and transacting elements that serve to modify the ability of RNA polymerase to carry out a productive initiation (28). Once initiated, transcription usually proceedsto the termination site unless premature termination occurs. InE. coli, such premature termination occurs in cases of attenuation (29) or polarity (30-34). Both events depend on concomitant translationof the mRNA inwhich the ribosomestalls or dissociates from the message. When ribosomal stalling occurs, the message to the 3' side becomes void of ribosomes, allowing termination to occur. Attenuators are rho-independent terminators, which contain the canonical GC-rich region with dyad symmetry, capable of forming a hairpin structure, followed by a run of U residues at the 3' end. During translation, the ribosomes prevent the hairpin structure from forming; but if the ribosome stalls upstreamof this site,the hairpin structure will form and ter-

Expression Gene

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minate transcription. Stalling may occur at a codonfor which the aminoacyl-tRNA is limiting, such as conditions of starvation for a certain amino acid. E. coli has developed the ability to exploit this phenomenon inthe regulation of amino acid biosynthetic operons(29). Polarity sitesare rho-dependent terminatorsthat become active when the mRNA is void of translating ribosomes.When rho binding sites are exposed on the mRNA, the terminationfactor rho can bind and mediate the release of the transcript from a paused RNA polymerase elongation complex. Such RNA polymerase pause sites occur naturally within some transcripts (35). The rho recognition site is believedto be C-rich and includes a potential hairpin structure with a CAA loop and preceded byan rho recACCCCA consensus(36)based on in vitro binding studies, but ognition of such sites in vivo has not yet been reported. This type of premature transcription termination is beneficial to the organism because polycistronic messagescontain gene productsthat are often needed for a common pathway. Ifan upstream gene cannot be made because ofa frameshift or ambermutation, the downstream gene products wouldoften be useless. Transcription termination prevents the cell from expending the energy required to make these useless gene products. In the expression of genes in E. coli, premature termination can be avoided by examining the sequence of the gene for both rho-dependent and rho-independent termination sequences. If these sequences occur, they will not necessarily leadto premature termination; however, they can be modified by making conservative changesthe in DNA sequence. Alternatively, an ahtitermination mechanism can be employed through utilization of the bacteriophage X N-nut system. In the context of wild-typeX gene expression, theNgene is the promoter proximal gene under PLcontrol. After the initial induction of PL,a transcript of geneNis produced which ends at the terminator immediately followingthe gene (37). Upon translation of this message, N proteinacts on the nut (N-utilization) site (38)located downstreamof the promoter. When RNA polymerase passes this site, it acquires the ability to continue transcription through both rho-dependent and rho-independent terminators (38-40).This system is especially useful in cases where there is a long,untranslated 5’ region in the cloned DNA. It has been reported that use of the N-mediated antitermination system caused a three-to eightfold increase in protein production, and this system leadsto increased expression even in the absence of a terminator (41). Some expression vectors currently available provide the N gene and the nut site engineeredto prevent premature termination within the cloned sequence(42).

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3.1 Promoters The most highly utilized promoters for overexpression have two features in common: they are (a) efficient and (b) repressible. Some promoters can direct protein synthesisto near 50% of total cellular protein. This is accomplished by growing cultures to optimal density, performingthe required induction, and expressing the protein for several hours. Repressible promoters are superior to unregulated promoters because continuous expression of plasmid-encoded proteins generally puts the hostat a selective disadvantage ina population where some cells have lost the plascoli promoters used mid or acquired deletions and other mutations. E.The for expression of heterologous proteins are shown with the promoter consensus sequence in Table 2 and their salient features are presented below.

3.7.7 Plac In the chromosome, this promoteris negatively regulated by binding of the trans-acting repressor protein, LacI, to the operator site, lac0 (43, 44).There are three operators to which LacI can bind:one near the start of transcription, one at a remote site upstream,and one at a remote site downstream (27). Binding of the inducerto LacI causesan allosteric transformation of the repressor, leaving it unable to bind the operator (45). Lactose isthe natural inducer for this operon,but the gratuitous inducer isopropyl-6-D-thiogalactopyranoside (IPTG) is more powerful than lactose becauseit is not metabolized. In addition to inducer, expression from the lactose promoter requires cyclic AMP and the catabolite activator protein (CAP)(46,47). In the presence of the IPTG, cyclic AMP, and CAP,expression of IacZ is increased 1000-fold(48). The complex regulationof the lactose operon is greatly simplified in cloning vectors where onlyone operator is present, the commonly used lacUV5 promoter is independent of positive regulationCAP by (49), and the gratuitous inducer IPTG is employed to bring about sustained induction. Many of the popular cloning vectors in current use contain the lac promoter, followed by a multiple cloning site within the sequence encoding the LacZa peptide, allowingfor easy screeningfor insertions. The LacZa peptide consistsof 39 amino acidsof the amino terminusof LacZ @-galactosidase), which is capable of restoring enzymatic activity to a mutant LacZ missing amino acids 11 to 41 (the w-fragment) (50). The w-fragment is encoded by the IacZA MI5 allele in the chromosome of the host cell. In the absence of an insert, the plasmid will direct the expression of LaczOr, resulting in a blue colony on agar containing IPTG

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and the chromogenic substrate for &galactosidase, 5-bromo-4chloro3-indolyl-fl-~-galactopyranoside (XG). If foreign DNA has been inserted into the multiple cloning site, the coding regionfor L a c 2 is disrupted and the colony appears white. Thus genes cloned in these vectors can easily be expressed following induction withIPTG. Plac should carry To maintain plasmid stability, host strains carrying a lacZ allele that allows for constitutive productionof the lac repressor. The lacZq allele will result in the production of 10-fold more repressor .than that normally found inthe cell (51,52), but this level of repressor is normally not sufficient when high-copy-number plasmids are being used. Another allele, lacP, produces 100-fold more repressor (51-53) and is better suited for expression systems. Some cloning vectors carry IacZq on them and produce higherlevels of repressor asa result of the higher gene dosage. Such constructs confer more efficient repression of the promoter and also allowfor a broader host range.

3.7.2 Ptrp The product of the trpR gene negatively regulatesthe trp promoter (54). The aporepressorform is a TrpR dimer which is unable to bind tightlyto the operator (55). When complexed with L-tryptophan, the TrpR dimer becomes a repressor that tightly binds the operator and prevents the binding of RNA polymerase(54). Induction is brought about by tryptophan starvation conditions when TrpR is in the aporepressor form. Another level of regulation of the trp operon occurs at the attenuator, resulting in premature transcription termination inthe presence of tryptophan (29). The trpR gene is autogregulated, and it has been demonstrated that in cells growing in excess tryptophan, approximately 50 molecules of repressor are available to bind the operators for trp, aroH, and trpR (56). The relatively high levels of TrpR and its autoregulationare well suited to an overexpression system in which multiple copies of the operator are capable of titrating out the repressor. However, may one expectthat there is a limit to the capacityof the cell to regulate when the copy numberapproaches several hundred. Indeed, problems have been encountered after attempts to clone genes encoding toxic proteins undercontrol of the trp promoter. It is not clear whether these problems arise because repression of the promoter is insufficient orif the repressor has beentitrated out. Some of the trp expression vectors also employthe translation initiation region of the leader peptide, but the attenuator has been removed (57). Host cells bearing plasmids withthe trp promoter should carry the wild-type trpR allele. Induction is brought about by tryptophan starva-

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tion, which can be accomplished through leaky mutations inthe trp biosynthetic genes or through the use of the tryptophan analog indolyl-3acrylic acid (IAA) in combination with mildtryptophan starvation (57, 58). On a pBR322-derived plasmid with a copy number of20 to 30, the trp promoter is capable of directing the production of 20 to 30% of the total cellular protein after 3 h of induction (58). The decision to use the trp promoter should take into consideration the tryptophan content of the protein to be produced. It has been observed that proteins with a high tryptophan content are not expressed well under the conditions described(58). One possible explanation that is ribosome stalling as a result of tryptophan scarcity exposes polarity sequences inthe mRNA and leads to premature transcription termination. 3.7.3 Ptac and Ptrc These hybrid promoters possess the efficiency of the trp promoter and the regulation of the lacUV.5 promoter by combining the - 35 region of former withthe - 10 region of the latter (5339). The difference between the two hybrid promoters isthat tac contains 16 nucleotides betweenthe -35 and - 10 regions and trc contains 17 nucleotides (Table2). Full induction of tac with IPTG has been reportedto yield fivefold greater expression of cloned genes than the lac promoter (45). Vectors carrying these promoters are available, and cassettes that will convert a lac promoter to a tac promoter are also available.

3.7.4 Bacteriophage h Promoters PL and PR In their normal context within a lysogen, both of these promotersare repressed bythe bacteriophage X protein cI, which is requiredfor maintenance of lysogeny (60-162). Production of c1 from the Pm promoter is autoregulated, and a lysogen will normally produce 200 repressor molecules per.generation (63). Wild-type repressor activity is regulated by the host protein RecA. Upon DNA damage by chemical treatment or ultraviolet irradiation, RecA activates autoproteolysis the of X repressor, leaving the repressor unable to bind the operator (64). Under these condito bind RNA polymerase and initiate tions, bothPLand PRhave the ability transcription that will eventually lead to lysis. About 80 to 90% of the repressor molecules must be inactivated to bring about lysis (65,66). Expression systemsthat utilize X promoters requirethe presence of the repressor supplied eitheron a plasmid oron a prophage. Two induction schemes have been employed. One method involves the use of nalidixic acid (67), which will activate RecAand alter the cellular physiology through

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activation of the SOS response. The c1 cleavage reaction is quite slow, taking approximately 30 minutes to inactivate 200 repressor molecules (68). This approach presents two problems: (a) the promoter does not become rapidly activated,and (b) the dependence on RecA for induction is sometimes in conflict with the desireto use a reCA host. These problems can be circumventedby using the second induction scheme, which employs the temperature-sensitive c1857 allele (68). At the permissive temperature (30°C)the repressor binds the operator and promoter activity is shut off; at the nonpermissive temperature (42°C)the repressor is inactivated (independentof RecA) and the hostRNA polymerase beginsto transcribe PR-or P,controlled genes. Although heat induction is the promoters, problems arise most commonly used method of utilizing Xthe from the concomitant induction of the host heat shock response. The E. coli heat shock response induces the production of a variety of proteins, most notably cellular proteases, which could confound efforts to isolate recombinant proteins. Two possible ways of circumventing this problem are by using a heat shockmutant host strain [HtpR-, available as temperature-sensitive lethalmutations (69)] or pH induction. The latter approach has been described as being specific for the c1857 repressor and is independent of RecA (70). When the cl gene is present in a single copy and the X promoters are present in multiple copies, repression is not complete. Therefore, inclusion of the cl gene ona compatible plasmid oron the expression vector itself is beneficial, especially in cases where complete repression is essential. The X PLpromoter is about 8- to 10-fold more efficientthan PIac (41,71) and therefore about twice as efficientas Ptac. 3.1.5 Bacteriophage 7 7 Promoter Use of the T7 promoter system is described in detail elsewhere (72,73); a brief summary is provided here. Unlike the promoters described above, the bacteriophage T7 promoter requires the T7 RNA polymerase. Several advantages accompany this system: (a) transcription is completely selective-TT7 RNA polymerase will not recognize the host’s promoters; (b) Ti’RNA polymerase elongates mRNA chains five times faster than E. coli RNA polymerase,and the accumulation of message risesrapidly, approaching 100% of the cellular RNA synthesis (host mRNA synthesis can befurther suppressed bythe addition of rifampicin, an antibiotic to which the T7 RNA polymerase is refractory); and (3) expression of the cloned gene is completely activatable and a repressor is not required.

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However, the presence of T4 RNA polymerase can potentially negate this advantage if its synthesis isnot strictly controlled. Various approachesto repression of T7 RNA polymerase synthesis and activity have been developed, and one's choice dependson the toxicity of by induction the gene cloned.T7 RNA polymerase can be delivered either of a chromosomal copy of the geneor by infection with a specialized bacteriophage X transducing phage that carries the gene. In the former apapproach, the host strain is a lysogenthat carries a copy of the T7 RNA polymerase gene on the prophage, along with l a d . The T7 RNA polymerase gene is underthe control of the lacUV5 promoter and repressed until IPTG is added to the growth medium.The natural basal levelof activation of the lac promoter will result in a low level T7 of RNA polymerase synthesis, which may not be tolerated by the host cell if the cloned gene is highly toxic. If the cloned gene is highly toxic, low-level synthesis can be decreased effectively by the presence of T7 lysozymetheincell. T7 lysozyme isa bifunctional enzymethat hydrolyzes the bacterial cell wall (74)and inhibits T7 RNA polymerase (75). When presentat low levels in the cytoplasm, the lysozyme doesnot have accessto the cell walland will onlyact to inhibit the polymerase. The lysozyme gene is carried on a low-copy-numbercompatible plasmid in the host cell. Two versionsthat differ in thelevels of lysozymeproduced are available. For a slightly toxic cloned gene, low levels of lysozymeare sufficient; but highly toxic genes may require higher levels of lysozyme. An added feature of this system is that the presence of the lysozyme willfacilitate cell lysis duringthe protein isolation stage. It is best to use the lower levels of lysozyme if possible because the presence of lysozyme results in an initial lag in protein production following induction. Delivery of T7 RNA polymerase by phage infection is an approach developed for the expressionof genes that are toxic even in the presence of higher levels of lysozyme.By this approach, theT7 RNA polymerase gene is under the control of the strong XPLand PI promoters on the phage and is expressedas part of a lytic infection. The phage carries the temperaturesensitive repressor allelec2857 and Sm7, which prevents cell lysis (73,76). Under nonpermissive conditions (high temperature), this phage is unable RNA polyto enter the lysogenic state and will produce high levels of T7 merase, whichwill activate expression ofthe cloned gene. Synthesis of the cloned gene is so high that it interferes with phage development. The purpose of the amber mutation in theX S gene isto prevent cell lysis during expression. If this approach is utilized,the host strain should not be supF (76).

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3.7.6 OtherPromoters There are examples in the literature of the use of other promoters, some of which will be described below. Some of these promoters have been used only for certain types of expression systems, while others recently have been appliedto expression systems,and their utility isnot yet supported by a strong track record. Use of the PGpromoter from a naturally occurringPseudomonasputida plasmid has recently been described (77). The promoter is available as a cassette that can be utilized for expression inE. coli and other gramnegative bacteria. This promoter regulates the expression of genes required for the oxidation of salicylate to acetylaldehyde and pyruvate(78). Induction is brought about by the presenceof salicylate and the positive regulatory genenahR, which is also included on the cassette. Two phosphate-regulated promoters havebeen employed in expression systems. The first, PphoA, has been used in conjunction with the early coding regionof the phoA gene to construct a fusion betweenthe PhoA (alkaline phosphatase) signal sequenceand a heterologous protein (79). The purpose of these constructs is to target the recombinant protein to the secretion system (see below). The second, Pugp, has recently been reported to be effectivefor controlled overexpression of cloned genes (80). Its efficiency was shownto be about 80% of thatof P tac. Regulation of phosphate promotersis complicatedand still not fully understoodbut is known to involve multiple positive and negative trans-acting regulators (81). It has been reportedthat in addition to phosphate regulationPugp is under the global regulationof CAP, although it is unclear whetherthe latter is director mediated byanother regulator. The promoter is repressed in the presenceof glucose and phosphate, and induction occurs when glucose and phosphate are limiting. These conditionsare convenient and inexpensive and should be easilyadaptable to fermentation systems. 3.7.7 WhichPromoter Is Best? There isno standard answer to this question. Currently,the most widely used promoters are tac, trc, PR,PL,and W .The lac promoter is still used frequently, but the hybridtac and trc promoters offer the same regulation with higher expression levels. Since its development,the T7 system has become very popular and appears to have become the promoter of choice for high-level expression. Before choosingan expression system, it may be useful to examine the literature for expression of protein similar to the one you wish to express.

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3.2 Terminators Many examples inthe literature describe expression systemsthat utilize strong promoters withoutterminators behind the expressed gene. However, it has been shown that placement of a strong terminator at the 3' end of the gene results in greater stability of the plasmid (4) and greater expression of the recombinant gene(82). In the absence of a terminator, transcription can proceed into the plasmid, causing overexpression of other genes carried on the vector, potentially causing harm to the cell. For instance, overexpression ofthe tet gene can decrease the stability of the plasmid(6,ll). The presence of a terminator may be particularly crucial when cloning DNAfrom certain organisms(83). Several vectors currently availablecontain terminators on both sides of the multiple cloning site.

3.3 MessageStability mRNA molecules are commonly degraded in the 3'- 5' direction, and resistance of a message to exonuclease activity is conferredby a 3' stemof these strucloop structure(see Ref. 84 and citations therein). Thus some tures may actually play two roles; theyasact simple transcription terminators and they increase message stability. Message decay is mediated by both endonucleases and exonucleases. The exonucleases RNase I1 and polynucleotide phosphorylase (productsof the mb and pnp genes, respectively) are functionally redundant(85). Endonucleases that cleave mRNA include RNase I11 (the mc gene product) (Sa), RNase E (me/ams) (8789), and RNase K (88-90). The latter two enzymes play a general role in the chemical decay of bulk mRNA and there is currently a question toas whether they are separate, interdependent, or identical. RNase I11 has been shown to cleave RNA at specific recognition sites that contain double-stranded structures.This enzyme acts on various stable RNA speciesfrom bothE. coli and its bacteriophages and may play a role (91,92) or increasing turnover of cleaved mRNAs, in enhancing translation depending on the specific message(93-95). It has been proposedthat the degree of message stability is related directly to the efficiencyof translation initiation. The logic of this model is that closely packed ribosomes would make the message inaccessible to cellular RNases. Although the model may be true in some cases, an example in which it does not apply is the ompA mRNA. This message contains a structural element within its long 5' untranslated region (UTR) that

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confers a fivefold longer half-life on the adjacent coding region during periods of rapid growth. The stabilizing effect of the 5'UTR was found to be independent of translation initiation rate and adjacent coding region (96). Moreover, it has been determinedthat the ompA 5' UTR is a substrate of RNase K, which may control the rate of ompA turnover in vivo (90). The rolethat mRNA stability plays an in expression system can probe found or insignificant.If a toxic gene is being expressed and there is leaky expression froman uninduced promoter, enhancing stability of the mRNA may be lethal. Conversely, expression of a nontoxic genethat is poorly induced or carries weak translation signals may benefit from the addition of the ompA 5' UTR or expression in acell that carries an RNase defect. Indeed, useof an rnapnp double mutant defective for RNase I and polynucleotide phosphorylase has been shown to be effectivein improving the yield of recombinant porcine leukocyte interferon (97).If a gene product is not being expressed and message instability is suspected, turnover should be measured. Message stability canbe determined by measuring mRNA levels at various time intervals following the addition of rifampicin to the growth medium. These determinations should be done under the same growth conditions employedfor expression. 4 TRANSLATIONSIGNALS

It is at the level of translation that some of the most irksome problems arise in attemptingto overexpress heterologous proteins. The translation process is sufficiently different in prokaryotic and eukaryotic cells that it is not unusualto find sequences in the message that are incompatible with levels of gene expression in E. coli. At the root of the problem are two a message to be translated issues: (a) it is not fully understood what causes efficiently, and (b) optimization often requires changing nucleotides within the coding sequence of the gene of interest. Translation involves multiple components and steps (for current reviews see Refs. 98 and 99) and the resulting implicationis that each step is a potential target for regulation. Indeed, E. coli regulates the relative levels of geneproducts from a polycistronic message by alteringthe rate of translation through various mechanisms. Of all the components involved, mRNA isthe only highly variable one, and it is therefore the component that exerts the mostcontrol over translation rate. Optionsfor improving expressionat the translation level have been developedand will be presented later in this section.

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initiation

Initiation, elongation, and termination are all potentially rate-limiting steps intranslation, and of the three, the mostis knownabout regulation of expression at the stage of initiation. Multiple cellular components, including the 30s ribosomal subunit, the initiator tRNA, several initiation factors, and messenger RNAcontrol the fidelity and efficiency of the initiation process. It is essentialthat the mRNA meet strict criteria ensuring that translation always begins at the proper AUG codon and not elsewhere. Comparison ofthe initiation regionof a number of E. coli genes (100-104)as well as mutational analysis (105-113)has made it possible to determine someof the “rules” involved inthe process that aredescribed below. Still, much remainsto be elucidated since thereare numerousexamples of messages that do not conform to our composite pictureof what determines a good initiation site and yet are efficiently translated(98,99, 114). There are also sequences (“nongenes”) that conform to the rules that do not act as initiation sites (103,115,116).It could be that our inability to predict translation efficiency reliably isa reflection of our inability to predict mRNA secondary structure accurately.

4.1.1 RibosomeBindingSite (RBS) The ribosome binding site (RBS) isfunctionally defined as the portion of mRNA that is protected from RNase digestion by ribosomes that have bound but not initiated translation (117). This region consists of about 30 to 35 nucleotides extending from about -20 to + 13 of the message (+ 1 being the first nucleotide of the initiation codon). The individual the , initiation components are the Shine-Dalgarno (SD) sequence (la) codon (105),the spacing betweenthe SD sequenceand the initiation codon (103,114),the second codon(102),and other elementsof the primary sequence that are notwell understood. Occlusionof the SD sequenceand/ or thestart codon hasa profound effect on translation efficiency (1 18-122).

4.l. 2 Shine-Dalgarno Sequence It is thought that the purpose of the SD sequence isto allow for proper alignment of the ribosome with the initiation codon (123). The SD sequence isa purine-rich sequence positioned5 to 12 nucleotides upstream of the initiation codonthat is complementary to nucleotides at the 3’end of the 16s rRNA (104): rRNA: 3’AUUCCUCCACUs‘ mRNA: S’UAAGGAGGUGA3’

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The length of theSD sequence on the message varies, but the average is five nucleotidesand the majorityof E. coli messages studied so far carry the central sequences (underlined above) (103,115). Hybridization between the twoRNAs has been demonstrated biochemically (124) and genetically (125,126). There are conflicting opinions on the effect of longer SD sequences on the efficiencyof translation (99,123). This issue has been addressed in several studies, and the answer is not clear-cut. For example, a single base change that resulted inGAAG to GGAG increases synthesis 10-fold (127), while increasing complementarity to 13 nucleotides decreased synthesis by a factor of 2 (128). Context probably plays a role in regulating these expression levels. Clearly, if therea isnegative effect from a longer sequence, it is not large. 4.7.3 Spacer Region Between the SD Sequence and the Initiation Codon The distance between the SD and the initiation codon ranges from 5 to 13 nucleotides (99) and depends on the SD sequence and perhaps other elements inthe mRNA. The variability in the distance implies a precise distance between the region of complementarity inthe 16s RNA and the site of the initiator tRNA bound to the ribosome. The nucleotide sequence 1 triplet, affects in the spacer region, specifically the three bases in -the the initiation rate up to 20-fold (112)the most favorable beingUAU and CUU and the least favorable UUC, UCA, and AGG. 4.7.4 InitiationCodon The most commonly used initiation codonis AUG, but occasionally,UUG and GUG are employed in E. coli (105). 4.7.5 SecondCodon A systematic study revealed that (a) the second-position codon choice deE. in coli, and (b) there is a wide viates strongly from overall codon choice variation in translation efficiency depending on the choice of codon in the second position(102). It was demonstrated that the most commonly used second codon, AAA, is also themost efficiently translated, and these data are consistent with another study showing a strong bias towardAs in the more efficient ribosome binding sites (129). There was considerable variation among synonymous codons, suggesting that the nucleotide sequence is responsible for the effect. This study examined the effect of changing the second codon within given a context (thelacZ gene) and does not necessarily apply to other genes. For example, changing the codon

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from GAUto AAA in one context increased protein synthesis 10- to 15fold (102), while in another,an increase of onlytwo- to threefold was found (4). It is clear that for a given amino acid in the second position, some codons will be better choices than others.

4.2 Elongation In contrast to initiation, there seems to be no effect from mRNA secondary structure on elongation. Ribosome stalling during elongation can occur if stretches of rare codons appear in the message or if stretches of a single amino acid are required and the cell is starved for that amino acid,as in the case of attenuation. Ribosome stalling resultingfrom the appearance in the coding region of a SD sequence isalso believed to play a role in the frameshiftingthat occurs duringtranslation of release factor 2 (RF2) (130).

4.3 Termination

Unfortunately, little is known about the existence of regulatory mechanisms actingon translation termination. It is conceivablethat even a slight stalling at the stop codon could affect proteinlevels dramatically by caussubsequent ribosomes to back up. Whether this situation ever arises in normal translated messages has not been studied to my knowledge; but given that it isa multicomponent mechanism, it would be difficult to imagine that E. coli has not expolited its regulatory potential. 4.4 Optimization of TranslationEfficiency

Designing optimal translation efficiency into an expression system has been described previously(1 14). Briefly, translation initiationcan be optimized if consideration is given to the following factors: 1. mRNA sequences that have intramolecular structures involving the SD or the initiation codon should be avoided. 2. A long (five to eight nucleotides) SD is favorable for translation. 3. The spacing between the SD and the initiation codon is important

but depends on the length and sequence of the SD. As a result of the precise distance between the 3' end of the 16s rRNA and the binding sitefor the initiator tRNA inthe ribosome, longer spacing is more tolerable than shorter spacing. 4. AUG is most favorable as an initiation codon.

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5. The gene of interest should have >25 nucleotides 5’ to the initiation codon.If this region is extensive, translation termination codons should be present upstream of the ribosome binding site(RBS). 6. Second codon choice should be optimized. Rules for nucleotide choice downstream of the initiator codon are difficult to define and may be somewhat irrelevant in designing an expression system due to the constraint imposedby specific amino acid requirements the in protein product and the difference in context. For these sequences, optimization is restricted to silent codon changes.

Several approaches have been developed for optimization of translation initiation. They include (a) cloning the gene directlyinto a vectorthat has a strong promoter followedby a SD sequence and initiation codon, (b) constructing an in-frame gene fusion that contains the 5‘ region of a gene knownto have goodtranslation signals fusedto the gene of interest, (c) site-directed or random mutagenesis of the early coding region followed by screening for high-level synthesis of the gene product, and (d) placement of a short cistron upstream of the gene of interest to increase the chances of reinitiation at the downstream RBS.

4.5 Direct Cloning into a Translation Vector Several families of vectors have been developed that contain a strong promoter followed bytranslation signals and carry a unique restriction site at the start codon. Use of such a vector is aidedby the fact that a large proportion of eukaryotic genes contain an NcoI site(C’CKGG) at their start codon (131). Other restriction sitesthat carry the ATG sequence are NdeI, BspHI, and Aj7III. For genesthat do not carry one of these sites, one can be introduced by site-directed or PCR mutagenesis. Table 1 indicates the presence or absence of translation signals within expression vectors. Vectorsthat utilize the direct cloningapproach generally havea + / - ” under the “hybrid protein” heading because theyoften provide the option of cloning into one of the ATG-containing restriction sites,or cloning into a downstream site that will result in one or more extra amino acids incorporated into the amino terminus of the protein. The direct cloningapproach does not take into account the role of the early coding region in translation initiation. Therefore, if expression is not achievedby direct cloning,the problem may be circumvented by one of the following schemes.

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4.6 In-frameGeneFusions

These constructionsare a useful way of avoiding problems withtranslation initiation and also provide other advantages (see Section 6). First, cloning is simple. Several families of fusion vectors are available that carry unique restriction sites in all three reading frames, eliminating the need for nuclease treatmentor site-directed mutagenesis. These vectors appear in Table 1with a “+” under the “hybrid protein” heading. Second, the translation initiation signals have been optimized. Third, the amino-terminal moiety may provide a simple assayfor expression, a means of affinity purification of the hybrid protein, protection against proteolysis, or competence for export out of the cytoplasm. The major disadvantageto using a fusion protein arises in cases where the hybrid protein is not useful as such and requires precise processing and purificationto yield an authentic protein product. However, methods for precise processing of fusion proteinsare being aggressively pursued and thereare some very well-designed schemes available that employ linker regions carrying protease recognition sites between the amino and carboxy terminalparts of the hybrid protein. 4.7 Site-DirectedandRandomMutagenesis If expression levelsare too low and production of a fusion proteinis not desirable, the early coding region can be mutagenized. Sometimes the problem may be an obvious secondarystructure causing blockageof all or part of the RBS that can be altered by site-directed mutagenesis. If the problem is not obvious, random “cassette” mutagenesis may be the method of choice. This approach involves the synthesisof complementary oligonucleotides in which random incorporation of the nucleotide in the wobble position of each codon is carried out. The heterogeneous mixture of nucleotides is then clonedinto the gene, and transformants are screened for production of the recombinant protein. Thisapproach has been utilized effectively in the expression of human glutathione reductase(132).

4.8 Two-CistronTranslationSystem

This approach (133) is applicable in cases where secondary structure within the mRNA occludes theRBS. The rationaleis based on numerous instances in E. coli where “translational coupling” (134-136) occurs. Occasionally, one of the downstream genes in a polycistronic message will have occlusion of the RBS due to mRNA secondary structure, resulting in the in-

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ability of the ribosome to bind and initiate translation. Ribosomes that are translating the upstream gene are capable of destroying the secondary structure in the downstream RBS if the stop codon of the upstream gene is closeto the start of the downstream gene.It is thought that such an arrangement leadsto “reinitiation” at the downstream gene immediately following termination of the upstream gene. The two-cistron system was shown to work in one casefor a gene that was transcribed and not translated, but it did not improve translation of a gene that was already efficiently translated (133). A two-cistron system was used in an attempt to express human glutathione reductase (132). In this case expression wentfrom undetectable to low levels but did not approach the levels expressed following cassette mutagenesis. In two other cases, such a system wasreported to yield high levels of recombinant protein(137,138). The applicabilityof this approach would seemto be limited to those cases where there isother no acceptable way of removing secondary structure at the RBS.

5 POSTTRANSLATIONALEVENTS Following synthesis ofa recombinant protein in E. coli a number of events may occur, some desirable, some benign, and some undesirable. These can be placed into two general categories, nonreversible, such as covalent modifications and proteolysis, and reversible, such as misfolding and aggregation. Onthe other side of the issue, thereare some posttranslational modifications that do not occur inE. coli that are part of processingand assembly operations that many eukaryotic proteins normally undergo in nature. Fortunately, in some cases the lack of these normal modifications does not diminish the biological activity of recombinant proteins. For example, recombinant proteins produced inE. coli do not become glycosylated, yet the biological activity of these proteins in some cases is the same or higher than that of the natural product (130). Moreover, it has been reported that the recombinant proteins have a longer half-life in the blood system (140,141). For this reason, recombinant proteins may be superior for some pharmaceutical purposes. 5.1 Nonreversible Events

This group of modifications includes reactions such as removal of the amino terminal fMet residue and proteolytic cleavage reactions. Other posttranslational modifications, such as methylation, acetylation, and

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fatty acylation, seem to be highly specificfor certain E. coli proteins and will not be addressed further. 5.1.1 Removal of N-Formylmethionine It is believed that the nascent polypeptide is first deformylatedand then the Met residue is removed by a Met-specific aminopeptidase. This view is based on the identificationof formylase activityfound in E. coli extracts and the lack offMet activity (142). However, the Met-specific aminopeptidase has not yet been found, and a full understanding of the process will have to await the identification and purification of this activity. The nature of the second amino acidaisdetermining factor in whether or not the fMet residue is removed from a polypeptide in both eukaryotes and prokaryotes(143). Those amino acidsthat favor removalof fMet are universally conserved in various organisms,and they are Ala, Ser, Gly, Pro, Thr, and Val. Those that disfavor removalare Arg, Asn, Asp, Gln, Glu, Ile, Leu, Lys,and Met. In some cases of recombinant protein production inE. coli, only a proportion of the protein product has had the fMet removed (144). This situation may result from overloading the cellular processing machinery by such a high levelof protein synthesis. 5.1.2 ProteolyticReactions E. coli has may proteases,both specific and nonspecific (for current reviews, see Ref.145 and 146). Specific proteolysis occurs routinely by signal and by signal peptidase peptidase Ias part of the general export pathway 11, in combination withother covalent modifications, in the localization of lipoproteins. In responseto various environmental conditions, specific proteolysis also plays a role, such that as medited by RecA as part of the SOS pathway. Numerous nonspecific proteases have been identified in E. coli and more are likely to be discovered in the future. The profile of nonspecific degradation changes with growth conditions. For instance, abnormal proteins that result from misincorporation of amino acids or truncation are routinely degraded under all growth conditions; (145). but under starvation conditions, normal proteins are also degraded Under most starvation conditions, proteolysis increases in the early stages but declines as starvation persists. Proteolysisis particularly high under nitrogen starvation. At least one protease, Clp, has been shownto have its highest activity during late exponential and early stationary growth phases (147). The most highly characterized E. coli protease is Lon, which acts on abnormal proteins but also cleaves normal proteins inthe regulation of

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capsular synthesis and cellular stress responses. For example,as part of the SOS response, the cell division inhibitor SulA is produced. Following DNA repair, it is necessary for the cell to rid itself of SulAto allow cell SulA through division to resume. The role of Lon in this case to inactivate is proteolysis (148). If Lon isabsent, SulA continuesto inhibit cell division, and eventually the cell becomes filamentous and dies. Thus strains that do not produce Lon are sensitive to treatments that induce the SOS response. Lon is also one of the proteases regulated by the heat shock response (149). This global control circuit is regulated by the alternate a-factor, HtpR (69,150-152), which is required at all temperatures,but higher levels are required at elevated temperatures. Recently, another protease, DegP/ HtrA (153,154), has also been identified as a heat-shock protease (155, 156). Some protease activity is confined to particular compartments. For instance, the periplasm contains DegP, protease I11 (157) [(product of the ptr gene (158,159)l and possibly theproduct of the sohB gene (160). The latter is homologousto protease IV,an enzyme that degrades cleaved signal sequences in the inner membrane (161). Protease V resides in both the inner and outer membranes ofE. coli and has specificityfor L-glycine and L-phenylalanine derivatives(162,163). The outer membrane contains protease VI, a serine protease (164), and protease VI1 (OmpT), which has been shown to have specificityfor paired basic residues(165). Even if a recombinant proteinis retained in the cytoplasm during synthesis, these extracytoplasmic proteases can create problems during the purification stages following cell breakage. The use of protease-deficient strains for the expression of recombinant proteins inE. coli has been described(166). Strains that carry nullmutations of Ion are availableand can be usedas hosts, or the mutation can easily be introduced into any strain by P1 transduction. Lon mutants have pleiotropic phenotypes stemming from the regulatory functions described above. One phenotype is mucoidy, resulting from unregulated capsule synthesis (167). This phenotype can create difficulties in harvesting cells from liquid culture, isolating colonies, and performing phage infections. Mucoidy can be controlledthrough careful choiceof media or by using secondary mutations that map within genes necessary for capsule synthesis (166). Another phenotype of Lon- strains is sensitivity to DNAdamaging agents stemming from the cell’s failure to degrade SulA. Several concerns arise from this situation. One is that a Ion- mutation shouldnot be used in combination withdam- mutations becauseit results in lethal

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induction of the SOS response, and it follows that ion- strains should not be used in combination with promoters inducibleby nalidixic acid. Another problem is the Zon- strains cannot be grown in media containing yeast extract (e.g., LB). These phenotypes can be avoidedby using S U M mutants (166). Mutations inhtpR have been used in expression strains. The advantage to inactivation of htpR is that it results in decreased synthesis of several cellular proteases, including Lon. The HtpR defect is most commonly employed by usingan amber mutation inhtpR in combination with a temperature-sensitive amber suppressor(69,168,169). At the permissive temperature the cells produce HtpR, and at the nonpermissive temperature HtpR will no longerbe produced. With these strains, cell lysis occurs soon after the temperature shift. Lon protease is not completely absent in htpR mutants, and therefore Zon htpR, double mutants can be used. Finally, factors relatedto the induction scheme may diminish or exacerbate the problem of proteolysisof recombinant proteins. Thermal induction of promoters in HtpR+ strains will increase proteolysis in all compartments of the cell. In addition, raising the temperature may lead to aggregation of the recombinant protein, which could either make it inof proaccessible to proteases (170,171) or it could increase the likelihood teolysis (172). Rapid, high-level expression of proteins sometimes leads to aggregation even at lower temperatures(173,174) and may simply overof the recombinant proteininto the load the cellular proteases. Secretion periplasm will allow it to escape cytoplasmic proteasesbut will expose it to periplasmic or outer membrane proteases. It should be pointed out that proteolysis is only one potential cause of low expression levels. Before any attempt is made to eliminate cellular proteases, a pulse-chase study should be doneon the proteinof interest to see if proteolysisis actually a problem.

5.2 ReversibleEvents The most common reversible reactions encountered in expression systems are the formation of insoluble aggregates (inclusion bodies) or the misfolding of proteins. Misfolding maybe avoided if the protein can be successfully secreted into the periplasm, especially if the protein contains intramolecular disulfide bonds(175-177). For these proteins, the E. coli enzymes peptidyl-prolyl cidtrans isomerase and protein disulfide isomerase may be limiting. This problem has been dealt with successfully

Expression Gene

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through the use of the pBR322-compatible plasmid pASK61, whichcarries the dsbA/pdi and ppi genes (178). Host cellsthat contain both pASK61 and the pBR322-based expression plasmidsare grown inthe presence of the thiol-reagent N-acetyl-L-cysteine to provide optimal conditions for homogeneity of disulfide bondformation. Even interchain disulfide bonds have been folded correctlythe in case of the antigen-binding fragment (Fab) of antibody (175,179). This feat was accomplished by cloning the genes for the two polypeptide chains together so that a dicistronic message was produced. Presumably, the polypeptides are synthesized and secreted with close proximity to one another, allowing them to assemble correctly. Other examples of simultaneous expression of interacting polypeptides improving stability through proper folding are the of cAMPdependent protein kinase (PKA) regulatory and catalytic subunits (172) and human hemoglobin( 180). Secretion of the recombinant protein in the periplasm doesnot always eliminate aggregation (173,174,181), but the addition of nonmetabolizable sugars to the growth medium has been shown to decrease periplasmic aggregation (173,182). In some cases, aggregationaisby-product of heat induction (170),171)or a high synthesis rate (73,174); however, many examples are also found where thesefactors do not leadto aggregation or misfolding. Aggregation can be beneficial in terms of making the protein less susceptibleto proteolysis and easier to purify from the bulk ofE. coli proteins. 5.3 InclusionBodies If a protein formsan inclusion body, sometimes it is best to isolate it as such and then solubilize it. For proteins secreted into the periplasm, a simple method involves harvesting the cell, resuspending them in chloro(183). The simplest form, and recovering the protein from the supernatant procedure for the isolationof inclusion bodies associated with other cell fractions involves harvesting the cells and resuspending the pellet inTris/ EDTA/lysozyme/Triton X-100 to solubilize the cell envelope followed by sonication to reduce the viscosity caused bythe presence of DNA.The addition of DNase is typically avoided because the enzyme requires magnesium, which may also activate proteases. Centrifugation of this preparation will pellet the inclusion bodies. Solubilization of inclusion bodies is typically done with 8 M urea or 6M guanidine-HCI, and refolding requires dilution or dialysis in a buffer suitable for the particular protein

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involved. Thereare several sources in the literature describing procedures for protein solubilityand folding (184-186), and commercial suppliers of expression vectors often provide detailed protocols for the isolation of recombinant proteins. Examplesof biologically active recombinant proteins that have been isolated from inclusion bodies include mouse interleukin-6 (IL-6) (187), human IL-6 (188), human interleukin-5 (IL-5) (189), and horseradish peroxidaseC (190). 6 SPECIAL DESIGNS: FUSION PROTEINS

In this sectionwe deal with some special designs for expression of recombinant fusion proteinsthat take into consideration many of the individual concerns described in previous sections (for a recent review, see Ref. 191). Fusion proteins constructedfor expression systems usually contain an E. coli sequence at the amino terminusand the protein of interest at the carboxy terminus. This class also includes proteins engineeredfor secretion (even if the finalproduct is not a fusionprotein, the primary translation product is). Some of the potential benefits of expressing heterologous proteins as hybrid proteins are: (a) The presence of an efficiently translated E coli protein moietyat the amino terminus eliminates concerns about construc(b) in some cases, especially with tion of an optimal ribosome binding site; small peptides, a fusion protein will be more stablethan a nonfused protein (192); (c) purification of the hybrid protein by affinity chromatography is possible even if there is no ligand available for the heterologous protein of interest; (d) a variety of cleavage options are available, including chemical cleavage,or there are specially engineered linkersthat contain specific protease recognition sites so that cleavage can be carried out to yield an authentic protein; and (e) if the amino terminus carries a signal sequence, the fusion protein may be secretedinto the periplasm successfully, greatly simplifying purification and providing a recombinant protein withan authentic amino terminus (for review a of secreting heterologous proteins inE coli, see Ref. 193). The potential disadvantages of fusion proteins are: (a) Except in the case of secretion, the amino terminusof the cleavageproduct may have extra amino acids attached; (b) there may beadditional sites on theprotein of interest that are susceptible to cleavage by the treatment used to remove the N-terminal moiety; and (c) conditions usedfor chemical cleavage may damage the protein of interest.

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6.1 StaphylococcalProtein

coli

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A Fusions

Protein A from Staphylococcus a u r w binds specificallyto the Fc portion by IgG Sepharose of IgG and therefore proteinA fusions can be isolated chromatography. The latest versions of the pRIT series of vectors (Table l), pRIT20 and pRIT30, are available in two types for expression of protein A fusion inE. coli. The pRIT20 series is usedfor secreted fusion proteins and is recommendedfor proteins that are normally secreted in their natural hostcells. The pRIT30 series is designed for use with proteinsthat are not secreted in their natural host. Both typesof vectors contain linkers for all three reading frames,and these linkers encode the recognition sequence for either enteropeptidase, collagenase,or factor X,proteases. Details of the constructionand use of these vectors have been described (194). A specialized expression system called EcoSec has been developed (195). This system is designed for the expression of peptide hormones in soluble form and has been used successfullyfor insulin-like growthfactors I and 11. The EcoSec system utilizes plasmid vector pEZZ18 (Table 1).

6.2 p-GalactosidaseFusions Both amino- and carboxy-terminal fusion of @-galactosidase have been used for years for a wide variety of purposes.In general, @-galactosidase fusion proteins are among the largest polypeptides produced inE. coli and therefore,gel filtration chromatography canbe utilized. Anti-@galactosidase affinity resinsare also commercially available. Two recent applications of @-galactosidase fusion technologyfor engineered expression systems are the pSS20 (196) and the PAX (197) series of plasmids. There has also been a series of low-copy-number vectors developed for the production of @-galactosidase fusions (198).

6.3 GlutathioneS-TransferaseFusions Fusions to gluthathione S-transferase (GST) can be purified on Glutathione Sepharose 4B. Two fusion vectors are commercially available, pGEX2T and pGEX-3X (Tablel), which carrythe recognition sitesfor thrombin and factorX,,respectively. Another vector,PGEX-~T,encodes ahistidine hexapeptide linked to GST for the expression of fusion proteins that can be purifiedon metal chelation resins (199).

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6.4 Maltose-BindingProteinFusions Vectors designedfor the productionof fusions to the E. coli maltose-binding protein, with or without a signal sequence, can be purified by affinity chromatography on cross-linked amylose (200). Vectors encoding the recognition sequencefor factor X,are also available (201).

6.5 SecretionSystems In addition to the systems described abovethat secrete fusion proteins, plasmid vectors that carry the E. coli signal sequencesfrom phoA (181, 200)or ompA (202,203) have been constructed. Successful secretion using these vectors is more likely if the protein is normally secreted in its natural cell type. All of the vectors currently available target the protein to the general export pathway inE. coli, even though there are several specialized secretion systemsthat also occur in this organism. Success has been good in getting proteins exportedto the periplasm, but there have been no major successes in secreting recombinant proteinsinto the medium. Sometimes large amounts of proteins are indeed found in the medium, but they probably get thereby leakage.

7 CONCLUDING REMARKS The expression of heterologous genes in E. coli has benefitted greatly from our expanding knowledgeabout the basic biology of this organism. Our deeper understanding of the steps in transcription has to the led availability of a number of promoters whose activity can be controlled very effectively using repressors and inducers. Use of the l7 promoter presents a new twist-an activatable promoter. Hopefully, the success of this system will encourage more experimentation withother activatable promoters, such as those that use alternate a-factors. Translation has been more difficultto understand in general, because distinguishing the basisfor codon choice (nucleotideor amino acid) confounds algorithmic analysis. Unlike transcription, the process relies on nucleotide sequences within the coding region theofgene, an area the researcher is not always at liberty to change. The choice for optimization of translation initiation always depends greatly on the intended use of the recombinant protein. Controlling posttranslational events isan area where much work needs to be doneat the basic level.To achieve more cost-effective recombinant protein production fromE. coli, it will be important to learn as much as

Expression Gene

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CO//

35

we can about proteolysis and locabation of proteins. The genetic approach willand prowill be helpful in achieving greater understanding in this area vide useful host strains. The physiology of recombinant E. coli is another area that should be studied further so that we can continue to improve our chances of designing host-vector systems that are able to thrive in continuous culture.

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13. Mashko SV, Mochulsky AV, Kotenko SV, Lebedeva MI, Lapidus AL, Mochulskaya NA, IzotovaLS, Veiko VP, Vinesky VP, Ketlinsky SA, Debabov VG.Use of a dual-origin temperature-controlled amplifiable replicon for optimization of human interleukin-lp synthesis in Escherichia coli. Gene 1991; 97:259-266. 14. Vocke C, BastiaD. DNA-potein interaction at theorigin of DNA replication of the plasmid pSC101. Cell 1983; 35:495-502. 15. Linder PG, Churchward G, Guisizn X, Yi-Yi Y, Car0 L. An essential replication gene, repA, of plasmid pSClOl is autoregulated. J Mol Biol 1985; 181: 383-393. 16. Skogman G, Nilsson J, Gustafsson P.The use of apartition locus to increase stability of tryptophan-operon-bearing plasmids in Escherichia coli. Gene 1983; 23:105-115. 17. Zurita M, Bolivar F, Soberdn X. Construction andcharacterization of new cloning vehicles. VII. Construction ofplasmid pBR327par, a completely sequenced, stable derivariveof pBR327 containing thepar locus of pSC101. Gene 1984; 28~119-122. 18. Tomizawa J-I, Itoh T. The importance of RNA secondary structure inColEl primer formation. Cell 1982; 31575-583. 19. Davison J. Mechanism of control of DNA replication and incompatibility in ColEl-type plasmids: A review. Gene 1984; 28:l-15. U). Tomizawa J-I. Control of ColEl plasmid replication:The process of binding of RNA I to the primer transcript. Cell 1984; 38:861-870. 21. Tomizawa J-I. Control of ColEl plasmid replication: Initial interaction of RNA I and the primer transcript is reversible. Cell 1985; 40527-535. 22. Cesareni G, Muesing MA, Polisky B. Control of ColEl DNA replication: The rop gene product negatively affects transcription from thereplication primer promoter. Proc Natl Acad Sci USA 1982; 79:6313-6317. 23. Covarrubias L, Cervantes L, Covarrubias A, Soberdn X, Vichido 1,Blanco A, Kupersztoch-Portnoy YM, Bolivar F. Construction andcharacterization of new cloning vehicles. V. Mobilization and coding properties of pBR322 and several deletion derivatives includingpBR327 and pBR328. Gene 1981; 13~25-35. 2 4 . Twigg AJ, Sheratt D. Trans-complementable copy numbermutants of plasmid ColEl. Nature 1980; 283:216-218. 25. Chambers SP, Prior SE, Barstow DA, Minton NP. The pMTLnic-cloning vectors, I. ImprovepUC polylinker regionsto facilitate the use of sonicated DNA for nucleotide sequencing. Gene 1988; 68:139-149. 26. Weinberg RA, DeCiechi PA, Obukowicz M. A chromosomal expressionvector forEscherichia coli basedon the bacteriophage Mu. Gene1993; 126:2533, 27. Collado-Vides J, Magasanik B, Gralla JD. Control site location and transcriptional regulation in Escherichia coli. Microbiol Rev 1991; 55:371-394.

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77. Yen K-M. Construction of cloning cartridgesfor development of expression vectors in gram-negative bacteria. J Bacteriol 1991; 1735328-533s. 78. Yen K-M, Serdar CM. Genetics of naphthalene catabolism in pseudomonads. Crit Rev Microbiol 1988; 15:247-268. 79. Craig SP 111, Yuan L, Kuntz DA, McKerrow JH, Wang CC. High level ex-

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T, Ljungqvist L, Holmgren E, Paleus S, Josephson S, Philipson L, Uhlen M. Production of specific antibodies against protein A fusion proteins. EMBO J 1986; 5:2393-2398. 233. Windass JD, Newton CR, De Maeyer-Guignard J, Moore VE, Markham AF, Edge MD.The constructionof a syntheticEscherichia colitrp promoter and its use in the expression of a synthetic interferon gene. Nucleic Acids Res 1982; 215639. 234. Tabor S, Richardson CC. A bacteriophage Ti' RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc Natl Acad Sci USA 1985;82:1074-1078. 235. Brown WC, Campbell JL, A new cloning vector and expression strategy for genes encoding proteins toxic to Excherichia coli. Gene 1993; 127:99103. 236. Wang RF, Kushner SR. Constructionof versatile low-copy-number vectors for cloning, sequencingand gene expressionin Escherichia coli. Gene 1991; 100:195-199. 237. Mandecki W, Hayden MA, Shallcross MA, Stotland E. A totally synthetic

plasmid for general cloning, gene expression and mutagenesis inficherichia coli. Gene 1990; 94:103-107. 238. Maniatis T, Ptashne M, Blackman K,.Kleid D, Flashman S, Jeffrey A, Maurer R. Recognition sequence of repressor and polymerase in the operators of bacteriophage lambda. Cell 1975;5:109-113. 239. Gilbert W. Starting and stopping sequences for the RNA polymerase. In: Losick R, Chamberlin M, eds. RNA polymerase. Cold Spring Harbor, N Y : Cold Spring Harbor Laboratory 1976:193-205.

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240. Pribnow D. Nucleotide sequence ofan RNA polymerase binding siteat an

early T7 promoter. Proc Natl Acad Sci USA 1975; 72:1069-1072. 241. Schaller H, Gray C, Herrmann K. Nucleotide sequence of an FNA polymerase binding site from the DNA of bacteriophage fd.Proc Natl Acad Sci USA 1975; 72~737-741. 242. Dickson RC, Abelson J, Barnes W M , Reznikoff WS. Genetic regulation: The Lac control region. Science 1975; 187:27-35. 243. Bennett GN, Schweingruber ME, Brown KD, Squires C, Yanofsky C. Nucleotide sequenceof the promoter-operatorregion of the tryptophan operon of Escherichia coli. J Mol Biol 1978; 121:113-137. 244. Maniatis T, Ptashne M, Barrel1 BG, Donelson J. Sequence of a repressorbinding site in the DNA of bacteriophage X. Nature 1974; 250:394-397. 245. Walz A, PirrottaV. Sequence ofthe P, promoter of phageX. Nature 1975; 254:118-121. 2 4 6 . Chang C N , Kuang W-J, Chen EY.Nucleotide sequence ofthe alkaline phosphatase gene of Escherichia coli. Gene 1986; 44:121-125. 247. Overduin P, Boos W, Tommassen J. Nucleotide sequence ofthe ugp gene of E. coli K-12: Homology to the maltose system. Mol Microbiol 1988; 2: 767-775. 248. Schell MA. Homology between nucleotide sequences of promoter regions of nah and sal operons of NAH7 plasmid of Pseudomonasputida. J Bacteriol 1986; 166:9-14.

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2 Gene Expression in Recombinant Bacillus Matti Sarvas National Public Health Institute, Helsinki, Finland

1 INTRODUCTION

Bacilli have many features attractive for a microorganism to be used as a host for the production of heterologous proteins; indeed, bacilli may be an optimal hostfor a numberof applications inboth industry and research. Intensive development on the expression of heterologous proteins during the last10 years has now made this mode of production feasible, and the first stepsto introduce the methods to established industrial technology have been taken. Bacilli have long been used for industrial production, the main products being secreted enzymes suchas amylases and proteases. This has provided much basic knowledge and practical technology for the culture, fermentation, and downstream processingof products. An important feature of all bacilli used in practical applications is their apathogenicity and the well-proved safetyof appropriate industrial processes using them. Most bacilli used in industry and research lack the cellular components or metabolic products toxic to humans or animals, an important feature facilitating the production of proteins of medical interest and an additional 53

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element of safety withthe technology of genetic engineering. In particular, as gram-positive organisms, bacillido not contain endotoxins (lipopolysaccharide), which are ubiquitous in all gram-negative bacteria, including Escherichia coli, and difficult to remove from many proteins in the process of purification. The prototypespecies of bacilli,Bacillus subtilis, has been characterized extensively both physiologically and genetically, second only to E. coli. This wealth of information provides a sound basisfor the genetic engineering of the bacterium for industrial applications, to develop novel modesof production, and to extend the exploitation of Bacillus as a model organism for the advanced understandingof bacterial cell biology. The long availabilityof effective genetic exchange methods, including physiological transformation, has providedan ideal systemfor the study of a broad range of genetic, biological, and physiological properties of Bacillus, resulting in a wealth of knowledge concerning the genetic map, the expression and regulation of bacillar genes,and the regulatory mechanisms associated withthe adaptation to environmental changes, including sporulation (for a comprehensive treatise, see Ref. 1). There is at present a multinational projectto sequence the entire genome ofB. subtilis (2). The development of host-vector systems has been expansive, and thereare now available a multitude of different types of plasmid for cloning and for constructing expression systems(3). The secretion of proteins, however, the is feature of bacilli that has attracted mostattention in biotechnologyand dominated their role as hosts for the productionof heterologous proteins. Most Bacillus spp. secretea multitude of proteins into the growth medium; many strains or species used in industrial processes secrete large amounts of many degradative exoenzymes, such as proteases, amylases, and nucleases. The prospect of exploiting this capacity in order to produce heterologous proteins by secretion has been the impetus for much of the research and development on Bacillus. The secretory production couldoffer attractive advantages for industrial production. Purification of a secreted protein is simpler and more economicalthan thatof a product produced intracellularly, the prevalent mode of production in most microbial production systems. A secreted protein, at least one without the embellishments may be found in eukaryotic proteins, couldbe expectedto adopt its nativeconformation even at a high levelof secretion, while effective intracellular expression often leads to the aggregation of the protein in a denaturedform; in such cases recovering the protein in an active or pharmacologically acceptable form may be tedious, if feasible at all. Secretory proteins can alsobe tailored

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to have the native N-terminalstructure more readilythan those produced intracellularly, as describedlater. Some proteins with biological activity harmful to the cell, if presentin the cytoplasm, may be produced only in a secreted form. this goal achieThe development of bacillar genetic engineering has made vable; most of the production systems developed for heterologous proteins in Bacillus are, indeed, based on secretory expression. Several production systems have been described that produce heterologous proteins Bacillus as a production host has also at high efficiency. However, using met major obstacles, the most prominent ones being the proteolytic degradation of many proteinsand the poor compatibilityof many nonbacillar proteinsfor secretion inBacillus. Much of the work on bacillar expression has dealt with development of the technology to tackle these problems. On the other hand, the secretory expression systems have'been developed exploitingthe expanding knowledge of the molecular biology of of Bacillus. The concomitant advances in gene technology have paved the way for the development of nonsecretory expression systems and have contributed much to the understandingof heterologous gene expression in other situations. in this chapThere is, therefore, much emphasison the secretion systems ter, which summarizes the technology of the expression of heterologous proteins inBacillus. Most of the discussion is focused onB. subtilis, the organism used in the vast majority ofthe studies described inthe literature, with somenotable exceptions. The well-developed methods of genetic engineeringare, in general, applicable onlyto B. subtilis, although it at present. In is not aBacillus species widely usedin industrial applications this chapter we describethe major production systems, their potential applications, and the constraints. Heterologous gene expression, the secretion of proteins inBacillus, and the genetic and molecular biological methods used in Bacillus have been the subject of several recent reviews (4-8).

2 SECRETION OF HETEROLOGOUSPROTEINS IN BACILLUS 2.1

Molecular Basis of the Secretion of Heterologous Proteins in B.subtilis

The overall pattern of protein secretion in Bacillus, studied almost exclusively in B. subtilis, follows the principle established initially for the secretion of eukaryotic proteins across the endoplasmic reticulum, and

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subsequently for E. coli, the most completely characterized bacterid model system. (For recent reviews, see Refs. 6 and 9,lO-12.) The proteins secretedare synthesized as precursors withan amino terminal extension, which contains the signal sequenceor the signal peptide. They are translocated acrossthe cytoplasmic membrane ofthe bacterial cell vectorially withthe concomitant removal of the signal sequence. Many, if not all, of the cellular components involved in this mechanism have already been identified E. in coli, with some understanding of their mode of function (for a review, see e.g. Ref. 9). The earliest event in secretion in E. coli is the interaction of the nascent polypeptideof the secreted protein with the cytoplasmic proteins belonging to the classof molecular chaperones. These includethe SecB protein (13-16) and heat-shock proteins, suchas GroEL (17-19) and DnaK (20), as well as the bacterialanalog of the signal recognition particle (11,21-23). These proteins affect the bondingof the precursorto keep itsconformation loose and compatible with the later stages of the process (9,24-26). One function of the signal peptide is to interact with the rest of the precursor molecule together withthe chaperons to achieve and maintain aconformation compatible with secretion. The peripheral membrane protein SecA (27-29), a protein with ATPase activity, specifically binds the signal sequence. This is the first step in guiding the polypeptide chainto the complex of integral membraneproteins that is known to contain a closely associated pairof SecY and SecE proteins and in addition at least the integral membrane proteins SecF and SecD (9,30-33). In a way not yet elucidated, the interaction of the signal sequence withthis complex'resultsin the translocation of the precursor polypeptide chain across the cytoplasmic membrane, possibly through a porelike structure containing the SecY/SecE protein complex. The signal sequence is cleaved off from the precursor on the outer surface of the membrane by a specific protease,the signal peptidase( h p ) (34), followed by the release of the secreted, mature protein from the membrane. Knowledge of the components of bacillar protein secretion is much more limited and has been obtainedand has been obtained rather recently (for a recent review, see Ref. 6). Protein homolog of SecA, SecY,and SecE of E. coli have been identified inB subtilis (35-40). Bacillar secA, secY,and secE genes complement the respective mutations of E. coli to a certain extent, implyingthat their functionsare similar. Thereis sequence homology betweenthe E. coli and B. subtilis protein counterparts; SecA ofB. subtilis has been shownto contain ATPase activity(36), and the predicted folding patterns of both SecY proteins are very similar. Bacillar SecA is

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somewhat shorter and SecE muchshorter than the homologous proteins of E. coli. B. subtilis homolog ofFfh protein.and the 4.5s RNA, compo(41,42). A gene, nents of the signal recognition particle, have been identified designated sips, encoding a protein with signal peptidase activity, with weak but significant sequence homology withLep theof E. coli, has been identified inB. subtilis (43). However, the deletion ofsips did not abolish secretion completely,and it was not indispensablefor survival, unlike its homolog inE. coli. A unique componentof the secretion machinery, the PrsA protein,with no known homolog E. in coli, has recently been characterized inB. subtilis (44,45). PrsA is a lipoprotein anchoredon the outer surface of the cytoplasmic membranebut predicted riot to be embedded in the lipid bilayer of the membrane. Mutation studies suggest that it functions in alate stage of protein secretion, possibly involved the in folding of the protein in a chaperone-like fashion after its translocation and in the release of the protein from the membrane (42). The overall cellular machinery of protein secretion B. in subtilis thus resembles that of E. coli, with presumed similar functional features. However, the bacillar components identifiedso far also show distinctstructural features; the bacillar system may thus have evolved and adapted in away that could be reflected in functional properties not yet elucidated. I With regardto the feasibility of the secretion of heterologous proteins in Bacillus, the role of the signal sequence is crucial.The presence of a functional signal sequence an is absolute requirementfor secretion to take place. It contributes to the folding of the nascent polypeptidechain, it is the targetof the binding and recognition by the translocator machinery, and it interacts withthe components of the translocator in the initiation of the translocationof the polypeptide across the membrane(9). On the contrary, the mature part of the exported protein, the part released from the cell after thecleavage of the signal sequence, has no known active function in the process (for a review, see Ref.46). Studies on exported bacterial proteins have virtually excludedthe possibility of any specific sequence serving asan export signal inthe mature part of the protein (see Ref. 46 and references therein). Biochemical and genetic studies involving the in vitro translocation of secreted proteinsinto vesicles of cytoplasmic membranes have not provided evidence of specific interactionof any part of the mature protein withthe components of the export machinery. The foregoing considerations suggest a model for the basic strategy in It was followed in allefforts to secrete heterologous proteins Bacillus. anticipitated that the fusionof a signal sequencefunctional in B. subtilis to the DNA sequence encoding the protein of interest would result in its

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secretion from the cell, provided that there were also functional expression signalsfor transcription and translation. Indeed,was it demonstrated very early that insulin was secreted across the cytoplasmic membrane of E. coli when fused to the signal sequenceof an exported protein, the periplasmic 0-lactamase (47). It was hopedthat a potentially large number of proteinsbemight secreted this way in Bacillus. Well-defined constraintswere also recognized. Many hydrophobic stretchesof significant length are expected to function as socalled “stop transfer”sequences (10) and arrest secretion; such sequences are likely to be found in membrane proteins,but they may also be found in some soluble cytosolic proteins. Other types of limitationsare exemplified by extensive studies on the cytoplasmic protein 0-galactosidase of E. coli. When this proteinis expressed as a fusion with a signal peptide of a secreted protein, it is arrested in the cytoplasmic membrane ofE. coli (48). At a high level of expression,the effect is also harmful to the cell. There is no defined segment(s) of 0-galactosidase, which hinders or slows down its traversion, but rather such structural features are distributed all along the moleucle (49). An interesting hypothesis has been presented by MacIntyre and Henning (46). They postulate that a crucial feature of cytosolic proteins is their rapid folding to tight conformationto prevent their unintentional export. a signal peptide were not Indeed, most cytosolic proteins provided with exported fromE. coli efficiently (50). A more subtle restriction on secretion may be posed by the presence and location of charged amino acid residues in the mature part of the protein. Basic amino acid residues at the amino terminusof the protein elicit especiallydramatic hindrance to secretion in bacteria(51-54). In agreement with this observation, secreted bacterial proteins tendto lack lysyland arginyl residuesat the amino terminus ofthe native protein, and acidic and basic residues often occur in a paired manner (935). Proline is never found at the N-terminusof secreted proteins, and when introduced, completely preventsthe cleavage of the signal peptide in E. coli (56,57). These restrictions clearly indicate that proteins that are secretory by nature are the primary targetgroup of heterologous proteinsto be produced withthe secretory mode inBacillus. Fortuitously, a large number of proteins of either industrial or pharmaceutical interest are secretory proteins. The foregoing principles have been applied in a number laboratories of to develop systems to secrete heterologous proteins inBacillus. In most studies the host organism has beenB subtilis, and combinations of variAs ous expression signals, signal sequences, and plasmids have been used.

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discussed in more detail in the following pages, these studies have demonstrated that the principle is applicableto the production of a number of proteins, both model proteins and those of practical interest,but the range of the types of proteins secreted efficientlyBacillus in is unexpectedly narrow.

2.2 Secretion Systems Based on the Genes of Major Exoproteins of Bacillus spp.

2.2.l

Vectors Based on or-Amylases

The most straightforward approach to achieving the secretion of a heterologous protein has been to insert the structural gene of the protein of interest downstream from the expression signalsand the signal sequence of a bacillar gene encoding an exported protein. The genes of exported amylases and proteases are an obvious choicefor this purpose and have been utilized in most approaches. Both types of enzymes are secreted at high levels in many species ofBacillus (58,60), implicating the presence of effective promoters, and signal sequences of high compatibility with the secretion machinery.Furthermore, due to industrial interest,mutant strains with increased production and enhanced level of expression of these genes have been developed and are available. The first practical system, also thoroughly explored, was the one based on the a-amylase of B. amyloliquefaciens. Many strains of this species naturally secrete high levels ofa-amylases and proteases (60). To develop a secretion system, Palva et al. (61) initially clonedthe amyE gene of an industrial strain encoding a-amylaseto the plasmid PUB1 10;the multicopy cloning vectorof staphylococcal origin used extensively as a vector for cloning and expression B. insubtilis (3).Up to 1 g/L of a-amylasewas secreted into the growth medium when this hybrid plasmid was expressed in B. subtilis (61). This is 2500-fold more than the endogeneous level of a-amylase secretion inB. subtilis, showing both the effectiveness and compatibility of the expression signals ofB. amyloliquefaciens in B. subtilis and the capacity of B. subtilis for highly increased levels of secretion. Furthermore, the hydrid plasmid was very stable. Insertion of DNA fragments of various origins into derivatives of pUBllO often results in unstable constructions (62), presumably due to the rolling-circle replica(3). However, inthe case of thisamyE intion of this family of plasmids sert, there was hardly any structural or segregational instability of the construct (63), even when grown over 50 generations in the absence of selection pressure. Similar stability was a characteristicof many derivatives of this hybrid plasmid constructed later (below).

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The feasibilityof basing the secretion of heterologous proteins on the expression and secretion signals of thisamy gene was demonstrated by fusing the segment of DNA encodingthe mature part of the periplasmic TEM P-lactamaseof E. coli downstream from the signal sequence in the hybrid plasmid above. A set of plasmids was constructed withthe fusion Of the P-lactamase gene with a linker sequence a few of codons (providing a cloning site) either directlyto the end of the signal sequence or a few codons downstream (64). The P-lactamase was well expressed from all constructions. Meeting expectations, virtually all of the enzyme was secreted into the growth medium; lessthan 5% of the P-lactamasewas cell bound in the form of the expected size of the precursor. The signal sequence was cleaved off from the secreted form; the cleavage took place in all constructionsat the end of the signal peptide,at the "natural" cleavage site. Levels of more than 20 mg of secreted P-lactamase in 1ofLthe culture supernatant were obtained inthe early stationary phase of growth in shake flask cultures ina rich laboratory medium; a good level of expression, although much lower than that of the a-amylase itself(see later for a discussion of possible reasons). P-lactamase was secreted inthe exponential phaseof growth and continued into early stationaryphase, fol-. lowed by a decrease inthe amount of the enzyme inthe medium (64,65) (for proteolytic degradation, see below and Section 2.6). This pioneering study encouragingly demonstratedthat a heterologous protein can be secreted efficiently B. in subtilis by usingthe elementary strategy of fusing to it a bacillar signal sequence and expressing it from a highly active promoter. Derivatives ofthe plasmid above have been developed to provide a set of secretion vectors withan appropriate cloning site (HindIII in most members of the set)and with properties enabling the ready expression of a gene or a DNA fragment encoding the protein of interest. The expression signals ofthe secretion vectorare in an insert of about 500 bp in the BamHI site of PUB1 10;the insert contains the promoter, the ribosome binding site, and the signal sequence of the amy gene of B. amyloliquefaciens. Upstream from the promoter, the fragment alsocontains an inverted rewhich prevents peat region functioningas a transcription termination site, any possible read through and interference with the expression upstream. Immediately at the end of the signal sequence there is a synthetic oligonucleotide with the cloning site, HindIII, with zero, one, or two extrabases between the linker and the signal sequence in different members of the set. Thus, by an appropriate choice, the gene insertedat the HindIII site will betranslated in the same reading frame with the signal sequence. The authentic cleavage site ofthe signal peptide, after its last Ala residue,is

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preserved. The linker oligonucleotide encodes three or four amino acid residues, depending on the particular construction or reading frame. In the basic setof plasmids, the HindIII linker is followed by PUB110 DNA, thus the translation and transcription termination signal should be provided by the insertat the HindIII expression site.In a second setthere is, downstream from the HindIII linker, another synthetic oligonucleotide with a translational stop codon in all reading frames(66). In a third set there is a further extension, a fragment with the transcription termination site of the amy gene (67). Efficient termination of transcription may, although not invariably, increase the efficiency of expression and even the stability of the plasmid. m e applicabilityof this secretion vector system, with some minor variation in the structure and the length of the linkage sequence betweenthe signal sequenceand the protein to be produced, has since been tested in several studies.A pattern has emerged which, by and large, has also been found withother secretion vector systemsto be described below. Interestingly, to date this secretion vector system has hardly been surpassed in terms of effectiveness and in the range of applicability. Penplasmic proteins of gram-negative bacteria, some of them of inductrial interest, such as pectin methylesterase ofErvinia chrysanthemi(68), or those of pharmaceutical interest, like somesubunits of the pertussis toxin (59,69), have been secretedrather efficiently, up to 500 mg/L of culture medium under of the signal optimal conditions. Inall of these constructions the cleavage peptide left someamino acids derivedfrom the linker on the N-terminus of the secreted protein.Partial proteolytic leavageof the productwas also found in some cases (69). Some periplasmic proteins were, however, either not expressedor not secreted,as demonstrated by very poor levels ofproduction of some of the subunits of the pertussis toxin (59). Not surprisingly, considering their closer relationshipto B. subtifis,and thus their expected better fit to the secretion machinery, extracellular or secreted proteins of gram-positive bacteria have been both expressed and secreted at high levels. For example,the thermostable a-amylaseof B. licheniformis is producedat levels morethan 500 times higherthan the amount secreted by the native host(70). However, even some secretory proteins of gram-positive bacteria, such as the inactive variant of diphtheria toxin, was produced and secreted onlyat levels of lessthan 5 mg/L, for reasons that were not obvious (71). The first eukaryotic protein expressed the with aid of this secretion system was human interferon-a2. It was secreted into the growth medium, processed by the cleaving of the signal peptide, and found to be biologi-

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cally active. Very disappointingly, however, and anticipating later findings with other secretion systems,the efficiency of its secretionwas verypoor, only in the range of 1 mg/L, withthe product being unstabledue to proteolytic cleavage (see Section 2.6 for more detailed discussion) (72,73). Another a-amylase-base secretion system has been constructed, based on the gene of the saccharifying amylaseof B. subtilis, by way of close analogy withthe foregoing setof vectors. The umyE gene from a hyperproducer strain of B. subtilis was cloned and a DNA fragment encoding the signal peptideand a long upstream segment containing the promoter and the so-calledumyR2 element, upstream from the promoter, was cloned in a derivative ofthe multicopy plasmidPUB110 (74). amyR2 was identiof fied in the hyperproducing strain as an element enhancing the activity the amyE gene (75); it has a palindromic sequenceand one could anticipate that it is a palindromic transcription termination site of the upstream gene and thus prevents transcriptional read-through by possibly interfering with the expression of umyE. Using the TEM P-lactamaseas a model protein, its processing and secretion with virtually no intracellular accumulation, were demonstrated, when the gene withoutthe signal sequence was fused downstream from the signal sequence of umyE of the vector (74,76,77). In some ofthe constructions, in additionto the signal sequence, there were about 10 N-terminal codons of the mature a-amylase, a "prosequence" foundin this a-amylase and nibbled offtheingrowth medium after cleavage ofthe signal sequence. This addition decreased the amount of secreted P-lactamase (to about half); the later finding showed that this prosequence hasno role inthe process of the secretion of a-amylase (78). Many "random" peptides of about 30 to 40 residues were also inserted between the signal sequenceand another model protein, thermostable bacillar a-amylase (79). Interestingly, some of them hardly affected secretion, suggesting much leeway the in designs of constructions for the secretion of a particular protein. The overall efficiency of this secretion vector was, however, much lower that that of the one based on the a-amylaseof the B. amyloliquefaciens. The maximal level of 0-lactamasewas only about 10% of the latter one in ordinary richlaboratory media (74). This probablyis a reflection of the lower rate of a-amylase secretion and thus presumably amyE gene activity in B. subtilis, even in the hyperproducing amyR2 strian. However, much improvementof the yield, up to 50 to 100 mg/L, was achieved in glucose and succinate rich media combined with growth under conditions of poor aeration usingan exoproteasedeficienthost (80). The maximum level of 150 mg/L of the same P-lactamase has been obtained with B.the

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amyldiquefaciens amylase-based vector in rich media with 6% of glucose (81). Efforts to produce two eukaryotic proteins, mouse interferon-/3 and a plant protein, thaumatin, with this system have been described (82-84). Again, the proteins were secretedand processed without cellularretardation, but at a very low level indeed (see also Section 2.4). Several constructions of mouse interferon were tested, with different NH2-terminal extensions. They were all secretedat similar levels, but there were severalfold differences betweenthe specific biological activities of the different constructions. In both secrtion systems baseon a-amylases, the maximal amount of the product is reachedat the end of the logarithmic growth phase in rich laboratory media such as L-broth. There is a decrease in the amount of the product in theculture medium inthe stationary phase, as indicated in detailed studies with the model protein &lactamase (80,85,86a). Unlike the case of P-lactamase, the production of homologous a-amylase, expressed from the same vector, continued for 10to 30 h after the cessation of growth (86). This suggests proteolytic degradation of the heterologous product by exoproteases abundant in theculture medium at stationary phase. The yield could be improved by usingmutant host strains with greatly (over 95%) decreased amounts of exoproteases (64,85) (see Section 2.6 for more discussion of the role of proteases). Furthermore, culture conditions suppressing the induction of exoproteases, suchas with high concentrations of glucose in the medium, can improve the yield further (81). An interesting feature of the secretion vector based on theamy gene ofB. amykhefaciens is the lack of catabolic repression by glucose when the promoter is fusedto a foreign gene (71,81).

2.2.2 Vectors Based on Proteases

Proteases are another class of bacillar exoenzymes secreted at high levels in manyBacillus species (60),thus providing a potential source of expression and secretion signalsfor the constructionof secretion vector systems. Like the a-amylase gene of a B. amyloliquefaciens,major exoprotease genes of this species have also been found to be efficiently expressed, with significant secretionlevels of the appropriate protein in B. subtilis (87,88). Several secretion vector systems have been constructed based on these genes. The activity ofthe genes ofthe major bacillar exoproteases is coupled to the initiation of sporulation. The synthesis and secretion of proteases does not start before the earlystationary phase of culture. This is a po-

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tential drawback for a secretion vector system based on these genes. The secretion of the heterologous protein follows this same pattern of temporal expression, resulting in the simulataneous secretion of the protein of interest and the exoproteases, with possible concomitant degradation. Therefore, the use of protease-deficient host strains for the production of most proteins (see Sections 2.6, 2.9) is highly desirable. On the other hand, the activity of protease genes is also regulated by several transcriptional regulatory factors, the degQ and degU among them (for a recent review, see Ref. 89). Certain mutations in these genes or their overexpression (degQ) (e.g., by cloning in a multicopy plasmid) stimulate the activity of exoprotease genes; the target sequences of these regulators are located in the region about lo00 bp upstream from the promoter. These regulatory genes provide a means to regulate, as well as to enhance considerably, the production of a heterologous protein, when secretion vectors based on protease genes are used. (deg genes are involved also in regulation of the genes of other exoproteins such as levansucrase, used as the basis of other secretion vectors; see below.) Unlike other bacillar exoproteins, the secretion of proteases in bacilli involves other extensions in addition to the signal peptide in the precursor, which are cleaved off in the process of secretion. The N-terminal segment of the precursor (often called alsopresequence), in the range of 30 amino acid residues, is a typical signal peptide with the appropriate function (for a review, see Ref. 6). Between the signal peptide and the N-terminus of the mature exoprotease, there is another extension, the prosequence, which varies in length from about 60 to 200 amino acid residues in the precursors of different proteases (6). The prosequence is essential for the maturation of the precursor to an active protease. It has recently been shown to function in the final step of folding of the protease, after its translocation across the cytoplasmic membrane but before release from the membrane, to promote its final, enzymatically active conformation (90,91). The prosequence is then cleaved off, mainly autocatalytically, releasing the active enzyme from the cell. Consequently, mutant exoproteases, which are enzymatically inactive, are not released from the prosequence and secreted. This correlates with greatly decreased synthesis (92,93). Although it was demonstrated subsequently that the prosequence does not have a function in the process of secretion of BaciZZus proteases (94), such a role was considered likely when many early and successful secretion vectors based on proteases were designed, resulting in vectors containing both a signal sequence and elements of the prosequence. However, as described below, segments of prosequence are dispensable for the construction of secretion vectors.

Gene Expression in Recombinant Bacillus

65

The first approach was taken with the neutral protease gene (npr)of B. amyloliquefaciens. In the initial construction a fragment of DNA encoding the signal sequence plus the entire prosequence (“preprosequence”), and the promoter region with upstream region of a few hundred base pairs was cloned in the plasmid PUB1 10 (88,97). The gene encoding the a-amylase of B. subtilis (without its signal sequence) was fused downstream from the prosequence, with the first 15 codons of the mature part of the protease intervening. Good expression of the a-amylase construction was seen in B. subtilis. Furthermore, the long prosequence (209 amino acid residues) was not present in the secreted a-amylase. Interestingly, the prosequence did not seem to interfere with the folding of a-amylase, from which was removed effectively despite the obvious lack of an autocatalytic process, presumably by exoproteases. An analogous construction based on the promoter region and preprosequence of the alkaline protease of B. amyloliquefacienshas also been described (95). Secretion of the protein A of Staphylococcusaureus was demonstrated with this system (96). However, in this case, the prosequence remained fused to the secreted product, although cleavage of the signal sequence took place. Reasons for the difference between these findings have not been elucidated. The secretion of two human secreted proteins, interferon-@(IFN-@) and growth hormone (hGN), has also been studied with the foregoing npr-based vector system. The parts of the appropriate genes encoding the mature protein were fused to the 3’ end of the prosequence as in the model proteins above (97,98). In good agreement with the studies of model proteins, both human proteins were secreted, and again there was no prosequence left in the secreted protein. However, the amounts were much smaller than those of either a-amylase or another model protein, P-lactamase. The amount of hGH was estimated to be about 1.5 mg/L of culture. Furthemore, proteolytic degradation of both proteins was evident. This secretion vector was developed further by the removal of most of the prosequence, resulting ultimately in a construct with the promoter and its upstream region, but with secretion signals composed of the signal sequence plus only the first 21 codons of the prosequence, followed by the structural gene encoding the protein to be secreted. In agreement with the lack of role for the prosequence in secretion per se, this vector performed similarly to the one with complete prosequence, with respect to the secretion of both a-amylase and hGH, in terms of the amount of these proteins secreted as well as the removal of the short N-terminal fragment of the prosequence (99,100). It was further demonstrated that the npr promoter of the vector was susceptible to the control of DegQ. Introducing

66

Sawas

another multicopy plasmid with the degQ of B. amyliquefaciens to the production host increased the expression of a reporter gene (cat) and the secretion of hGH moderately, about 1.5-fold (101). The use and development of this secretion system for the efficient secretionand production of hGH is described in Section2.4. Severallaboratories have described secretion based both on the neutral protease geneof B. amyloliquefaciens and on the alkaline protease gene of the same species, where the only secretion signal is the signal sequence. The signals of npr have been used in the vector constructions described in Refs. 95 and 102-105, the apr in Refs. 95 and 102 and a hybrid with npr promoter and the signalsequence of the subtilisinin (106). The plasmids were multicopy plasmids, either replicating onlyin Bacillus, or shuttle plasmids replicating also in E. coli (102). Some of these plasmids, like those based on either npr or apr in Ref. 102, were tailoredto secretion vector systems with a convenient cloning site immediately downstream from the cleavage site of the signal sequence, allowing the construction of fusions whose processing during export could be expected to release a secreted protein withthe native N-terminus of the heterologous protein. Studies of model proteins of bacterial origin, protein A of S. hureus (95) and the extracellular form of the a-amylaseof B. lichemifonnis (103), showed that the signal peptide alone was a good secretion signal resulting in efficient secretion,up to 3 g/L of protein A, and 140 mgof a-amylase under optimal culture conditions. Whether the signal peptidewas cleaved exactly as intened, at the native cleavage site,was not possible to determine. The secreted proteins had ragged N-termini, most likely to dueproteolytic cleavage inthe culture medium, which was quite evident in most cases. As far as bacterial proteins are concerned, the efficiency of secretion by vectors based on the protease genes of B. arnyloliquefacens is, by and large, similarto that observed with vectors based on a-amYlase described above, although there are data only on proteins from grampositive bacteria. The secretion and production of a number of eukaryotic and human ~ U ~ U ' I proteins have been explored in these secretion systems, including atrial natriuretic factor (hANF) (107), human growth hormone @GH) ( l a ) , bovinepancreaticribonuclease (76), prochymosin and human IL-IP (105) (for details, see also Table 1).The secretion of these Proteins was achieved,but as described in Section 2.4, the efficiencyWaS generally poor. The feasibility of the secretion of a eukaryotic protein with a native N-terminus and biological activity was demonstrated both for the Pan-

Gene Expresston In Recombinant Bac///us

67

creatic ribonuclease and for hGH, with yields of about 5 and 8 mg/L, respectively (thelatter increased to 40 mg/L in highdensity cultures).

2.3

Other Secretion Vector Systems

A set of secretion vectors and production system has been constructed

exploiting the expression and secretion signals of the levansucrase gene Ivs of B. subtilis (108-113).The primary emphasis ofthis set has beento introduce the elements of inducibilityand the activity inthe exponential phase of growth to the expression system. This has beenthe main goal in using the heterologous expression and secretion signals of the staphylokinase of S. aureus, for both secretion and intracellular expressionsystems (1 14,115). These production systems, and their use and benefits for secretory expression,are discussed in Section 3. Some secretion vectors have been based the on signal sequences ofthe a-amylase of the B. amyloliquefaciem or the apr exoprotease of B. subtilis, but fusedto promoters of other genes. A few constructions have been described, each, however, usedto express onlya single protein. The signal sequence ofamy has been combined with a promoter of the phages SPOl (1 16)and SP02 (1 17), and the signal ofapr with a strong vegetative promoter of B. subtilis cloned for the purpose (1 18). An analogous construction was made by fusinganother strong, vegetative promoter ofB. subtilis, P43, to the signal sequenceof the levansucrase ( h ) gene (1 19). The performance of these vectors, especially in comparison with the vectors based on the appropriate native genes, is difficult to evaluate, becauseno common reporter proteins have been studied. Three constructions were tested with an eukaryotic protein. The peak productionswere the secretion of 5 mg of a single-chain antibody molecule and 10 mg of a plant galactosidase per liter of culture, with combinationsof the P43 promoterIvs signal (1 19) and the SPOZamylase signal (1 17), respectively. The E. coli P-lactamase was reported to have been secreted very efficiently with the vector combiningthe secretion signalof apr with the vegetative promoter, but data on absolute amounts were not reported (1 18). An early secretion vector construction was based on the gene of the plactamase (penicillinase) ofB. licheniformis.This proteinis an exported lipoprotein, anchoredon the outer surface of the cell membrane by three fatty acid molecules covalently boundto the N-terminus ofthe molecule (for a review, see Refs. 120 and 121). Considerable amounts of the protein domain of this moleculeare released in a soluble and enzymatically active exoform inthe growth medium inthe stationary phase of growth

68

Sawas

by a proteolytic cleavage near the N-terminus. When clonedB.in subtilis, the protein is well expressed, but there is proportionally much less ofthe exoform as in B. licheniformis(122). To construct a secretion vectorthe promoter and signal sequence of the penP gene were used; the performance of the vector was studied by fusing these expression signals to DNA sequences encoding preproinsulin, human @-interferon, and growth hormone (123). There was a very low level of secretion of insulin together with cell-bound protein, cell-bound interferon, and an undefined level of secretion of growth hormone.The performance of this vector system has not been tested with reporter a protein secreted efficiently with some other secretion vector system.

2.4 Secretion of EukaryoticProteinsin BacNus

As described inthe sections above, heterologous proteins of bacterial origin are, as a rule, secreted inB. subtilis with the aid of the secretion vectors. Those of gram-positive speciesare secreted very efficiently, as are many of thosefrom gram-negative bacteria. Incontrast, eukaryotic proteins, particularly those of human origin, even when they are secretory proteins by nature, are invariably secreted at much lower levels. Many eukaryotic proteins areof pharmaceutical or industrial interest, and much work on the bacillar secretion vectors hastherefore been on eukaryotic proteins. None of bacillar secretion vector-host systems have found been to be clearly superiorto the others for the productionof eukaryotic proteins, but inthe course of these studies, elementsfor optimizing the secretoary modeof production have been identified, resulting in improvements in the efficiency of secretion and yields suitablefor the industrial production for some proteins. In the following pages, some findings relevant to the efficiency of secretion of eukaryotic proteins in Bacillus will be discussed, the constraints identified, and the means for optimizing the vector-host systems and the conditions of production presented. The potential of secretion vector systemsto produce eucaryotic proteins has been tested with interferon (IFN) in many laboratories. Interferons are naturally secreted proteins and of relatively small size(about 20 kDa); botha- and @-interferons are nonglycosylated. These are all features expectedto be favorablefor secretion in a bacterial system. Furthermore, there would be much practical interest to produce these proteins in a bacterial production system other than E. coli (for references, see Ref. 72). To study the secretion vector based on the a-amylase B. amyof loliquefaciem, Palva etal. (72) and Schein et al.(73) inserted the segment

Expression Gene

in Recombinant Bacillus

69

of DNA encodingmature secreted humaninterferon-a2 (IFN-a2) inthe vector. In the first construction, there were a few linker-derived codons between the cleavage site of the signal peptide and the N-terminus ofIFNa2; in the latter construction, the IFN-a2 insert was fused directlyto the last codonof the signal sequence. Both constructions were expressed, and secretion ofIFN into the growth medium was clearly demonstrated. More than 90% of I F N synthesized wasfound in the culture medium.Furthermore, in both cases there was accurate cleavage of the signal peptide at its C-terminus. Thus B. subtilis is clearly capable of secreting this eucaryotic protein. However, the amount of IFN inthe growth mediumwas only in the range of1 mg/L of culture, while expression levelsup to 1 or 0.1 g/L had been obtained in similar growth conditions.(L-broth, shake flask culptures) inthe case of homologous a-amylase or heterologous, bacterial lactamase (TEM of E. coli)respectively (61,64,65,73). The strainused for the experiment wasa mutant with much reduced secretion of exoproteases; in agreement with protease sensitivity of the interferon, hardly any activity found in culture media when the host strain was wild type. Quantitatively similar results were obtained in experiments designed to secrete humanand mouse IFN-fl inB. subtilis using secretion vector systems basedon the neutral protease geneof B. amyloliquefaciens [human IFN-p (97)] or the a-amylase gene of B. subtilis [mouse IFN-b (82,83)]. Again, active interferonwas secreted, but far below the secretion capacity of the system,as judged by the level of secretion of homologous or heterologous bacterial exoenzymes expressed from the same signals (74,97). The amount of IFN secreted in the production system of Palvaal.et(72) was not higher than that in the others, despite its much higher inherent expression capacity. No secretion, onlya low level of intracellular accumulation of murine INF-a7, was achieved with a secretion vector based on the levan sucrase promoter and signal sequence(108). Thus rather similar levels of secretion were found for these eukaryotic proteins, although the efficiency of the promoters used varied considerably and the signal peptides were of differentamino acid sequences. Adjustingthe fusion of the murine IFNto the signal peptide in such a way that its predicted native secondary structurewas maintained also did not improve secretion. Clearly, there are severe obstacles, either for the secretionor expression of IFN itself in these systems. A somewhat different pattern was observed by Breitling et al. (114) with a secretion vector based on staphylokinase (above). Human IFN-a1 was secreted up to a level of 15 mg/L of culture in ordinary L-broth. This is about 10-fold higherthan obtained with the a-amylase secretion system;

70

Sarvas

IFN-a1 is closely related to IFN-a2 in its sequence and properties. Furthemore, the level of IFN secretion was only slightly below the level of secretion of staphylokinase itself using these same expression signals in B. subtilis under similar growth conditions(about 25 mg/L). As pointed out above [p.67 and in (114)], expression from the sak vector takes place effectively in the exponential phase of growth mitigating the effect of exoproteases, which might contribute to the better yield observed (but see also the role of degradation below). Alternatively, the combination of the sak signal peptide with IFW is especially favorable. It will be interesting to see whether this vector systemcan be adjusted for even higher levels of secretion of IFN or other heterologous proteins,up to the level of industrial significance. To date, the secretion B. in subtilis of a large numberof other eukaryotic proteins of different origin has been studied withdifferent secretion vector sytems. Table1 shows a partial list of representative experiments. As in the case of interferons,the gene of interestwas inserted into these secretion vectors eitherto result in a fusion with an exact joint to the signal sequence,or often with a few intervening codons derived from linkers with cloning sites (usually with little or no effect on the secretion). In most of these studies, the production host was also a mutant with decreased amounts of exoproteases, and production of the secreted product was reported for shake flask cultures in richlaboratory media. The overallpattern is not much different from that seen abovefor interferons, with a few notable exceptions. Regardless of the size, origin, or natureof the protein(e.g., glycosylation ofthe native protein), the level found in the culture supernatant at peak level of secretion was low in the majority of cases, from traces (indicated bya + in Table1) to a few milligrams per liter of culture. Again, the amount was always far below the amount of a homologous proteinor a bacterialreporter protein secreted by the same system; a difference of upto 1000-fold could be estimated. As a rule, the signal sequencewas absent from the product secreted; depending on the system, the heterologous protein was released witha native N-terminus or with a few additional or missing N-terminal amino acid residues (in most studiesthere was no effort to trim the fusion for exact cleavage). Most proteins with assayable biological activity had retained that activity. Rather consistently,the presence of a cell-bound precursor with an uncleaved signal peptide was either demonstratedor deduced, but the amountwas small in relation to the potential expression capacity of the system. Althoughthe overall level of secretion of the heterologous proteins was low, there were more than 2000-fold differences in secretion levels

~ecrgtionvector s ~ s t e ~ 67 Viral ~ e ~ b r a n47~ ~lycop~otei~se Bovine 40 pro~h~osin Sin~le-ch~n 26 rgc~~binant

+ + and 1 0.1 5

, a-amylase

+ +

P43 and ss of

.rap$gene of B. U m Y i o ~ ~ ~ M e ~ ~ ~ i e ~ P and ss of amy of ~20,221 B. f f ~ y i u ~ ~ ~ u e f u ~ i ~ ~ 106 P of the npr and ss of apr of B. s ~ b t i ~ ~ P 119

-

-;I 111.

0

tib body

Human lysozyme

14

0 or 0.2

Bovine pancreatic ribonuclease H u ~ a ~ p roinsulin man atrial na~ri~retic factor Plant protein t~au~atin

13

1-5

140, @-lactamase P and ss of npr, and P of spol or P1 with ss

+

116

of umy of B. s u b t ~ ~ ~ s 76 P and ss of u p of B.

a~y~~li~uefu~~ens 9

0.1

3

0.5

22

+

-

Protein Plant galactosidase

Size (kDa) 40

Amount secreted mg/L of cultureb 10

Human IL-1/3

18

7

Human IFN-P

18

+

Human growth hormone

22

50

Biological activity secreteC(mg/L)

+

Secretion vector system based ond Refs. SP02 promoter, ss of 117 amy of B.

amyloliquefaciens

+ 1O00, neutral otease , neutral protease

P and ss of npr of B. subtilis P and ss of npr of B.

105

97

amyloliquefacien P and ss of npr of amyloliquefaciens

bCulture in rich laboratory media in shake flasks if not indicated otherwise. ‘Judged by the secretion of a homologous or bacterial model protein under similar or nearby similar conditions. a +indicates the secretion of a small amount detected either with immunoblotting or by enzymatic activity. dDesignations of the genes: m y , a-amylase; apr, alkaline protease (subtilisin); npr, neutral protease. P and ss stand for promoter and signal sequence, respectively. %xtra membraneous domains of vesicular stomatitis virus membrane glycoprotein G and Semliki Forest virus glycoprotein E l .

Expression Gene

in RecombinantBac///us

73

among different proteins. Onecannot single out any obvious parameters correlating withthe efficiency of expressionand the secretion of a particular protein. It is thus interesting, and also of practical significance, that there were also cases of remarkably efficient secretion. Good production of human growth hormone (hGH) has been achieved (99,101) with the secretion vector based on the neutral protease gene (npr) of B. amyioliquefaciens (above), seemingly by maximizing the level of transcription and optimizing growth conditions. The insert encoded the mature hGH, with a short oligonucleotide (derived from the prosequence of npr) between the end of the signal peptide and the N-terminal codon of hGH. This construction resulted initially inthe secretion of the peak level of about 10 mg/L in a protease-deficient strain of B. subtilis. Again, this rather modest level isfar below the potential of the system, representedby hundreds of milligrams of the neutral protease B. of amyioliquefaciens under similar conditions(88). However, about a five-fold increase was obtained when the degQ gene ofB. amyloliquefaciens was introduced into the production host as a chromosomal insertion. Finally, by shifting production to fermentor cultures and applying a procedureof transferring the cells to fresh mediumat late stationary phase of growth (101), up to 200 mg of hGH per liter of culture was secreted, hGH comprising about 30% of total extracellular proteins. The majority of the protein was of the size of but native hGH (22 kDa), withthe signal peptide presumably cleaved off leaving a ragged N-terminus. Despite the use of a low-exoprotease strain, there was a considerable amount of a degradation product of about 15 kDa. The same vector system was also trimmed to fuse hGH gene directly to the end of the signal sequence(104). This modification didnot affect the level and pattern of expression significantly:about 10 mg ofhGH/L was found in the late stationary phase in shake flask cultures. In this construction, need for the termination of transcription of the inserted gene was also clearly demonstrated. Without a termination sitethe yield was less than 10% of the one above. The secreted hGH had the specific activity ofthe native hormone,and most important, its N-terminal sequence matched the authentic one. The authors achieved production up to 40 mg/L of culture in a high-density culture. Human interleukin-3 (IL-3),a 13-kDa glycoprotein, isan example of another eukaryotic protein, which has been produced successfully at high levels in the culture medium of Bacillus by choosing the proper production strain and culture conditions (124). The hostwas an industrial strain of B. licheniformis, which is an effective secretor of a-amylase, devoid

74

Sawas

of the major exoproteasesand nonsporulating. The secretion systemwas a derivative of the pUBllO plasmid, with the expression cassette composed of the signal sequence ofthe a-amylase of B. licheniformis, combined either with the promoter of the same gene or with a promoter of was introduced the plasmid PUB110. The sequence encoding native IL-3 into the vector by precise fusion to the signal sequence. Upto 200 mg/L of IL-3 protein and about 100 mg/L of nondegraded IL-3 was found in in the culture medium in a fermentor batch; virtuallyno IL-3 protein remained in the cellular fraction. Secreted IL-3was fully active biologically although it remained unglycoslated. A purification procedure compatible with industrial production was developed, and the purified product has now entered clinical trials.Van Leen etal. (124) also studied and evaluated the production of IL-3 in E. coli, Saccharomyces cerevisiae, and mammalian cell production systems.In terms of yield, quality, and production cost, they judged the B. licheniformis system to be the most suitable one for practical production.

2.5 ConstraintsontheSecretion of Heterologous Proteins in Bacillus The overallpattern emerging from the studies described above is that secretion of many heterologous proteins of bacterial origin with the secretion vectors isquite efficient, but some bacterial proteins and most of the eukaryotic proteins are produced with low and variable yields. The studies performed so far give few indications as to whether this high degree of variation is relatedto the type of protein or someof its specified properis not very high and most ties. However, the number of proteins studied of the eukaryotic proteins had biological activityof pharmaceutical interest; they may comprise a biased group. Reasonsfor the inefficient production and some possible ways to improve it are discussed below.

2.6 ProteolyticDegradation of Heterologous Proteins Secreted in Bacillus B. subtilis and other Bacillus species typically secrete proteasesinto the growth medium (for a review, see Ref. 58). In B. subtilis, the alkaline, serine protease, subtilisin,and the neutral metalloprotease comprise more than 90% of all secreted exoproteases. They are efficient enzymes with low substrate specificity, especially subtilisin. In addition, there are several minor proteases (see below). The expression of proteases is temporally

Gem Expression in Recombinant Bacillus

75

controlled; the maximal synthesis takes place in the late exponential phase of growth, concomitant withthe initiation of sporulation. Most ofthe heterologous proteinsof interest, which are candidates for production inBacillus, are sensitive to these exoproteases,and generally much more sensitive than bacillar exoproteins. An obvious and anticipated reason for the low extracellular level of heterologous proteinsproduced with secretion vector systems could thus be (rapid) proteolytic degradation in the growth medium. This has actually been observed to varying degrees with all heterologous nonbacillar proteins secreted with any of the secretion systems described (see,e.g. Refs. 125 and 126). A clearcut, even decisive rolefor proteases in the decrease of the yield was shown for some of the model proteins. Ulmanen et al.(65) found that the peak level of P-lactamase in theculture supernatant was about 10 to 20 mg/L when secreted from the vector based onthe gene of the a-amylase ofB. amyloliquefaciens.This level then decreased rapidly inthe stationary phase of growth, suggesting proteolytic degradation. The rate of degradation correlated with the amount of the exoproteases secreted by the host strain (85). A similar decline has been shown for the same 0-lactamase in other secretion vector systems (127). Even at the peak level of secretion the amount was only inthe range of 10% of the amount of homologous a-amylase expressedfrom the same vector. However, pulse labeling experiments showedthat the rate of secretion of both proteins was identical, even in late stationary cultures, where the ratio of P-lactamaseto a-amylase (assayedas protein) was about 1500. Pulse-chase experiments demonstrated degradation of P-lactamase in the culture medium at a rate comparable to its decreased steady-state level. Thus, in the case of this model protein, the sole reasonfor the low yield compared to the homologous proteinwas clearly proteolytic degradation. Another documented example of significant proteolysis of a heterologous proteinthat is effectivelyand efficiently secreted per se B. in subtilis is the protein A of S. aureus. This proteinwas secreted at a very high level from another a-amylase vector, but wasit found in the culture supernatant to be largely in a fragmented form (128). At least apartial contribution of proteolytic degradationfor the low yieldOf many other heterologous proteinis indicated by the decreased amount of the product after extended growth, concomitant with the appearance of exoproteases in the culture medium, as describedfor some proteins in previous sections. There is thus little doubt that extracellular proteolytic degradationis a

76

Sanras

major problem for the production of heterologous proteins sensitiveto in many laborabacillar exoprotease. There has therefore been much effort tories to construct host strains devoidof proteases. Exoprotease-deficient mutants of B. subtilis have been isolated after mutagenic treatment andused as hosts for the productionof secreted proteins. Some mutations have been shown to be present in the known genes of (apr)and neutral protease(npr) the major exoproteases, alkaline protease (96,101). Others are not characterized genetically, yith single or multiple mutations affectingthe level of exoproteases, perhaps via the regulatory network affecting the level of expression of exoenzymes, as well as possibly sporulation and competence (85,97,114,129). Well-characterized spo mutants such as spoOA which affect the secretion of multiple proteases can also be used as low-exoprotease hosts(107). Most of these mutants have residual exoproteolytic activity,up to a few percent of that of the wild type. Efforts to lower that level further by mutagenesis often decreased viability, presumably via the regulatory system above. The cloning and identification of the structuralgenes of the exoproteases ofB. subtilis was a major step forward, providing the means for their precise deletion. Such deletions, even those encompassing several protease genes, turned out not to affect optimal growth. Nor did they impair sporulation, contrary to previous beliefs concerning their “essential” role in this process. However, the culture supernatant of multiple protease-negativemutants may contain more cell-derived proteinsthan that of the wild type, indicating cell lysis and hampering the purification of the secreted product(IPalva, . personal communication). The deletions, or inactivationby point mutations, of the two genes of the major proteases of B. subtilis, apr and npr, simultaneously (130-133) decreased the level of proteolytic activity inthe culture medium to 10 to U)070 of the wild-type level, dependingon the particular B. subtilis strain used. Since becoming available, mutants defective with respect to both of these major exoproteases have been used extensively in studies and applications of the secretion of heterologous proteins (Table1). A further decrease of proteases has been obtained by introducing deletions proof tease genes inspo mutants (1 14,132).However, the residual extracellular proteolytic activityof the mutants defective only inthe major proteases similar or even lower levelscan also be achieved is not particularly low, and in mutants constructed with random mutagenesis (64,85). The residual protease activityafter elimination of the major proteases was believed to be due to minor exoproteases (see, e.g., Ref.109). Indeed, up to five such proteases have been identified, with subsequent cloning

Expression Gene

in RecombinantBacillus

n

and deletionof the respective genes. There are three serine proteases: Epr extracellular protease (encoded by epr (134);bacillopeptidase F, which has also unique high esterolytic activity (encoded by the gene designated bps in Ref. 135 and bpt in Refs. 130 and 136), and another minor extravpr (137). Furthermore, there is a minor cellular serine protease encoded by metalloprotease, Mpr (138), and a novel neutral protease, NprB (1 12, 139). Elimination of these proteases by deletion mutations decreased the level of exoprotease activity,but the removal of any single gene doesso only due to the commoderately (134,137,140).The modest effect is probably pensatory increase in the amount ofother minor exoproteases, someof them possibly still to be identified (112,137).Deletion of one protease may either increasethe expression of the remaining protease genes, or a new proteolytic activityis provided by a protease, which is degraded by a deleted exoprotease. For example, deletion of bpr caused about a 20fold increase in extracellular Mpr (1 35). Multiple deletions of exoprotease genes invarious combinations reduced the exoprotease level significantly below 1% of the wild-type level(1 12,137,141).However, eventhe simulsix exoprotease genes left some active residual protaneous deletion of the tease, presumably due to some unidentified protease(s). Releaseof some intracellular proteases may also contribute to the residual extracellular protease activity;the deletion of the gene for the major intracellular serine protease, ips (142),has therefore been combined with exoprotease deficiency in many strains. Studies on model proteins have demonstratedthat the use of exoproteasedeficient host strains improves both the yield and stability of secreted heterologous proteins. The most widely used model protein with very representative results in such studies has the been TEM P-lactamaseof E. coli. Its peak level, expressed from different secretion vectors, increased severalfold and its degradation in late stationary cultures decreased dramatically when the production host lacked the two major proteases (apr and npr genes) (e.g., Refs 85 and 118). Further improvement in stability was achieved by using the strain deficient in six exoproteases (AnprE, AaprE, Aepr, Abpf, Ampr, AnprB);a secretion vector basedon theP43 promoter and the signal sequence ofthe levansucrase was usedin the studies, in the presence of multiple copies ofdegQ (see also the section above) (1 12). Comparable stability of a eukaryotic protein,a single-chain antibody molecule secreted by this vector system and this strain, was achieved at the level of5about mg of protein per liter of culture (1 19). The eliminationof only the two major proteases increasedthe stability only slightly compared with the produc-

78

Sanras

tion host with all wild-type exoproteases (119). The proteasedeficient strains of B. subtilis are clearly the obvious choicefor the production of most, if not all, heterologous proteins secreted B. in subtilis. As pointed above, hosts devoid of the major proteases have already been used in most of the experiments to produce eukaryotic proteins of practical interest (Table l), with favorable effectson yield and stability. There are, however, no reports in the literature that clarify whetherthe use of multiple deletion mutants could further improve the production of these proteins to the same degree as has beenfound with the model proteins.

2.7 Other Factors Influencing the Secretion Heterologous Proteins

of

There are indications that proteases may not be the only cause of poor performance of secretion vectors. In particular, the elimination of exooften left thepeak levelof the proteases downto a very low residual level secreted heterologous protein disproportionately below the optimal capacity of the secretion system, as judged by the expression of a homologous reporter protein. A high rate of transcription hasbeen found in such cases (e.g., Refs. 65, 73 and 108), strongly suggesting a hindrance in or incompatibility of the heterologous proteins with the secretion machinery itself. Schein etal. (73) addressed this directly by studying the secretion of IFNa2 with the secretion vector based on the a-amylaseB.ofamyloliquefaciens (64). Under conditions where only a minimal amountof exoproteases was produced (a low-protease host strain and early stationary phase of growth) and where no measurable degradation of interferon occurred in the growth medium, several-fold less interferon than a-amylase was secreted from the bacterium, as judged by pulse-chase labeling. Moreover, there was significant cellular accumulationof the precursor with an cleaved signal peptide. Clearly, the interferon so produced is either retarded in its passage or translocation across the cytoplasmic membrane, or it is aggregated in the cytoplasm. A cell-bound form of a poorly secreted protein has been 1, suggesting that retarreported in several of the studies shown in Table dation in the secretion of heterologous proteins inB. subtilis is not an exceptional occurrence.Very suggestive ofa block in secretion the is cellular accumulation,and harmful effecton growth, of the human natriuretic a-factor when expressed from an effective secretion vector (107). A block at a very early stage of secretion was demonstrated for,the outer membrane proteins OmpA and OmpF of E. coli. When expressed as a

Expression Gene

in Recombinant 8acillus

79

fusion to the signal peptide of the a-amylase of B. amyloliquefaciens,they accumulated intracellularly with an uncleaved signal peptide, showing no indication of engagement with the secretion machinery B. ofsubtilis (143). Translocation acrossthe cytoplasmic membraneand cleavage of the signal sequencetook place, however, when the level of expression was low (144). As such, these findingsare rather unexpected, becauseouter membrane proteins are translocated across the cytoplasmic membrane in E. are secreted effectively coli just like periplasmic proteins, most of which in Bacillus (above). A differentpattern of the blockage of secretion was found for human serum albumin (145). There was no secretion fromB. subtilis using a secretion vector based on the a-amylase geneof B. amyloliquefaciens, strain B, but low-level secretion was achieved from protoplasts after removal of the cell wall. However, even in cells with intact cell walls, there was both low-level synthesis of albuminand some cleavage of the signal peptide ofthe construction (albeit partial), indicating translocation across the cytoplasmic membrane. The cell wall maythus be a hindrance for the secretion of a heterologous protein. Interestingly,both the efficiency ofthe cleavage ofthe signal peptide and the secretion of albumin from protoplasts was inversely of difrelated to the efficiency ofthe synthesis of albumin, when vectors ferent levels of expression werestudied. Unfortunately, virtually nothing is knownabout theway even naturally secreted proteins of Bacilli traverse the cell wall after they have crossed the cytoplasmic membrane and are released from it, or on their interaction withthe components of the cell wall during the process of secretion (for a review, see Ref.146). So far, human albumin has remained the only heterologous protein, the secretion of which has been clearly demonstrated to be blocked at the level of the cell wall. However, the mode of its interaction with the cell wall has not been described. Similar problems with some other heterologous proteins might, however, be anticipated. Taken together, the foregoing findings pointto an incompatibility of varying degree between the components of the cellular secretion machinery of Bacillus and many heterologous proteins, especially those of eukaryotic origin. At present,this is a formidable problem efforts in to optimize the secretion of proteins of interest, requiring identificationof the limiting factors as well as characterization of their interaction with the secreted protein. The incompatibility could materialize at any stage of the secretion. First, the heterologous protein may be retarded during translocation through

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the cytoplasmic membrane;the few pulse-chase experiments performed, indicating slow export and processing (above), would be consistent with a hindrance at this stage. However, very little is known about this process at the molecular level inBacillus. As discussed above, not even all of the cellular componentsof the secretion machinery have been identified, and any studies on specific interactions of the machinery with the secreted protein itself are seriously hampered Bacillus in by the lackof an in vitro assay for translocation. Second, the precursorof the secreted protein. has to be folded properly and maintained in the conformation compatible for export before the step of translocation. As pointed out in previous sections, this involves the interaction of several chaperonesand the recently characterized bacterial signal recognition particle. Our understanding of the specificity of these factors is poor. The pattern of intracellular precipitation inan expressiondependent manner is indeed very suggestive ofinappropriate folding due to the lack of interaction with a chaperone. At least one chaperone, the SecB proteins of E. coli, is known to be specificfor a defined set of exthat incompaported proteins (fora review, see Ref.26). It seems possible tibility with bacillar chaperones could be a common reason for the poor export of heterologous proteins.This also suggests that the effect of introducing new chaperonesto the production hostto improve secretion should be explored.

2.8 Influence of Junction Between the Signal Peptide and the Mature Region Much effort has gone into studying the optimal way in which the signal sequence of the secretion vector shouldbe joined to the N-terminal end of the protein to be produced. A major role of the signal peptide isto keep the precursor ofthe secreted protein in a loose, export-compatible conformation, by interacting with the mature part of the exported protein. Mutation studies showthat a mismatch betweenthe signal peptideand the Nterminal portionof the mature region can affect the efficiency of secretion by introducing fast, tight folding of the precursor(147); and optimal amino acid sequenceand/or conformation at the junctionbetween the signal peptide and the heterologous protein can be anticipated to favor secretion. A further constraint on thenature of the junction is the need to preserve the cleavage site ofthe signal peptidase, with astructure facilitating the

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the optimalrate of translocation and release of the protein from the cytoplasmic membrane. For many but not all applications, an additional goal is the release of the protein in a mannerthat generates a product with a homogeneous, native-like N-terminus. Direct fusionof the last amino acid residue of the signal peptideto the N-terminal residue ofthe protein of interest has usually resulted in both secretion of the protein and cleavage of the signal peptide in all the secretion systems described. However, it should also be noted that the cleavage may be blocked or severely hindered by positively charged residues immediately downstream fromthe signal peptide; such residues are found of some eukaryotic proteins(148). In among the first N-terminal residues a pattern consistent with the cleavage of signal peptides of natural proteins (148,149) the specificity of the cleavagewas found to be largelyindependent of the residues on the side ofthe mature region. The cleavage may thus produce a product with the native N-terminus. However, this has not always been the case. A mixture of cleavage products differing in afew N-terminal amino acid residues in length has also been found. One possible reason may be proteolytic cleavage, or exoproteolytic nibbling after the correct cleavage of the signal peptide and after the release of product to the medium. In some cases, the N-terminal sequences of the product show that cleavage hasnot taken place after the signal peptide,but at sites conforming to the specificity of the signal peptidases and in the vicinity of the correct cleavage site (e.g., Refs. 68, 111 and 150). This is in agreement with the experimental evidence that the length of the C-terminal domain of bacillar signal peptides canbe varied with little influence on the efficiency ofthe cleavage of the signal peptide. The change in length can redirect the cleavage to a nearby site with a proper sequence(79,127,148). Accordingly, it may be reasonable to modify the junction between the signal peptide and mature region to contain only one signal peptidase cleavage site, resulting in exactly the product desired. The efficiency of the secretionthe of heterologous protein may also be affected by insertingan additional peptide between the signal peptide and the protein to be produced, often the consequence of introducinga (multiple) cloning site in the secretion vector. A systematic analysis of the effect of relatively long peptides of "random" sequence 20 to 70 (amino acid residues) between the signal peptideof the a-amylase of B. subtilis and the reporter protein, a-amylase of B. licheniformis, showed moderate

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decrease, drastic decrease, or moderate (about 1.5-fold) increaseof the secretion, depending on the particular peptide(79). In another study, the structure of the signal/mature junctionwas varied by extendingthe signal sequence by up to five codons of the mature region of the a-amylase (the protein on which the vector was based on), using additional codons derived from the cloning site constructions (70,151). The maximal level of secretion ofthe reporter proteinswas achieved with constructions of about four intervening amino acid residues; the length of the peptide,but not its sequence, seemedto be the crucial determinant. The level of secretion was about twofold in comparison with a nearly “direct” fusion; other insertions decreased secretion down to 50%. In agreement with these findings, short peptides derivedfrom a cloning-site oligonucleotide nextto the signal sequence have turned out in other secretion vector constructions eithernot to affect secretion or to decrease it moderately. Interestingly, extendingthe secretion signal inthe secretion vector to include even significant segments of the N-terminal region of the appropriate mature secretory protein has not improved the performance ofthe vector nor decreased secretion, although such constructions would be expected to contain an ideal signal peptidase cleavage siteand possibly optimize the match between the signal peptide and the (fusion) protein to be produced (12,64,9!3,101). One more strategyto optimize the region isto construct a hybrid secretion signal, where the N-terminal part of the signal sequence derives from the gene ofthe promoter of the secretion vectorand the C-terminal part from the gene of the protein to be produced, extending directlyto the mature region of that protein. The rationale for this construction is to create mRNA with a native-like 5‘ part in order to optimize expression, and as native a fit as possible with the signal peptideand the mature region of the protein. Indeed, such a construction was up to fourfold more effective than direct fusion when tested with a periplasmic galacturonase and using the secretion vector based on the a-amylase of B. amyloliquefaciem (1 52). One can conclude that there is considerable gainto be achieved bytuning the amino acid sequence at or around the junction between the signal peptide ofthe expression systemand the N-terminus ofthe secreted, heterologous protein. However, there is clearly no single optimal strategy or structure, and the optimization remains an empirical task; none of the strategies discussed have been reported to have been applied to more than one model protein on a comparative basis.

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2.9 Bacillus brevis as a Host for the Production of Secreted Protein An original approach to the design ofan efficent host-vector system devoid of some of the constraints of B. subtilis has beenthe development of B. brevis for the secretion of heterologous proteins. This has been a longterm pursuit inthe laboratory of S. Udaga, alsoreviewed recently bythe same group(153,154). The host systemis based on two B. brevis strains as 47 (155)and HPD31(156). These isolated by this laboratory, designated strains have a cell-wallstructure with unique features. Onthe surface of cell there is a layer of two proteins in an hexagonal lattice, each about100 kDa ( M W P , middle cell-wallprotein, and OWP, outer cell-wall protein). In an unusual way, these proteins are shed fromthe cell inthe stationary phase of culture. However, the synthesis of the cell-wall proteins continues and they are secreted to the culture medium for hours, resulting in high extracellular concentrations, 10 and 20 g/L in laboratory media in the strains47 and HPD31, respectively. This indicates that this B. brevis system has a high intrinsic capacity for protein secretion and that the genes ofthe cell-wall proteins are highly active. The cell-wall proteins are encoded bya two-cistron operon (157,158), whose promoter region5'-and cistron secretion signals (encoding M P ) are also obvious candidatesfor the construction of an expression vector. A further major attraction of the B. brevis system isthe very lowor nearly nonexistent level of extracellular proteases. In late-stationary-phase cultures, exoprotease activity of 1.6% (strain 47) and less than 0.02% (MPD31) of the amounts in the culture supernatant of B. subtilis have been reported (159). This suggests that B. brevis could be an exellent alternative host to exoproteasedeficient mutantsof B. subtilis. However, data on the stability of notoriously protease-sensitive eukaryotic proteins in the system are not yet available. A set of secretion vectors with the promoter region (158)and the signal sequence ofthe 5' proximal geneof the operon (the MWP) have been described. The plasmids used are either derivatives ofthe PUB1 10or of an endogenous high-copy-number plasmid of B. brevis (160). Initially, the capacity of the system was studied,and demonstrated with exoproteins from relatedBacillus species. These include thermostabile a-amylases of B. licheniformis and B. stearothermophilus, both secreted at very low levels by the respective species and both of obvious industrial interest, and an esterase of B. stearothermophilus and the sphingomyelinase ofB. cereus (156,161). Allof these enzymes were indeed expressed and secreted,

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with proper cleavage of the signal peptide. The maximal level of synthesis varied from0.1 to 2 g/L of the protein of interest in the culture supernatant; the highest values were obtained with the strain HPD31 in a very rich medium. The amounts of these proteins at arethe level of industrial interest, although theyare one order of magnitude belowthe yields ofthe secreted cell-wall proteins themselves. The feasibility ofthe system for the productionof eukaryotic proteins has since been demonstrated. However, not unlike in the case ofB. subtilis secretion system, the production was much lessefficient than for Bacillus proteins. The best results were obtained with human epidermal growth factor (hEGF), with the partof the gene encoding the mature protein fused directly to the signal sequence of the M W gene. Upto 250 mg/L of hEGF was secreted under optimal conditionsand after 3 days of growth (159) (see also Section2.4). The productwas a major protein of the culture supernatant, although the amount of contaminating MWPand O W proteins concomitantly secretedby the host were not measured. In agreement with the virtual lack of proteolytic activity of this strain, there was no evidence of degradation of hEGF. The amountof hEGF compares favorably with the best yields obtained with B. subtilis secretion systems, with spectacular stability of the product. Regrettably, such high yields were not obtained with other eukaryotic proteins of higher molecular weight, although their secretion was shown to be feasible. Usinga noncharacterized mutantof strain 47 with enhanced production capacity,60 mg/L of human salivary a-amylase was secreted. Swine pepsinogen, 10 mg/L, was secreted in native-like conformation and was activated autocatalytically under acidic conditions(see Table 1 for references). TheB. brevis system is clearly an elegant and interesting approach with much potential. But studies on the B. brevis system also showed that the poor compatibility of eukaryotic proteins with the secretion machinery inBacillus is not limitedto B. subtilis or B. Iicheniformis hosts. The extremely high levels of cell-wall proteins secreted concomitantly withthe produced proteininto the growth medium inthe B. brevis system complicate purification; secretion of cell wall proteins may also compete withthat of the protein of interest. One obvious step for further development of this system would be deletion ofthe chromosomal MWP and OWP genes. 3 CONTROLLEDGENEEXPRESSION

Despite their inherent efficiency, production systems based on the a-amylases and exoprotease genes of Bacilli have undesirable properties. As

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pointed out above, protease genes are naturally active onlyafter the exponential phaseof growth, when there also are rapidly increasing amounts of exoproteases in the culture medium. This problem is avoided in the case of a-amylase-based vectors; the expression signals of the cloned amy gene ofB. amyloliquefaciens are active in both the logarithmic and stationary phases of growth (e.g., Ref. 65). Gene of a-amylasesand the exoproteases are under catabolic repression and repressed by glucose, see above for the abocomplicating the use of very rich media. (However, lition of the glucose repression of the secretion vector based on the a-amylase of B. amyloliquefaciens, and Ref. 71) Glucose in the medium therefore cannotbe used to repress the initiation of sporulation, a convenient way to decrease the amount of exoproteases. Finally, the expression of a-amylases and exoproteases is not inducible in away practical either under laboratory conditions or in large-scale applications. This excludesthe possibility of using these expression signals for products, which,at a high level ofproduction are harmful to the cell, but whose production would be feasible ifthe expression of the gene could be limited to a short production phase at high cell density. Such a production mode could also increase the stability of some expression plasmid constructions wherethe very synthesis ofthe protein to be produced may contribute to the structural instability of the plasmid.On the other hand, it should be pointedout that secretory expression as such is oneway to avoid harmful effects that may be caused by the intracellular accumulation of the product. The inducibility of secretory production as such may result only in a modest improvement in the process. A protein effectively secreted is not expectedto be harmful to the cell. Furthermore, the typical mode of expression in an inducible system is ashort period of production at a high cell densityand at a very high level of expression. This is not feasible with secretory production, becausethe maximal capacity of cellular secretion isthe rate-limiting factor in all present applications. The feasibilityand usefulness of inducible expression have been amply demonstrated by the multitude of such production systems developed and routinely available for E. coli. Development along similar lines has now resulted also inthe availability of bacillar expression systems with a more favorable mode of expression than those basedon a-amylases or exoprotease genes. There are already several vector systemsfor inducible synthesis basedon various homologousand heterologous genes and genetic elements. As described below,the majority of these have been developed with the initial or explicit goal to construct a secretion vector system with improved properties; they have been mainly studied and characterized with such applications in mind. On the other hand, many of them canor

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have been easily modified for the expression of heterologous genes for other purposes.

3.1 Expressionwith Bacillar GeneticElements The shift to the exponential phase of growthand insensitivity to glucose was achieved bya constructionof Wong et al. (1 18). A strong vegetativephase promoter froman unidentified gene ofB. subtilis was cloned, characterized, and fused to the ribosomal binding siteand signal sequenceof the subtilisin gene B. ofsubtilis in aPUB1 10-derived plasmid. When tested with a reporter gene (the TEM 0-lactamaseof E. coli), a good level of expression and secretion was achieved. The combination of a very rich of medium containing3% of glucose further increased the amount secreted 0-lactamase, rendering it stable without any degradation, even during extended timeof incubation (upto 100 h), with a high density of cells in the culture and without sporulation. A very different principle has been applied in the laboratory D. Behnke A production to achieve expression in the exponential phase (1 14,129). system based onthe heterologous gene(sak)of bacteriophage 42D of S. aureus encoding the secreted staphylokinasewas developed. This gene is active inB. subti&, where it is expressed constitutively throughout growth (1 14,129).The protein is secreted efficiently inB. subtilis. A “portable” expression/secretion signalwas constructed by fusing a fragmentof about 400 bp containing the promoter and the signal sequenceof the staphylokinase geneand a polylinkerto the end of the fragment in a set of hybrid plasmids derived originally from Streptococci. The applicability of the production system was demonstrated with the gene of human interferon a1 (IFN-el). In a proteasedeficient B. subtilis strain, there was secretion of IFN-al, beginning in the expotential phase of growthand extending constitutively to the stationary phase; activity of the promoter was also evident inthe late stationary phase. The sak expression system thus shows flexibility for different modesof production. However,the peak level for I F N was only about 15 mg/L of culture with indications of proteolytic degradation. Thereare no data for any well-expressedor secreted protein produced with the aid of this vector. One way to achieve an inducible secretion and expression system has been to start with a gene that is readily inducible. Among few secreted proteins of Bacilli with inducible expression,the levan sucraseof B. subtilis is the one best characterized. The synthesis and secretion of levan sucrase are induced by exogenous sucrose, also in the exponential phase

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of growth (162,163). The activityof the correspondingsacB gene is regulated by several well-characterized regulatory mechanisms and genetic elements, which can be utilized inthe construction of a secretion system not only to achieve inducibilitybut also to further enhance and control the production. A region calledsacR between the promoter of the gene and sacB (the structural gene of levan sucrase) contains a transcription terminator (a stem-loop structure), which ismodulated by sucrose via the function of the product of sacY (in sacs locus), involving its presumed phosphorylation and binding to sacR, thus providing antitermination and induction of sacB (164,165). Further control is provided bythe set of the pleiotropic, mainly positive regulatory genes, which affect the activity of a number of genes encoding secreted degradative enzymes. These include degQ, degU, and prtT. The well-characterized targets of these regulatory systems are upstream of the sacR region (89,162,166). Expression systems utilizing these features have been constructed in several laboratories. The Rapoport group (108,110,162) has used sacRB the region of B. subtilis to construct inducible secretion vectors based on the multicopy plasmid pC194. The expression signals were in a DNA fragment of about 550 bp, which contains the sacR region, the promoterof sa&, and the complete signal sequenceof the levan sucrase. DNA fragments encoding the protein to be secreted have been fused to the 3’ end of the fragment, providedby unique cloning sites either for the exact fusion of the DNA fragment of the “N-terminal” codonto the cleavage site of the signal sequence, or by inserting afew amino acid residues between the signal peptide and the N-terminus. The secretion of three heterologous proteins with the aid of this basic construction has been reported. The desired sucrose-dependent expression and secretion were initially demonstrated with the TEM 0-lactamase as a reporter; the sacs locus was the one in the chromosome. Although the amountof 0-lactamase secretedwas described only in arbitrary units, the presence of inducibility was shown bythe absence of P-lactamase activity in cultures without sucroseand a steady increaseof activity in the exponential phase of culture in the presence of sucrose (162). It should be noted that although thesacR region was present in multiple copies, there was only one chromosomal copy sacY/sacS; of this imbalance did notseem to interfere with induction. More quantitativedata were reported for the secretionof a bacillar exoenzyme, the cellulase ofB. thermocellum, presumed to be protease resistant (1 11). Again, therewas no detectable secretionof the proteins in un-

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induced cultures, and secretion was increased to the level of about 0.5 mg/L of cellulaseby induction in culturesof wild-typeB. subtilis in stationary phase (1 10) (but see also below). The same level of secretion was found when the signal sequence of levan sucrase was replaced by the native signal sequence ofthe cellulase. In the case of a third construction, the reporter was murine interferon-a7 (108). There was no secretion in B.subtilis, but transcriptionof IFN-a7 inducible with sucrose was demonstrated. Wong (109) has constructed another secretion vector based on sacB. He cloneda slightly modified segment about of 600 bp of B. subtilis containing the upstream sequences from the 3’ end of the signal sequence of sacB as a cassette ina Bluescribe plasmid inE. coli. The matureTEM plactamase ofE. coli was then fusedto the end of the signal sequence and the construction inserted in a derivative of the multicopy plasmid PUB1 10 to study the expression inB. subtilis. A rapid increase in P-lactamase secreation was seen following the addition of 2% sucrose to a culture of super-rich media with3% glucose. Again, there were negligible amounts of P-lactamase in the absence of sucrose. A significant advantage of expression systems basedon sacBR is the possibility of enhancing expressionby increasing the transcriptionof the fusion genes via the activity of degQ, degU, and degS genes (formerly sacQ, sacU, and sacs, respectively). Increased levels of thedegQ regulatory protein(e.g., from multiple genes in a plasmid or due to anup-promoter mutation) or degU (Hy) and degS (Hy) mutations (for a review, see Ref. 89) increase transcription of sacB. Indeed, significant enhancement of secretion has been achieved by taking advantage of this regulatory system. Usingthe sacB secretion system above, Joliff et al. (110) demonof heterologous strated abouta six- and eight-fold increase in the secretion cellulase indegS (Hy) and degU (Hy) mutants of B. subtilis, respectively. In both mutants, the expression was still inducible by sucrose. Up to 5 mg/L of cellulase (in contrast to 0.5 mg in the wild-typestrain) was secreted in the early stationary phase when the expression cassette was ina plasmid with a copy number ofabout 20. Furthermore, increasing the copy number of the expression cassetteto the extraordinarilyhigh number of 250 (see also Section 4), by integrating it tandemly in the chromosome ina degU (Hy) mutant (Ref. 111 and Section 4), resulted in a fourfold increase in secretion relative to the plasmid system. Wong (109) has studied the feasibility of enhancing expression based on sacRB by increasing the expression of the cloneddegQ gene ofB. subtilis in the production host.He cloned degQ (sacQin 109)in asacB-based

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secretion vector expressing the P-lactamase as a reporter. The copy number of degQ was thus matched withthat of the expression signals.A high level of degQ expression was provided by placing the initial promoterless degQ gene under the strong P43 promoter.*This promoter is highly active in both the exponential and stationary phases of growth. Introduction of the degQ gene enhancedthe secretion of P-lactamase 17-fold. The author also studied the effect of degU(Hy)mutation in the host strain no addiand founda %fold increase in P-lactamase secretion. There was tional effect of degQ(Hy) in the strain carrying the plasmid with cloned P43-degQ fusion. A potential drawbackof enhancingthe production by hyperactivating any degsystem is the simultaneous increase in the secretion of exoproteases, even ina protease-deficient host. Overexpression of degQ increases the activity of secreted minor proteases inapr and npr mutants (138). It has been shownto decrease significantly the half-life E.ofcoli P-lactamase expressed from a secretion vector, even ina mutant defective in several exoprotease genes (112). However, in some secretion vector systems, as in the oneused by Wong (109) and in the secretion vector system based on the neutral proteaseof B. amyloliquefaciens (IOI), overexpression of degQ was not foundto increase the exoproteaselevel or the rate of degradation of a secreted model protein. Zukowski et al. (167,168) have constructed a sacB-based inducible expression vector explicitly for intracellular protein production B. in subtilis. The vector,a derivative of pC194, hasan insert comprising either the native sacR regionor the fusionof a synthetic bacteriophage T5 promoter with truncated sacR region (containingthe stem-loop regionbut not the promoter). The T5 promoter, although of gram-negative origin by nature, is well recognized inB. subtilis and, when fusedto a promoterless reporter gene (chloramphenicol acetyl transferase of S. aureus), can mediate very efficient synthesis of the respective protein to(up 25% of total cellular proteins) (169). TheVIE gene-of Pseudomonaputida (encoding intracellular catechol 2,3-dixoygenase), with its ribosomal binding site, was by a fragment fused to the 3’ end of the truncated sacR region, followed containing the transcription termination siteaof phage ofE. coli. Expression of the reporter proteinwas moderately inducible by sucrose in both cases. Slightly higher noninducedand induced levels ofthe reporter were observed whenthe expression plasmid contained an insert with the sacY/ sacs region, resulting additionally in presumed overexpression of the positive regulator sacY (the antiterminator). However, the maximal levelof expression was only10 to 20% of the very high constitutive level obtained

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from the T5 promoter in a construction without the stem-loop region of the sacR region, in agreement with the similar difference observed between the maximal sucrose inducible level of expression of sacB+ and that of a stem-loop deletion(168).

3.2 Induced Expression with lac Elements of E. coli In E. coli, inducible expression based on the P-galactosidase (IacZ)gene, the lac repressor (lacl), and induction by IPTG (isopropyl-o-thiogalactoside) has been the basis of a number of practical and widely used expression systems. These elements have also been introduced to control gene expression in Bacillus in several systems. Indeed, the earliest inducible expression of heterologous proteins inB. subtilis was based on this principle in the laboratory of D. Henner, as described below. Yansura and Henner (170) constructed two hybrid expression signals based on bacillar promoters, designatedPpac-Z and Pspac-I, and fused to a lac operator sequence (a sequence of 35 bp containing the target of site lac repressor), followed downstream by a cloning site for the gene to be expressed. This cassettewas combined withthe lacZ gene brought under the control of an active Bacillus promoter, the one from the B. licheniformis 0-lactamase gene(blaP).The pacZ promoter isa derivative of the foregoing blaP promoter, comprising a fragment of about 100 bp with the lac operator sequence, situated between the - 10 region and ribosomalbinding site(RBS) of blaP. The spac-Z promoter is composed of a fragof the B. subtilis bacterioment of about 200 bp from the promoter region phage SPOl, the lac operator sequence,and a synthetic ribosome binding site fused downstream ofthe - 10 region, with cloning sites between the RBS and the initiation codon at the 3' end of the fragment. The expression vectors described in Ref. 171 containedtheinsame vector thePpacZ or Spac-Z cassette and thelacZ cassette; the vector was an E. coli-B. subtil& shuttle vector. The functionality of the mac-Z was demonstrated with the P-lactamase ofB. licheniformis and that of SpacZ with human interferon-a. In bothcases there was a 50- to 100-fold difference in enzymatic or biological activity inthe presence and absence of IPTG in the growth medium, respectively. et al. (172) to inThe lac operator and lacZ were also used by Le Grice troduce inducibilityinto a promoter, which was modified from theE. coli bacteriophage T4 promoter, lac operator and a RBS derived from phage T5, in an E. coli-B. subtilis shuttle vector. The IacZgenewas introduced into theB. subtilis production strain ina second multicopy plasmid under

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the control of a promoter adapted for expression in B. subtilis. Expression in this system was also shownto be dependent on IPTG induction (172); synthesis ofthe protein of interest couldbe induced inthe late exponential phase for efficient production (173). Interestingly, the hybrid tac promoter of E. coli, derived from the lac and try promoters, is also weakly active inB. subtilis (169) and repressed by lacI(174). The promoter was inducible byIPTG; due to the low level of expression,the repressibility was high, suitable for cloning genes with detrimental expression (174). 3.3 Temperature-DependentControl Rapid induction ofthe synthesis ofthe protein to be producedby a temperature shift might be advantageous in many systems, compared with induction by chemical agents, although a definedlevel of production is more difficultto achieve and the high temperature may be harmful to some proteins of interest. Two systems, both analogousto the widely applied thermoinducible systems of E. coli, based on temperature-sensitive repression of lambda phage and the appropriately controlled promoter, have been described for B. subtilis. Two laboratories have constructed dual vector systems with the expression signals of an early promoter of the B. subtilis phage $105. In both systems, one plasmid containsthe native promoter and operator of this phage gene fusedto the heterologous geneto be expressed. Inthe second plasmid, pE194,there was an insert containing the represser gene of this early promoter. pE194 is nonreplicating at the restrictive temperature of 45°C (175,176).The repressor gene described in Ref. 175 is wild type, and temperature induction of the promoter is achieved by nonreplication the of plasmid after the shift to 45"C, with subsequent slow diluting out of the repressor in the course of further growth. In the second system the repression gene encodes a temperature-sensitive mutant repressor (176); more rapid inductionof the synthesis by the promoter at the temperature shift is anticipated. Upto a 20-fold induction ofthe synthesisof a reporter prois relatein was obtained in the latter system. However, the 6105 promoter tively weak and thesystem as such is not suitablefor high-level production. Breitling et al. introduced the elements of the temperature-sensitive induction of theE. coli X phage system inB. subtilis (177). They constructed an expression plasmid containing two inserts: one, the expression cassette, contains theP, promoter of the X phage with its upstream region, the second insert contains the temperature-sensitive X C1857 repressor, brought under thecontrol of the sak42D expression cassette of S. aureus

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origin (see above,and Ref. 129). The plasmid is a multicopy plasmid of streptococcal origin. The system has been tested using the secreted a-amylase of B. amyloliquefaciens as a reporter, the gene being linked to the end of the expression cassette with its signal sequence. Impressive activation of a-amylase secretion greater than 1400-fold was achieved with a temperature shift from 30°C to 42"C, althoughthe maximal level of secretion was only modest.

4 CHROMOSOMAL EXPRESSION SYSTEMS Expression systems based on the use of plasmids are not an optimal choice for large-scale applications because they are inherently unstable. Plasmids can be lost by segregational instability without selection pressure. Moreover, there can be structural alteration during extended growth, with concomitant lossof the activity of the gene to be expressed. Such changes may be favored if the protein to be produced affects the growthof the cell. Plasmid instability isan annoying problem, especially with the Bacillus cloning and expression vectors. Those most widely used are derived from S. aureus, and due to their mode of replication, are especially liable to a high level of plasmid instability (3). The maintenance of plasmids with the prevailing method,that is, by using antibiotics for selection, is undesirable in industrial applications because of the high cost involved and for the possible environmental and medical risks that can result from increasing the prevalenceof antibiotic resistance in microorganisms. The maintenance of the plasmid inthe expression system with selection not based on antibiotics and feasible in industrial growth media has also been described (178). Integration of the heterologous expression cassette into the chromosome of the production host provides a way to stabilize its inheritance. The rate of recombination of chromosomal DNA is significantly lower than that in plasmids, greatly improving the stability of the inserted gene. There are methods availablefor the tandem duplication of the integrated gene to achieve high levels of production.A number of integration vectors have been developed and described for the purpose. Theseare (usually) plasmids of E. coli, amended with a (antibiotic) resistance gene active in B. subtilis and with a segment of DNA homologous to a region of the chromosome but devoid ofa replicator activein B. subtilis. An exemplary and widely usedconstruction inthe plasmid pHJ101, a derivative of pBR322 with a chloramphenicol resistance gene (from the S. aureus plasmid pC194) conferring resistance inB. subtilis and E. coli (179). If provided withan

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insert containing sequence homology with chromosomal DNA,it can confer chloramphenicol resistance when transferred to B. subtilis by integration of the entire plasmid in the chromosome at the site of homology. The integration takes place by Campbell-type recombinationat high frequency by one crossing-over event. The same principle has been applied using a number of derivatives of E. coli plasmids containingdifferent antibiotic markers selectable B. in subtilis (86,111,145,180,181). Homology with the chromosome can be provided by a chromosal fragment in the plasmid as above,or by preinsertion of heterologous DNA present in the integration plasmid into the chromosome of the B. subtilis host strains (181-183).

A crucial consequence of Campbell-type integration isthe generation of of directly repeatedDNA sequences flanking the inserted segment DNA. Such segmentsare inherently proneto multiplication or amplification in the course of replication of the chromosome. Amplified structures can readily and conveniently be selected, if, as usual, theantibioticresistance gene in the integrated plasmid shows a gene dosage effect and thus amplification of the structure confers resistance to higher concentrations of the respective drug. Amplification of inserted segment DNA of can readily be achieved by the (gradual) selection of increased resistance. Typically, upto 10 to 30 copies of the amplifiable segment have been obtained by straightforward selection for chloramphenicol, kanamycin, or erythromycin resistance conferred by the respective genes in the plasmid. Furthermore, a desired level of amplification canbe designed and maintained to a certain degreeby adjusting the concentrationof antibiotic in to adjust and reguthe growth medium(181,184). This makes it possible late thelevel of synthesis ofthe protein to be produced bythe choice of a production strain with the appropriate level of amplication of the expression cassette. The highest level of amplificationthat can be obtained dependsboth on the antibiotic used, and in a way not yet clarified, on the repeated sequence flankingthe structure (181,182). An antibiotic resistance geneexpressed through a relatively weak promoter will provide favorable conditions for a high level of amplification selectedfor resistance. Multiple copies of an integration plasmid have also been introduced into the-chromosome by multiple integration steps, resulting ainset of strains witha well-defined and well-controlled integrant copy number (185). Integrated and amplified heterologous structures in the chromosome of B. subtilis can be very stable, although not invariably. Virtually no loss

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of or decrease in the level of amplification has been observed during the 30 to 100 generations of growth without selection pressure in the case of various integrated structures, even with copy numberstoup30 (181,183, 186). This was still true when the integrated and amplified plasmid (copy number 5 to 9) encoded a protein through an effectively expressed promoter; there wasno detectable loss of the vector ina 40-h batch culture (185). On the other hand, some integrated structures, possibly depending on the flanking, repeated segment, are less stable and may eventually be lost without selection pressure (185). The stability may also be affected unfavorably by a high level of expression ofthe heterologous gene in the construct (182). A molecule of DNA containing heterologous sequences can also be integrated into the chromosomeas a consequence of two crossover events, each in one of two regions homologous with the chromosome and flanking the heterologous sequence. The integrated molecule replaces the chromosomal segment between the regions of homology,and a stable structure is formed if the integration does not replaceor inactivate an essential gene (187,188). As in Campbell-type recombination, the moleculeto be integrated is usually introduced into B. subtilis as a part of a nonreplicating plasmid containing a selectable marker in the segment inserted. For a more detailed description of the methods of plasmid transformation in Bacillus, see for example, Ref. 189. Amplification of the inserted fragment can also be achieved readily under selective conditions (e.g., for antibiotic resistance) if the insert is constructed to contain sequences of direct repeatsat both ends. Both the levels of amplification and the stability of the amplified structures are comparable to those obtained witha single crossover(181). On the other hand, becauseno repeated sequences are generated by integration with double crossover, a fragment devoid of repeats is not amplified,a property utilized for the stable single-copy integration of heterologous genes, as 189). well as a methodof mutagenesis andan aid in cloning (see, e.g., Ref. A third method of chromosomal integration utilizes the staphylococcal plasmid pE194, which has a temperature-sensitive replicator(3). At permissive temperatures, it replicates inB. subtilis as an autonomous plasmid, but the plasmid also integrates in the chromosome at multiple sites without any reciprocal sequence homology. At elevated temperatures (above 45"C), such integrants can be selected for erythromycin resistance conferred by the integrated plasmid, which isno longer replicating autonomously. Stable strains with multiple integrationa nontandem in fashion of can be the plasmid and any heterologous genes inserted into it constructed

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(3). pE194 derivativesare also a convenient alternativefor nonreplicating plasmids whenthe goal is integrationat a site of homology, by eitherthe Campbell-type or double-crossover mode. The plasmid is initially transferred to the host at the permissive temperature. Dueto its autonomous replication, transformantswith a high plasmid copy numberare obtained even if the effectiveness of the transfer is low, as in electroporation. A subsequent shift to a nonpermissive temperature under antibiotic pressure then integrates the plasmid into the chromosomeat a high frequency, with permanent loss of the autonomous plasmid. Several expression systemsfor heterologous proteins basedon expression cassettes integrated in the chromosome have been described. Saunders et al. (186) and Fahnestock et al. (180) deliberately constructed strains with a very low level ofamplification (from 1 to 2-4 copies) of staphylococcal genes encoding secreted P-lactamase and protein A, respectively. The constructions were stable and the proteins were synthesized and secreted at high levels in the case of proteinA. Both studies demonstrated that the possibility of constructing an expression system with a stable low copy number of the expression cassette may be very useful. It was not possible to express either gene from a multicopy plasmid, presumably due to the harmful consequences to the cell resulting from hyperexpression. Both human albumin and dihydrofolatereductase have been synthesized inB. subtilis with the aidof an expression cassette in the chromosome, human albumin from the expression and secretion signalsof aamylase and neutral protease genes of B. amyloliquefaciens (145), and dihydrofolate reductasethrough the cat promoter of the plasmid pc194 (183). Kallio et al. (86,185) compared the expression of the a-amylase gene of B. amyloliquefaciens in B. subtilis when integrated ina stable manner in multiple random sites from twoto eight copies, or carried by pUBllO the plasmid witha copy number ofabout 50 per chromosome. Interestingly, both the secretion of a-amylase and the level oftranscription in thestrain with two copies nearly equaled those of the strain carrying the plasmid construction. A further threefold increasewas obtained by increasingthe number of chromosomally integrated genesto eight.

5 PRODUCTION OF HETEROLOGOUSPROTEINS INTRACELLULARY IN B. SUBTlLlS Most efforts to produce heterologous proteins Bacilli in have focused on the extracellular mode of production, as described above. However, in

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many respects bacilli would compare favorably with alternative production hosts, even if synthesis of the protein were to take place intracellularly. Of particular importanceare the extensive experience with Bacillus in large-scale industrial applications,and its nonpathogenicity and nontoxicity, which greatly facilitates downstream processing. The main problems hampering the production of heterologous proteins in secreted form are inherent in that mode of production and may be expected to be circumvented if the proteins remain intracellular.As pointed out in Section 3, the expression systems developedfor the secretion of heterologous proteins could be applied, with simple modifications, to the intracellular mode of expression. Moreover, if high levels of intracellular production were to be achieved, resulting in inclusion bodies as with many proteins produced in E. coli, it would compensateto a degree for the lack of secretion by providing an easy first stepof purification. The feasibility ofthat approach has, indeed, been demonstrated in a number of studies. High-level productionof heterologous intracellular proteins B. in subtilis was first reported by Zukowski and Miller (167) for a reporter protein, the catechol 2,3-dioxygenase of Pseudomonas putida. Expression of the protein was from the xylE gene, with its own RBS, fused alternatively to one of three B. subtilis promoters. The gene constructions were cloned in a multicopy plasmid. A very high level of expression ofVIE was obtained with two ofthe promoters, the sacR promoter of the levansucrose gene in adeguh (designed sac@ in Ref. 167) mutant of B. subtilis and with a synthetic pro(see above) moter, with a consensus sequence of bacteriophage T5 promoter in a wild-type strain. In the deguh mutant, up to 25% of the soiuble cellular protein was estimated to be catechol 2,3-dioxydenase protein; the protein showedthe correct enzymatic activity. These data point to a considerable potential for B. subtilii as a host for the intracellular production of proteins. Intracellular production in B. subtilis was extended to a human protein of potential pharmacological interest. Wanget al. (173) have synthesized human tissue-type plasminogen activator (TPA) in B. subtilis, using a shuttle plasmid system, in which the expression ofthe heterologous protein in B. subtilis was based on an inducible hybrid promoter containing E. coli lacregulatory elementsand bacteriophage T5 promoter.TPA is a natural secretory protein, it is not compatible with the secretory machinery of B. subtilis. The stretch of a cDNA clone of TPA devoid of its signal sequence and encoding the 60-kDa mature TPA was inserted into the expression vector. IPTG-induced cultures of B. subtilis con-

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taining this expression vectorwere found to synthesize immunoreactive TPA of mature size. Estimates basedon immunoblotting indicatedthat the amount of TPA was in the range of 20 mg/L of culture, an amount clearly smallerthan thatof the catechol 2,3dideoxygenase described above, although encouraging with respectto practical applications. The reason for this comparatively small yield was not elucidated. All ofthe TPA was found in the pellet fraction of the broken cells, suggestingthat the protein was aggregated, possibly in the form of an inclusion body, despite the low level of expression (lessthan 0.5% of the mass of packed cells). However, the finding is consistent with the amounts of intracellular heterologous protein found in cases of some proteins forming inclusion bodies in E. coli (190,191). A higher levelof intracellular synthesis B. in subtilis of another human protein, interleukin-10, has been achieved by Bellirii et al. (105). Again, a semisynthetic stretch of DNA encoding the mature, secreted form of IL-10 (ca. 17 kDa) was fusedto an efficient promoter in a shuttle plasmid for expression. Analysis of the total cellular protein of the production strain indicated the presence of immunoreactive IL-10 in amountsabout of 5% of the total cellular protein. However, despite this relatively highintracellular concentration, all of the IL-1P was present in the soluble fractions of the cell. Furthermore, the biological activity of the proteinwas indistinguishable fromthat of authentic IL-10. It is thus likely that unlike TPA, the protein formed its native conformation in the intracellular environment of B. subtilis. The authors also described a purification scheme from high-density cultures of B. subtilis with yields of 35 mg of highly purified active IL-10 from 1 Lof culture. The possibility of replacing secretion with the intracellular expression of heterologous proteins has been studied extensively and applied to two types of potential vaccine proteins of gram-negative bacteria, the pertussis toxin fromBordetellapertussis,the agentof whooping cough, and a number of outer-membrane proteins, including one fromNeisseria rneningitidis, the causative agent of bacterial meningitis and other invasive infections. Pertussis toxin is a multimeric protein encodedby a five-cistron operon (192). Each subunit is predicted to be a secreted periplasmic protein by nature and synthesizedas a precursor with a cleavable signal peptide. However, only the subunit S1 was secreted effectively inB. subtilis (59) (Table l). Only insignificantamounts of subunits S2 to S4 were found extracellularly, with low intracellular accumulation of the precursors, indicating poor compatibility with the export mechanism ofB. subtilis (59). To test the intracellular expressionof these proteins, the secretion vector based

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on the a-amylase of B. amyloliquefaciens(64) was modified bytruncating most of the signal sequence ofthe expression cassette ofthe plasmid. However, the codons of the seven N-terminal amino acid residues were preserved; this stretchof nucleotides was found to be essential for expression of the protein (193). All pertussis toxin subunits (devoid of their native signal sequence) were expressed from this modified vector. Consistent with the efficiency of the a-amylase promoter, remarkably highlevels of all subunits exceptS1 were produced. The highest level achieved (for the subunit S4) was upto 500 mg/L and about 20% of the total cellular protein in shake flask cultures,and up to 3000 mg/L in batch cultures in a fermentor (194). There was no indication of degradation even during extended periodsof growth, althoughthe optimal yield was achieveda few hours after the exponential phaseof growth underlaboratory conditions. Consistent with the lack of the signal sequence inthe expression vector, all subunits were tracellular in B. subtilis, presumably inthe form of inclusion bodies. The expresion vector system described above has also been to study used the intracellular productionof a setof outer-membrane proteinsof gramnegative bacteria, includingboth model proteins,the major outer-membrane proteins OmpF and OmpA of E. coli, and a prospective vaccine antigen, the classI or P1 protein of N. meningitidis, a major protein of that organism. As described above, these proteins were synthesized at low levels and not secreted inattempts to produce them with a secretion vector system inB. subtilis, although theyare proteins normally exported across the cytoplasmic membrane in their native host. theOn other hand, . being integral membrane proteins embedded extensively in the lipid membrane in their native environment, theyare very different from the periplasmic pertussis toxin or the soluble, eukaryotic TPA. Expression of these three outer-membrane proteins from the a-amylase promoter in the vector described above produced consistent results, very similar to those obtained with the pertussis toxin subunits. Again, each 20% of the total protein was produced at a high level, in the range ofto10 cellular protein, and all were cell bound (194,195,197).It seems remarkable that this high concentration of intracellular heterologous protein was not significantly harmful to the cell. The growthrate of the strains producing outer-membrane proteins,as well as those producing the pertussis toxin subunits, was unaltered. However, moderately increased plasmid instability was indicated(M. Nurminen, personal communication).This suggests that growth had a slight effect on plasmid stability, in contrast to the high (63). stability of the variant of the plasmid expressing secreted a-amylase

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The pertussis toxin subunits and the three outer-membrane proteins were insoluble inB. subtilis, being found in the fast sedimenting fraction after breakage ofcells (193-195), consistent with the presence of inclusion bodies. Intracellular globules of electron-dense material were seen in cells expressing each of the outer-membrane proteins (194,195); these structures were very similarto the inclusion bodies observed inE. coli (196), and left no doubt of their presence in B. subtilis. The productionof a heterologous proteinas an inclusion body provides of the advantageof an easy initial isolation step, using simple procedures cellular breakage and low-speed centrifugation, as amply demonstrated with numerous proteins in E. coli (196). Using this procedure, the pertussis toxin subunits and the outer-membrane protein inclusion bodies could be isolated easily in gram amounts on laboratory a scale (193), with considerable enrichmentof the protein of interest (193-195,197). Isolation by centrifugation and enrichment was also achieved in the case of intracellular aggregatesof TPA, although the intracellular concentration was relatively low (173). The intracellular mode of production of heterologous proteins in B. subtilis can thus be highly efficient, makingB. subtilis competitive with E. coli as a hostfor the productionof a wide variety of proteins. The high initial yield,the ease of isolation ofthe inclusion bodies,and the proven industrial fermentation technologyof B. subtilis make this approach attractive, even for the production of low-cost bulk products. A potential drawback of producing proteins as intracellular inclusion bodies is, however, the formof the protein. Typically, although not invariably, the proteins are paracrystallic and in a nonnative conformation. Furthermore, they are usually solubilizable only with strong detergents orchaotrophic agents, a process leaving the protein fully denatured (190). This could discourage practical applications if the goal is a biologically active product of high purity, which can be achieved onlythrough a complex process of solubilization and renaturation of questionable efficiency. The inclusion bodies of the heterologous proteins in B. subtilis were highly insoluble and none of them were reported to be solubilized without complete de'naturation (173,193-195). However, conditionsfor effective renaturation have been developed. TPA in inclusion bodies, after solubilization and full defolding with guanidine HCL, was subsequently diluted to allow refolding. Purification with affinity chromatography then produced a protein with the native enzymatic activity of plasminogen activator (173). An effective way to renature the outer-membrane proteins produced as inclusion bodies

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has also been described; proteins solubilized with detergents or quanidine HC1 werenonnative, as judged by their immunogenic properties[PI protein of N. meningitidis (195)] or by their inabilityto bind phases [OmpA or OmpF proteins of E. coli (194)]. However, complexing these proteins with lipopolysaccharide(LPS), with the simple procedure of dialyzing the solubilizing agent away in the presence of restored the phage-binding LPS, activity of OmpA and OmpFand, very significantly, the ability of the P1 protein to elicit protective immunity an in animal model(195,197). In the case of P1 protein, LPS has subsequently been replaced with nontoxic phospholipids (Muttilainen, S., personal communication). The formation of inclusion bodies and the loss of biological activity are notalways an inevitable consequence of the heterologous intracellular expression of proteins B. in subtilis. The catechol 2,3dioxygenase of Pseudomonas, a native intracellular protein, was obviously not associated with aggregates andwas recovered as a soluble protein with full enzymatic activity in cell extracts, although produced at very high levels,as described above. Reverse transcriptase of a T-cell lymphotropic virus also retained its biological activity when produced intracellularly in B. subtilis (172) using a high-level expression system containing elements of phage T5and lac operator (169). Two human proteinsof pharmaceutical interest havealso been expressed intracellularly without loss of activity: human IFN-a2, from staphylokinase sak420 transcription and translational signals (11 9 , and human IL-lp from a strong constitutive promoter(105). The yield of human interferon, however, was low, with an indication of extensive proteolytic degradation. Indeed, the level of recovery was several orders of magnitude lower than the amount of this protein secreted inB. subtilis when (1 14). the same expression signals were used in a secretion vector system In contrast, more than 2 mg of IL-10 per gram of cells (wet weight) was found intracellularly in high-density cultures (105) and was purified at a high yield without loss of biological activity. This compares favorably with the extracellular production of the same protein, using a secretion B. subplasmid withthe expression and secretion signals of proteases of tilis; about 0.4 mg of interleukin per gramof cells was found in the culture supernatant (105). 6 PRODUCTION OF PROTEINS OF GRAM-POSITIVE BACTERIA

By and large, genes from a large variety of gram-positive species may be expressed inB. subtilis and in other Bacillus spp. from expression signals

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of their own (5,199). This applies especiallyto proteins of other bacilli, staphylococci, and streptococci, butalso to proteins of species less closely related, such as Listeria (see below). This enables the design and construction of a simple expression system, suitable for many purpose, by cloning an apspecies to a bacillar propriate large fragment of the DNA of the heterologous plasmid vector and looking for direct expression ofthe proteinof interest. Direct expression in E. subtifis can be used to produce a protein for biochemical, enzymological, structural studies often more readily than the native speciesof the protein. The native species may befastidious for growth. If the gene is clonedon a multicopy plasmid,the level of expression canoften, but not invariably,be high enoughfor ready purification, even on a large scale,due to the gene dosageeffect. Direct expression in E. subtifis of (exo)enzymes with properties of industrial interest may be the host straightforward initial step for the development of a large-scale production system. Other potential and proven applications of direct expression include dissection of the individual components of a complex system to study of their role and possibly their mode of function via their expression in E. subtilis. Interestingly, this approach has been applied successfully even as Listo the virulencefactors of pathogenic gram-positive bacteria such teria (200), suggesting a possible simplified method of analyzing other complex function of gram-positive bacteria. Expression inE. subtifiscould also provide a means to study species- or gene-specific regulatory mechanisms. All genes of bacillior other gram-positive bacteria are not expressed in E. coli, genes involved in sporulation and the development of competence among them (3). Utilization of direct expression E. in subtifis is (3). Examfacilitated bythe development of efficient direct cloning systems ples of the use of these approaches follow. Major exocellular enzymes of bacillar species used in industrial biotechnology have been expressed efficiently from expression signals of their own after shotgun cloning, in most cases on multicopy plasmids derived from pUBl10. These include a-amylaseof E. amyfofiquefaciensand E. licheniformis (61,201), and alkaline and neutral proteases of the foregoing species (87,88,202). In all these cases,the level of production was very high, comparable with or even manyfold higher (88) than that in'the donor organisms of the gene used previously in industry. subtifisof degradative bacilThere has been successful expressionE. in lar exoenzymes with propertiesof practical interest such as thermostability [a-amylases (201,203), proteases (204-207),and favorable pH range (a-amylase) (208)] or with other activities of interest [e.g., cellulases (209)

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and penicillin acylase(210)]. Many of these enzymesare secreted onlyat low levels inthe native host, and their expression inB. subtilis or other species ofBacilli for industrial production couldbe a good alternativeto traditional strain improvement with mutagenesis and other classical methods. Examples of good expressionin B. subtilis of the genes of another genus are those of streptoformase (DNAase) from Streptococcus (211) and of staphylokinase, nuclease, a-hemolysin, and P-lactamase from S. aureus (129,206,212,213). Although the method is widely applicable, some constraints forlthe direct expression of heterologous proteins from other gram-positive species can be noted. All of the cases reported to date are secretable proteins; they were also secreted in B. subtilis. This bias is clearly caused by the wide industrial interest in this class of proteins but leaves unclear whether the expression of other types of proteins (intracellular, membrane bound) might in some cases be harmful to Bacillus and thus limit the usefulness of this aproach. Some proteins were also expressed B. in subtilis at levels disproportionately low in comparison with the copy number of the cloning vector, althoughthe level might have been high or favorable in comparison withthat produced in the native host. Such proteins, which include the 0-lactamase and hemolysin ofB. cereus (214,215,216) streptodornase (211), the xylosidase ofB. pumilis (217), and an amylase of B. stearotheermophilus (203), suggest poor performance of some gram-positive expression signals inB. subtilis. High-level expression of some proteins may be harmful, excluding the use of high-copy-number vectors, but such proteins may be produced with low-copy-number vectors (213,218). There may also be a needto coexpress a gene- and a species-specific regulator gene (205).

7 FUTUREPROSPECTS Due to the successful development of secretion and expression vectors, the productionof heterologous proteins inBacillus for practical purposes is now feasible. Bacillus, epecially B. subtilis, has become a realisticand readily available alternative asa production host for a number of proteins; for some applications,Bacillus can be expected to be the optimal host. The use of bacillar secretion vectors isat its best in production of proteins of nonindustrial strainsand species ofBacillus and of other bacterial proteinsof a secretoryor periplasmic nature.A broad range ofthis type of protein is expected to be secreted effectivelyand thus produced efficiently. As several examples abovedemonstrate, the construction of a

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production systemfor such proteins may be simple and straightforward, thus providing all ofthe benefits of well-established bacillar production technology. This method can be expectedto replace, to a large extent, useof the species of the origin of the protein for production in fermentation processes, especially if the original species is fastidious inits growth condition and difficult to handle, as is nearly always the case for pathogenic microorganisms. The use of effective secretion and expression vector systems is also likely to be far superior in improving the efficiency of industrial production processes compared with the classical methods for improvement of the production organism.It should be notedthat secretion vectors and the technology used for improving their efficiency can also be used to enhance secretionof the homologous secretory proteinsproduced at present in bulk in industry. Although exact figures are not available in the published literature, the present production technology is believed to rely on genetically engineered expression systems in mosttheof processes. Efficient productionof eukaryotic proteins has remained the major challenge for development ofthe expression of heterologous proteinsBacilin lus. However, some eukaryotic proteins have been produced in significant amounts with the present secretion vector systems.It is interesting and encouraging for the future prospects ofthe technology, that these proteins are of pharmacological significance, and in one of these cases, theBacillus production systemwas found to be optimal among those explored. These successful applications, together withother experience, confirmthe possibility of obtaining high yields aofprotein poorly expressed initiallyby the meticulous fine tuningof the appropriate gene constructions,and by determining the optimal growth conditions. The principal obstacleto the secretory productionof eukarytic proteins is their apparent incompatibility with the bacillar secretory machinery. To improve their productionand to extend this technologyto a broader range of eukaryotic proteins it is necessary to introduce more compatibility for the secretion of various kindsof proteins inBacillus. This is a task made quite formidable by the lack of knowledge concerning the molecular biology of protein secretionBacillus in and its unique features, especiallyincomparison to eukaryoticmechanisms.However,many improvements are expected in this regard dueto concerted efforts of research into the mechanism of bacillar protein secretion in several laboratories, together with rapidly progressing studies on the genetics and physiology of this organism.Major components of the secretory machinery

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of B. subtilis have already been identified and characterized, with considerable understandingof the possible or probable bottlenecksof the secretion of heterologous proteins. The identification and cloning of the appropriate gene homolog inBacillus and in the organismof the source of the protein of interest have not only provided tools to identify the source of the incompatibilitybut also suggest suitable means for expression systems of improved compatibility by the addition or replacement ofappropriate components. This progress is also expected to benefit efforts to improve the efficiency of the secretion of those heterologous proteinsalready producible withthe present systems for secretory expression. The way to go is indicated, for example, by the enhanced secretion of exoproteins of B. subtilis achieved by increasing levels ofthe bacillar PrsA protein, therecentlyidentifiednovelcomponentoftheirsecretionmachinery (45), and the inceased level of export of periplasmic proteins in E. coli caused by the hyperexpression ofthe translocation complex proteins SecY and SecE (219). The availability of effective expression vectors has also created conditions for the use ofBacillus hosts for the production of heterologousproteins intracellularly, a possibility so far much neglected in favor ofother production organisms. Experience with a number of model proteins amply demonstrates that Bacillus can served as a host for a very high level of intracellular protein production. The repertoire of present expression vectors is wideand is expectedto provide a modeof production suitablefor any particular protein of interest, with the choice of different temporal Bacillus modes of expression and inducibility. Intracellular production in would share many ofthe features of similar heterologous production systems, but adding the obvious advantages of welldeveloped industrial fermentation technology. The low-level or nonexistence of toxicity and lack of biological activity of bacillar cellular components are further benefits, adding an element of safetyto the fermentation process, contributingto a less demanding purification process,and making proteins produced in Bacillus especially suitable in basic research for studies of their effects and interactions in eukaryotic cellular systems. The frequent formation of inclusion bodies, and thus the nonnative conformation of the protein producedat high levels, isan inherent problem in any intracellular production system. Several proteins have, however, been successfully renatured with methods feasible for practical production, although there are no generally applicable procedures available. The rapid advanceof knowledge ofthe propertiesand structure of manyproteins of interest is also expectedto further such pursuits.

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ACKNOWLEDGMENT I thank Ilkka Palva for critical reading of the manuscript.

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in heat-shock genesgroES and groEL on protein export in Escherichia coli. EMBO J 1989; 8~3517-3521. 19. Lecker S, Lill R, Ziegelhoffer T, Georgopoulos C , Bassford J, Philip J, Kumamoto CA,Wickner W. Three pure chaperone proteins ofEscherichia coliSecB, trigger factor and GroEL-form soluble complexes with prescursor proteins in vitro. EMBO J 1989; 8:2703-2709. 20. Wild J, Altman E, Yura T, Gross CA. DnaK and DnaJ heat shock proteins participate inprotein export inEscherichia coli. Genes Dev1 m 6 1165-1 172. 21. Luirink J, High S, Wood H, Giner A, Tollervey D, Dobberstein B. Signalsequence recognition by an Escherichia coliribonucleoprotein complex. Nature (London)1992; 359:741-743. 22. Miller JD, Bernstein HD, Walter P. Interaction of E. coli Ffh/4.5S ribonu-

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160. Takagi H. Yamagata H, Udaka S. Gene expressionin Bacillus brevisunder control of theLac repressor-operator systemfrom Escherichia coli. J Ferment Bioeng 1992; 74:l-6. 161. Tamura H, Tameishi K, Yamagata H, Udaka S, Kobayashi T, Tomita M, Ikezawa H. Mass production of sphingomyelinase of Bacillus cereus by a protein-hyperproducing strain, Bacillus brevis 47, and its purification. J Biochem 1992; 112:488-491. 162. Edelman A, Joliff G, Klier A, Rapoport G. A system for the inducible secretion of proteins from Bacillus subtilisduring logarithmic growth. FEMS Microbiol Lett 1988; 52:117-120. 163. Nagarajan V, Borchert TV. Levansucrase:A tool to study protein secretion in Bacillus subtilis. Res Microbiol 1991; 142:787-792. 164. Shimotsu H, Henner DJ. Modulation of Bacillm subtilislevansucrase gene expression by sucrose and regulation of the steady-state mRNA level by sacU and sacQ genes. J Bacteriol 1986; 168:380-388. 165. Crutz A M , Steinmetz M, AymerichS, Richter R, Le Coq D. Induction of levansucrase inBacillus subtilis: An antitermination mechanism negatively controlled by the phosphotransferasesystem. J Bacteriol 1990, 172:10431050. 166. Palva I. Production of medically important proteins. Ann Clin Res 1986; 18:337-343. 167. Zukowski MM, Miller L. Hyperproductionof an intracellular heterologous protein in a sacUh mutant of Bacillus subtilis. Gene 1986; 46947-255. 168. Zukowski M, Mille L, Gogswell P, Chen K. Inducible expression system based on sucrose metabolism genes of Bacillus subtilis. Genet Biotechnol Bacilli 1988; 217. 169. Peschke U, Beuck V, Bujard H, Gentz R, Le Grice S. Efficient utilization of Escherichia coli transcriptional signals in Bacillus subtilis. J Mol Biol 1985; 186547-555. 170. Yansura DG, Henner DJ. Development of an inducible promoter for controlled gene expressionin Bacillus subtilis. In: Ganesan AT, HochJA, eds. Genetics and Biotechnology of Bacilli. New York: Academic Press, 1984: 241. 171. Yansura D G , Henner DJ. Use of the Ekcherichia coli lac repressor andoperator tocontrol gene expression in Bacillus subtilis. Proc Natl Acad Sci USA 1984; 81:439-443. 172. Le Grice SFJ, Beuck V, Mous J. Expression of biologically activehuman T-cell lymphotropic virus type I11 reverse transcriptase in Bacillus subtilis. Gene 1987; 55:95-103. 173. Wang LF, Hum WT, Kalyan N K , Lee SG, Hung PP, Doi RH. Synthesis and refolding of human tissue-type plasminogen activator in Bacillus subtilis. Gene 1989; 84:127-133. 174. Leonhardt H, Alonso JC. Construction of a shuttle vector for inducible expression in ficherichia coli and Bacillus subtilis. J Gen Microb 1988; 134:605-609.

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175. Dhaese P, Hussey C, Van Montagy M. Thermo-inducible gene expression in Bacillus subtilis using transcriptional regulatory elements from temperate phage 4105. Gene 1984; 32:181-194. 176. Osburne MS, Craig RJ, Rothstein DM. Thermoinducible transcription system for Bacillus subtilisthat utilizes control elements from temperate phage 4105. J Bacteriol 1985; 163:llOl-1108. 177. Breitling R, Sorokin AV, Behnke D. Temperature-inducible gene expression in Bacillussubtilis mediated bythe I857-encoded repressor of bacteriophage X. Gene 1990; 93:35-40. 178. Diderichsen B. A genetic system for stabilizationof cloned genes in Bacillus subtilis. In: Ganesan AT, Hoch JA, eds. Bacillus Molecular Genetics and Biotechnology Applications. New York: Academic Press, 198635-46. 179. Ferrari FA, Nguyen A, Lang D, Hoch JA. Construction andproperties of an integrable plasmidfor Bucillussubtilis. J Bacterioll983; 154:1513-1515. 180. Fahnestock SR, Saunders CW, Guyer MS, Lofdahl S, Cuss B, Uhlen M, Lindberg M. Expression of the staphylococcal protein A gene in Bacillus subtilis by integration of the intact gene into the B. subtilis chromosome. J Bacteriol 1986; 165:lOll-1014. 181. Janniere L,Niaudet B, Ehrlich P, Ehrlich SD. Stable gene amplification in the chromosome of Bacillus subtilis. Gene 1985; 40:47-55. 182. Declerck N, Joyet P, Le Coq D, Heslot H. Integration, amplification and expression of theBacillus licheniformisa-amylase gene inBacillus subtilis chromosome. J Biotechnol 1988; 8:23-38. 183. Prozorov A, PoluektovaE, Sachenko G, Nezametdinova V, Khasanov F. Various means of integration of the expressible human dihydrofolate reductase gene into the Bacillus subtilis genome. Gene 1987; 57:221-227. Bacillus subtilis: The establishment of 184. Young M. Gene amplification in multiple tandemly-repeated copies of a heterologous DNA segment in the bacterial chromosome. In: Ganesan AT, Hoch JA, eds. Genetics and Biotechnology of Bacilli. New York: Academic Press, 1984:89-102. 185. Kallio P, Palva A, Palva I. Enhancement of a-amylase productionby integration and amplifying the a-amylase gene of Bacillus amyloliquefaciens in the genome of Bacillus subtilis. Appl Microbiol Biotechnol 1987;27: 64-71. 186. Miller J R , Kovacevic S, Veal LE. Secretion and processingof staphylococcal nucleus by Bacillus subtilis. J Bacteriol 1987; 169:3508-3514. 187. Niaudet B, Janniere L, Ehrlich SD. Integrationof linear, heterologous DNA molecules into the Bacillus subtilis chromosome: Mechanismand use ininduction of predictable arrangements. J Bacteriol 1985; 163:lll-120. 188. Shimotsu H, Henner D. Construction of a singlecopy integration vector and its use in analysis of regulation of the trp operon of Bacillus subtilis. Gene 1986; 43:85-94. 189. Errington J. Gene cloning techniques. In: Harwood CR, Cutting SM, eds. Molecular Biolow Methods for Bacillus. London: Wilev. 1 9 9 0 :

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190. Marston FAO, Hartley DL. Solubilizationof protein aggregates. Methods Enzymol 1990, 182:264-276. 191. Hartley DL, Kane JF. Properties of inclusion bodies from recombinant Escherichia coli. Biochem SOCTrans 1988; 16:lOl-102. 192. Locht C, Keith JM. Petussis toxin gene: Nucleotide sequence and genetic organization. Science 1986; 232:1258-1264. 193. Himanen J-P, Taira S, Sarvas M, Saris P, Runeberg-NymanK.Expression of pertussis toxin subunitS4 as an intracytoplasmic protein inBacillus subtilis. Vaccine 1990; 8:600-604. 194. Sarvas M, KontinenVP, Himanen J-P, Saris P, Taira S, Runeberg-Nyman K. Secretion and production of foreign proteins in Bacillus. In: Heslot H, Davies J, Bobichon L, Durand G, Penasse L, eds. Proceedings of the International Symposium on Geneticsof Industrial Microorganisms 1990 (GIM90), Societe Francaise de Microbiologie, 1990. 195. Nurminen M, Butcher S, Idhpaan-Heikkila I, Wahlstrom E, Muttilainen S, Runeberg-Nyman K, Sarvas M, Makela HP. The class 1 outer membrane protein of Neisseria meningitidisproduced in Bacillus subtilis can give rise to protective immunity. Mol Microbiol 1992; 6:2499-2506. Williams DC, van Frank RM, Muth WL, Burnett JP. Cytoplasmic inclu1%. sion bodies in Escherichia coli producing biosynthetic human insulin protein. Science 1982; 21k687-689. 197. Puohiniemi R, Butscher S, Tarkka E, Sarvas M. High level production of Escherichia coli outer membraneproteins OmpA and OmpFintracellularly in Bacillus subtilis. FEMS Microbiol Lett 1991; 83:29-34. 198. Sarvas M, LehtovaaraP, Sibakov M, KZirihen L. Secretion ofEscherichia coli P-lactamase from Bacillus subtilis by the aid of a-amylase signal sequence. Proc Natl Acad Sci USA 1982; 795582-5586. 199. Hager PW, Rabinowitz JC. Translational specificity in Bacillus subtilis. In: Dubnau DA, ed. The Molecular Biology of theBacilli. New York: Academic Press, 1985:l-29. 200. Wuenscher MD, Klijler S, Bubert A, Gerike U, Goebel W. The gene iap of Listeria monocytogenesis essential for cell viability, and its gene product, p60, has bacteriolytic activity. J Bacteriol 1993; 175:3491-3501. 201. Ortlepp SA, Ollington JF, McConnell DJ. Molecular cloning in Bacillus subtilis of a Baciltus licheniformisgene encodinga thermostable a-amylase. Gene 1983; 23:267-276. 202. Vasantha N, Thompson LD, Rhodes C, Banner C, Nagle J, Filpula D. Genes for alkaline protease andneutral protease fromBacillus amyloliquefaciens contain a large open reading frame betweenthe regions coding for signal sequence and mature protein. J Bacteriol 1984; 159:811-819. 203. Diderichsen B, Poulsen GB, Jdrgensen PL. Cloning and expression of an amylase gene from Bacillus stearothermophilus. Res Microbiol 1991; 142: 793-796.

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204. 205. 206. 207.

208. 209. 210. 211.

212. 213. 214. 215. 216.

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van den Burg B, EnequistHG, van der HaarME, Eijsink VGH, Stulp BK, Venema G . A highly thermostable neutral protease from Bacillus subtilis and characterization of the gene product. JBacteriol 1991; 173:4107-4115. Kubo M, Imanaka T. Cloning and nucleotide sequence ofthe highly thermostable neutral protease gene from Bacillus stearothermophilus. J Gen Microbiol 1988; 134:1883-1892. Fairweather N, KennedyS, Foster TJ, Kehoe M, Dougan G. Expressionof a cloned Staphylococcus aureus a-hemolysin determinant in Bacillus subtilis and Staphylococcus aureus. Infect Immun 1983; 41:1112-1117. Fujii M, Takagi M, Imanaka T, Aiba S. Molecular cloning of a thermostable neutral protease gene from Bacillus stearothermophilus in a vector plasmid and its expressionin Bacillus steurothermophilusand Bacillus subtilis. Bacteriol 1983; 154:831-837. Diderichsen B, Christiansen L. Cloning of a maltogenic a-amylase from Bacillus stearothermophilus. FEMS Microbiol Lett 1988; 5653-60. Jdrgensen PL, Hansen CK. Multiple endo-P-l,4-glucanase-encodinggenes from Bacillus lautus PL236 and characterization of the celB gene. Gene 1990; 93:55-60. Kang JH, Hwang Y,Yo0 OJ. Expression of penicillin G acylase genefrom Bacillus megateriumATCC 14945 in Escherichia coli and Bacillus subtilis. J. Biotechnol 1991; 179-108. Wolinowska R, Ceglowski P, Kok J, Venema G. Isolation, sequence and expression in Escherichia coli,B. subtilis and Lactococcus lactisof the DNase (streptodornase)-encodinggene from Streptococcus equisimilisH46A. Gene 1991; 106~115-119. Kovacevic S, Veal LE, Hsiung HM, Miller JR. Secretion of staphylococcal nuclease by Bacillus subtilis. J Bacteriol 1985; 162521-528. Wang P-Z, NovickRP. Nucleotide sequence and expression of the B-lactamase gene from Staphylococcus aureus plasmid p1258 in Escherichia coli, Bacilltls subtilis and Staphylococcus aureus.J Bacterioll987; 169: 1763-1766. Wang W, MCzes PSF, Yang YQ, Blacher RW, Lampen JO. Cloning and sequencing of the 0-lactamase I gene of Bacillus cereusS/B and its expression in Bacillus subtilis. J Bacteriol 1985; 163:487-492. Sloma A, GrossM. Molecular cloning and nucleotide sequenceof the type I P-lactamase gene from Bacillus cereus.Nucleic Acids Res 1983; 11:4997. Kreft J, Berger H, HWlein M, Muller B, Weidinger G , Goesel W. Cloning and expression in Escherichia coli and Bacillus subtilis of the hemolysin (cereolysin) determinant from Bacillus cereus. J Bacteriol 1983; 159681689. Panbangred W, Fukusaki E, Epifanio EC,Shinmyo A, Okada H. Expression of a xylanase gene Bacilluspumilus of in Escherichia coli and Bacillus subtilis. Appl Microbiol Biotechnol 1985; 22:259-264. Shivakumar AG, Vanags RI, Wilcox DR, Katz L, Vary PS, Fox JL. Gene dosage effecton theexpression ofthe &endotoxin genesof Bacillus thurin-

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Sawas giensis subsp. kurstaki in Bacillus subtilis and Bacillus megaterium. Gene 1989; 79:21-31. Perez-Perez J, Marguez G, Barber0 J-L, Gutierrez J. Increasing the efficiency of protein exportin Escherichia coli. Bio/Technology 1994; 12: 178180. Lundstrom K. Expression of the vesicular stomatitis virus membrane glycoprotein gene in Bacillus subtilis. FEMS Microbiol Lett 1984; 23:65-70. Lundstrom K, Palva I, KaLiiiinen L, Garoff H, Sarvas M. Pettersson RF. Secretion ofSemliki Forest virus membrane glycoproteinEl from Bacillus subtilis. Virus Res 1985; 1985:69-83. Novikov AA, Borukhov SI, Strongin AYa. Bacillus amyloliquefaciens aamylase signal sequence fusedin frame with human proinsulinis properly processed by Bacillus subtilis cells. Biochem Biophys Res Commun 1990; 169:297-301. Vasantha N, Filovla D. Expression of bovine pancreatic ribonuclease A coded by a synthetic gene in Bacillus subtilis. Gene 1989; 7653-60.

3 Gene Expression in Recombinant Yeast Albert Hinnen,” Frank Buxton, Bhabotosh Chaudhuri, Jutta Heim, Thomas Hottiger, Bernd Meyhack, and Gabriele Pohlig CIBA-GEIGY AG, Basel, Switzerland

1 INTRODUCTION A large number of molecular and genetic toolsand techniques have been developed over the past decade which allow the production of a variety of heterologous proteins in the yeast Saccharomyces cerevisiae. Many techniques have been-developed to deliver foreign genetic information to the yeast genome usinga variety of vectors. It is possible to manipulate specific regulatoryand signaling sequencesto ensure propergene expression andto deliver the protein productto the desired location. Thereare essentially no limitations to manipulating this organism at the genetic level. In addition, numerous possibilities existto influence the metabolic flux with the aim of obtaining better production yields. Almost anynew insight into the genetics, biochemistry, and cell biology of this organism has an immediate impact on the work of the yeast genetic engineer. In particular, recent advances in our understanding of yeast cell biology have provided us with a wealth of information and options that can be integrated into the day-to-day approach of the “expression specialist.” ~

*Present uffiliution: Hans-Kndll-Institut fur Naturstoff-Forschung, Jena, Germany.

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Of course, all of this has only been possible as a result of a detailed understanding of yeast geneticsand biochemistry, which was pioneered by a few leadinglaboratories in the 1950s and 1960s, demonstrating the potential of S. cerevisiae as an ideal microorganism for biological studies. S. cerevisiae cells are nonpathogenic, grow rapidly, and have stable haploid and diploid states, which makes genetic analysis straightforward. This, combined with a versatile systemfor unrestricted manipulationof the genome, meansthat S. cerevisiae combines all the virtuesof biological model organism with the long-proven record of a commercial production system. Designing an experimental pathway toward the successful expression of a heterologous gene requires basic knowledge in a number of fields. Even this knowledge is sometimes not sufficient. Predictabilityof success is often frustratingly poor and pragmatic approaches are needed. This means exploring alternativesand combining rational strategies with “gut feeling.” We hope that this chapterwill assist inthe search for new solutions and that it will open upways to explore novel ideas in this still rapidly moving field. We have divided this contribution into two parts. In the first part, we review briefly the basic background in the area of heterologous protein production. Inthe second part we provide data from our own laboratory to illustrate specific points with pertinent examples. Previous reviews have dealt with this subject and related topics. One has been published by Tuite (l), and a valuable monograph dealing with various aspects of yeast genetic engineering has been edited by Barr et al.(2). More basicinformation in Enzymoon yeast geneticsand molecular biology is given Methods in logy, Vol. 194 (3), in the two volumes of The Molecular Biology of the Yeast Saccharomyces (4), and in The Yeasts, Vol. 3 (5). 2

GENERAL

BACKGROUND

2.1 Yeast Vectors Almost all yeast vectorsare artificial hybrids between sequences derived from Escherichia coliand S. cerevisiae. The E. coli part serves as a cloning and amplification deviceand consists of a replication originand a selection marker for this organism. Convenient cloning sites add to the versatility of these vectors (e.g., pUC series). The yeast part varies. A minimal requirement isthe presence of a marker,which allowsfor selection in yeast and a sequence homologousto the yeast nuclear genome. Dependingon

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the additional yeast elements present,the hybrid vectors exhibit specific characteristics with respectto mode of replication, copy number, and genetic stability inS. cerevisiae cells. The general strategyto follow isto find a vector that guarantees a maximal gene dosage without sacrificing genetic stability. Since heterologous gene expression usually represents an additional burden on the cell, the optimal solution is often a compromise.

2. l . 1 IntegrativeVectors Integrative vectorsdo not contain a functional replication origin for yeast (Fig. 1, pFBY4). To be maintained in a growing population they need to integrate into a replicatingstructure (i.e., a chromosome or a yeast plasmid). Integration occurs exclusively by homologous recombination (6). Two types of eventcan.be distinguished: circular donorDNA creates tandem duplications of the target sequence, and linearized DNA leads to an exact replacement of chromosomal or plasmid sequences, provided that both DNA ends are homologous to sequences at the integration site(7,8). As a consequence, integrationcan be directed to any DNA locus byintroducing a double strand break into the donor DNA, resulting in gene replacement (9). Transformation by integration guarantees the highestlevel of stability. Delivery of extra DNA to a yeast chromosome by gene replacement should make such a foreignDNA sequence indistinguishable from a host sequence provided that the host cell is not discriminated against by a significant growth disadvantage. Since circular DNA donorwill create tandem duplications,this will giye riseto looping-out reactions,, low-frequency eventsthat seem to correlate approximately with the length of the duplication (0.1 to 1070 loss inan overnight culture). The copy numbers resulting fromintegration events are 1 for gene replacements andfrom 1to a few for the integration of circular plasmids. Due to the low copy numberattained with integrativetransformation, this mode of gene transfer is not the preferred onefor the expression of heterologous genes. However,there are situations where gene doses have to be low because the foreign gene expression is deleteriousto the host cell, or because overexpression leads to other metabolic effects which reduce the overall efficiency of expression. In thesesituations a single copy integraton can bethe best compromise between gene dosage and genetic stability. An interesting approach involves the creation of multiple insertion even& is applicable only by the use of yeasttransposons (10). Again, this strategy if the foreign gene isnot deleterious to the host cell. Compared withthe 2-pm vector (see below), these multiple integrants should exhibit g higher degree of stability.

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c EcoRl

I 2000

c Sac1 c EcoRI

ECOW

P a t 1 c SphI

Figure 1 (a)

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Figure 1 Yeast vectors for gene cloning and heterologous expression. pFBY4 is an integration vector with the yeast URA3 gene as a selection marker. pDP34 is a 2-pm vector with 2-pm specific functions(REPI, REP2, REP3, FLP, IRl, I=), and URA3 and dLEU2as selection markers for yeast. pGH5 is a centromere vector with CENI4 and ARSl as centromere and replication originsand URA3 and ZLV2-SMR as selection markers for yeast. The vectors above contain pUC18- or pUC19derived E. coli plasmids with the amplicilline resistane(AMP) gene. ILV2SMR is a sulfometuronmethyl-resistant allele of the ILK2 gene.

2.1.2 2-pm Vectors These vectorsare the preferred vehicles for achieving a high level of foreign protein production (1 1). The 2pm vectors are true shuttle vectors that can replicate autonomouslyin E. coli and S. cerevisiae cells (for a detailed overview, see Ref. 12). In addition to the integrative vectors described previously, they containa replication origin derived from the endogenous yeast 2-pm plasmid (Fig. 1, pDP34). This plasmid, with contour a length

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of 2 pm, is a resident of most yeast cellsand its biology has been studied in great detail(13). It is stably maintained without givingan obvious advanDNA”). tage to the host,and it does not have a selectable marker (“selfish The main structural features of 2-pm circlesare the replication origin (OM), a cis-acting stability locus(STB or REP3), and four trans-acting loci (REPZ, REP2, FLP, R A F ) , which are important in plasmid replication/amplification and segregation at cell division (13). Two inverted repeat structures allow switching from one isoform to the othervia a FLP-mediated recombination event (A-form, B-form). Most 2-pm vectors are composed of an E. coli plasmid part, the 2.2-kb EcoRI fragment of the B-form, which REP3 locus, and one of the codes for the replication origin, the cis-acting two inverted repeats. These vectors need a wild-type 2-pm plasmid inthe trans. Withsame host cell(cir’ cells), which provides the other functions in out these functions plasmid stability is poor the andaverage copy number is low. Alternatively, all 2-pm functions (with the possible exception of RAF) can be provided on one plasmid which is then stably propagated without resident 2-pm plasmids (cir’ cells). Stability of the endogenous 2-pm plasmid is very high and segregational loss can not be detected under most growth conditions.All hybrid 2-pm vectors,on the other hand, have markedly reduced stability(1 to 10% plasmid loss after overnight growth). Even higher instabilitycan be observed, depending on the additional genetic load of the vector. The copy number of wild-type 2-pm plasmid isabout 60 per haploid cell, hybrid plasmids have copy numbers ranging from a few (lessthan 5 on average)to more than 50. Obviously, hybrid 2-pm vectors have a large range of copy numbers even under selective conditions. It seems that yeast cells exhibit a high degree of flexibility to adapt to the best tolerated copy number,which is the resultof a positive selection pressure for plasmid retention via the complementing geneand a negative selection pressure for plasmid loss exerted by expressing the foreign gene (“metabolic burden”)(14). For foreign geneproducts that donot affect cell growth, a number of genetic trickscan be appliedto boost copy number. The best known system makes use of a defective allele ofLEU2 which acts as a complementing marker in aLEU- host (11). This LEU2 gene (leu2-d allele) is devoid of its own promoter and is transcribed from a read-through transcript orifor growth, ginating in>pm sequences. To provide sufficient gene product the gene dosage (i.e.,the plasmid copy number) has to be increased(15). The net resultof this selection pressure is the preferential growth of cells with increased copy number. A similar system has been by devised Unternahrer et al. (16) on the basis of a temperature-sensitive CDC9 allele.

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Yeast cells witha deleted CDC9 gene are transformed with a 2-pm vector containing the temperature-sensitive cdc9-l allele (17). Under low-temperature conditions the copy numbernotisaffected since even a low gene dosage of cdc9-l is sufficientto support growth. When the temperature is shifted to above 30°C,only cells with higher copy numbers can survive. This selection pressure leads to a higher average copy number the in growing population because a higher copy number results in more active CDC9 gene product(DNAligase), which inturn can compensatefor the destabilized protein. Surprisingly enough, these high copy numbers are maintained over many generations even in the absence of selective pressure, probably becausethere are sufficient trans-actingfactors available to support a high level of replication and amplification. An additional advantage of this system isthat the plasmid cannot be lost from the cell because yeast cells cannot grow without DNA ligase. As indicated above, all these vectors contain E. coli plasmid sequences which are needed for cloning and amplification in E. coli but do not serve 3 we address this point and any function in the final yeast host. In Section describe an interesting strategy to remove these dispensible sequences via a homologous recombination event in vivo. The resulting vectors have better stability than that of conventional hybrid vectors.

2.1.3 OtherVectors There are a number ofadditional yeast vectors available, but their role in biotechnological applications is relatively minor. The most prominent ones include centromere vectors (Fig. 1, pGH5) and artificial chromosome vectors. In both cases a functional yeast centromere is included, which leads to a copy number of approximately1 (18,19).They are most useful for the isolation and functional analysisof yeast genes (centromere vectors) (20) and mammalian DNA sequences (artificial chromosome vectors) (21). 2.1.4 SelectionSystems Most vectors utilize genes involved thein biosynthesis of amino acids and nucleotides. Usually, the vectors carry the wild-type genesand the hosts have the corresponding auxothrophic mutation. Stable mutations (deletions, double and triple mutations) are available for all commonly used loci. The obvious disadvantage of these vectorsthe is need for the introduction of auxothrophic mutations into the strains of interest. A few dominant resistance genesare in use, but most scientists preferthe amino acid and nucleotide complementing genes since selection can be made abso-

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lutely tight, whereas spontaneous resistant clones usually arise with dominant resistance markers.The most commonly used markers are summarized in Table1. Vectors carryingURA3 or L YS2 are very useful because yeast mutations inthe URA3 or LYS2 gene can easily be obtained by selecting for resistance to 5-fluoroorotic acid(41) or a-aminoadipate (42), respectively.

2.2 Transcription Efficient and faithful transcription can only be obtained from yeast transcriptional control sequences. These include promoter sequencestranand scription termination signals, most conveniently arrangeda transcripin tion cassette. If additional control sequences are placed within the cassette (e.g., protein coding regions as signalsfor protein targeting), the more general term expression cassette is used. Genetranscription depends on the 5’ and 3’ sequences flanking the protein coding region. Since relatively little is known about factors that stabilize/destabilize the mRNA transcript (43) and since the foreign DNA coding sequence is in most cases fixed, our discussion will concentrate primarily on transcriptional elements that determine transcription initiation. For the sake of their utility for heterologous protein expression,one can distinguish between constitutive promoters and regulated promoters.

2.2.1 Promoter Elements and Transcription Terminator Elements Many ofthe elements of yeast promoters are typical for eukaryotic genes (Fig. 2; for a review, see Ref.44).Starting from the protein-initiating ATG (position + 1to + 3) and moving upstreaminto the promoter region, these elements include the mRNA start site (-30 to -60), a mRNA selection site (about 5 to 15 bp upstream fromthe start site), a TATA box(-60 to -160) and one or more upstream activation sequence (VAS) elements (about 60 to 100bp upstream fromthe TATA box). UAS elements are cis-acting elements that mediate transcription activation. Theyare analogous to the enhancer sequences found in higher eukaryotes. The spacing between these elements and their sequence content appear to be less stringent in yeast than in higher eukaryotic promoters. This allowsfor a larger flexibility in fusing the foreign DNA sequence to the yeast control sequences. For convenience most fusions are done between the corresponding mRNA start sites and the beginning ofthe protein coding regions. This is accomplished by providinga yeast promoterarm, which ends with a restriction site closeto the initiating ATG.

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Table 1 Genetic Markers Used for Yeast Transformation

Refs. Yeast genes complementing auxotrophic mutations LEU2

HIS3 TRPl u . 3

Vector (examples)

pYeleulO pJDB219 Yep13 pDP34' YEpL3 pYehis3 YEP6 pYc 1 YRp7 PLC544

-

Pa2 pDP34' YEP24 pFBY4' L YS2 ADE2 Yeast genes conferring resistance phenotype CAN1

CUP1 TUNR

IL V2-SRM CHY2 E. coli genes conferring resistance phenotype cat hYg kan %e Fig. 1.

pYRp3R pASZlO TLC-l =p36 pJR41 pGHS'

22, 6 11 34 23 24

25 26 27 28 29 30 31 23 26 This laboratory 32 33

pRCl

34 35 36 This laboratory 37

p m 11 pLG89 pRC2

38 39 40

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,+-- AAA

H

60

L. .I

60-160

AUG UCU ---+mRNA

b

Figure 2 Structural elementsof an efficient yeast promoter and the corresponding ATG context region.UA'S, upstream activating sequence;TATA, typical eukaryotic transcription-initiation controlregion;CnTn,pyrimidine-richtrack; ATG, initiation codon.

Transcription termination is recommended to ensure contained transcription. Although many reports exist that demonstrate efficient expression without added transcription termination signals, it is not advisable to rely on it. 2.2.2 ConstitutiveExpression

The most straightforward approach to express a heterologous gene in yeast is to take a strong promoter, which is efficiently expressed in the presence of glucose. These include promoters controlling the genes coding for the glycolytic enzymes 3-phosphoglycerate kinase (PGK) (49, gly(GAP/TDH3)(W,enolase (ENO) ceraldehyde-3-phosphate dehydrogenase (47), triose-phosphate isomerase( T ' Z ) (48), and the fermentative enzyme alcohol dehydrogenaseI (ADHZ)(49). These genesare often called constitutive; however, one has to keep in mindthat expression is repressed to various levels when cells are grownon nonfermentable carbon sources. For many applications constitutive expression is the best solution, since the fermentation process is easier to handle and expression titers are largely growth related. Combined with high-copy-number 2-pm vectors, expression levels of several percent total of soluble protein can be obtained. We will not cover these promoters in any detail since extensive coverage of the most prominent representatives (PGK, GAP/TDH3) is available (50,51).

2.2.3 RegulatedExpression Regulated expression is necessary for products that interfere with cell growth. It is usually not possibleto determine the point of interference,

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and various terms have been used to describe this phenomenon of growth inhibition (toxicity, metabolic burden, metabolicload). Expression from a regulated promoter allows uncoupling of growth from production and thus may delay synthesis untillater in the growth phase and/or reduces the time periodof production to a few hours. Since most of the available regulated promotersare less efficientthan the constitutive promoters described above, expression levelsare usually lower. Furthermore, the fermentation process may becomerather complicated and the system may be fully exploitable only with sophisticated feeding regimesand control devices. Shake flask experiments will often not reveal the full potentialof these expression systems. The most commonly used regulated promoters are galactokinase/galactose epimerase(GALI, GALZO) (52), acid phosphatase (PH05)(53,54), the regulated alcohol dehydrogenase gene (ADH2) (S), and the CUP1 system (56) (see also Section 4). In addition, hybrid promoters are available that combine promoter elements from strong conconstitutive promoters(downstream elements) with UAS elements from regulated promoters (upstream elements) (23,55,57). a. The GAL System. This systemis designed to metabolize galactose and has been studied extensively (58). Several structural genes (GAL2, GALI, G A L 3 coding for a permease and various conversion enzymes are needed to direct galactoseinto the glycolytic pathway. The GAL system is regulated twofold:it is repressed by glucose and induced by galactose. The best studied genes are GALI and GALZO, organized in a diverA number of regulatory loci control gene gently transcribed gene cluster. expression. The GALA protein binds at a UAS region between GAL1 and GALZO and, in the presenceof galactose and absence of glucose, induces transcription. In the absence of the inducer galactose, GAL80, a negative.factor, bindsto the activation domain ofGALA and thus abolishes transcription. The GAL system is tightly regulated and can be induced up to 1000-fold (48,59). Under high-copy-number conditions there seems to be a limitation inthe positive regulatoryfactor GALA, which can be GAL4 at the same time. Since overexpresovercome by overexpression of sion of GALA increases the background expression under repressed conditions, yeast strains have been constructed withan integrated GAL4 gene regulated by the GALZO promoter (60). b. The p H 0 System. Phosphatases in S. cerevisiae are organized ina gene family consisting of individual members which are regulated bythe is PH05, presence or absence of inorganic phosphate. The best studied gene coding for a secreted acid phosphatase(61,62). In termsof transcriptional

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control the system exhibits obvious similarities to the GAL system described above. Two positive factors, PH02 and PH04, are needed to activate transcription.PH04 is negatively affected byPH080, which may interact directly with PH04 to abolish transcription in the presence of high levels of inorganic phosphate. Additional regulatory genes include PH08.5 and PHO81. Regulation by phosphate is an interesting option since it allows transcriptional control irrespective of the carbon source (23,54,62). As with the GAL system,there is a need to control the depletion of the repressor (glucose or phosphate, respectively). This is best achieved under controlled fermentation conditions.An alternative is the use of a yeaststrain with a temperature-sensitive activator, pho4’”. At the restrictive temperature 37”C, the pho4’ gene product is inactive, and at lower temperature, 23”C, induction can take place. In the presence of a pho80 mutation, induction occurs irrespectiveof the presenceof inorganic phosphate in the medium by a temperature downshift(63). c. The CUPI System. S. cerevisiae contains a single metallothionein protein that is encoded bythe CUPl gene (64). This locus is responsible for conferring copper resistance on yeast cells by a combinatio of CUPI gene amplificationand CUP1 transcriptional induction following the addition of exogenous copper. A UAS promoter region has been defined where the positive factor ACEl (CUP2) binds.ACEl itself binds copper in a highly cooperative fashion. The CUPl system is the only natural yeast system in whichtranscription is activated by the addition of a small molecule, in this case Cu2+, tothe medium. It is somewhat surprising that this system has been used only rarely for the production of heterologous proteins (56). In Section 4we provide an example of hirudin expression under control of the CUPI promoter.

2.3 Translation of Heterologous mRNA Inspection of the 5’ untranslated leader region of yeast genes reveals that this sequence is nonrandom. There is a strong bias for A residuesand almost no G residues are found. It is probable that this fact is important for the expression of heterologous proteins since the 5‘ sequences seem to influence expression levels of foreign proteins dramatically without affecting the steady-state levels of mRNA (65). This is in agreement with (CYCI),where modificationof the 5’ data from homologous yeast genes leader sequences (addition of G residues) leads to inhibition of translation (66).

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In higher eukaryotic genes the ATG context region is very important in controlling the efficiency of translation initiation (67). This is also true 5’ AAAfor yeast, but the consensus sequence is substantially different: AUG-UCU 3’ (68). In particular, the -3position seems to be very important. As a whole, however, the need to conform to the consensus is less stringent in yeast compared with higher eukaryotic cells. S. cerevisiae genes showa codon bias(69) which is most prominent for highly expressed genes.It is speculatedthat this has consequencesfor the expression of foreign genes, since codon bias seems to parallel the abundance of tRNA isoacceptors. Data in the literature are very scarceand do not allow firm conclusions to be drawn. Only in a very few cases have careful comparative studies pointed to an effect of codon exchanges(70). There is, however, general agreement that rare codons (usuallyG/C rich) should be avoided(CGA, CGG, CGC, GGA, GGG, GCG, CCG, CCC, GUA, CUC), although yeast hasthe capability to translate all 61 amino acid-specific triplets (71). For a review on yeast protein synthesis, see Ref. 72. 2.4 Secretion of HeterologousProteins

One major attraction in choosinga fungus for heterologous protein production isthe potential of these microorganisms to secrete large amounts of protein productsinto the medium.Protein secretion uses the synthetic capacity of the cell and at the same time removes its product from the synthetic machinery. This is ofparticular importance for toxic products that interfere with structural or enzymatic components in undesirable ways, which isprobably the caseat high expression rates. Since commercial applications tend toward ever-increasing titer demands, these considerations almost always apply. As a eukaryotic organism,S. cerevisiae shares allthe major characteristics of the mammalian secretory pathway (73). It is therefore likely that proteins of mammalian originwill have a good chance of being properly secreted from yeast cells. There are, however, specific differences between lower and higher eukaryotes aswell as within these twobroad classes of organisms. The most important, from a practical point of view, is the rather different capacityfor posttranslational modifications (see below). 2.4.1 Secretion Signals Proteins destinedfor secretion have N-terminal extensions the in form of signal sequences (presequences), which range from approximately 15 to 30 amino acids, microbial signals usually being at the lower end of this

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size range. These signal sequences may be followed by prosequences which have large size variations. Additional sequences may be present as C-terminal extensions. Commonto all these sequences is their transient nature. While the role of praequences is that of a primary signal for passage through a membrane, prosequences may have a multitude of functions, ranging from auxiliary signal sequences to folding or antifolding moieties and enzymatic inhibitors. In eukaryoticcells membrane passage takes place at the level of the endoplasmic reticulum, where initial processing steps and protein folding occurs. The folded protein is then transported through a series of membrane compartments until it is finally released to the exterior of the cell. Due to the close similarity of the secretion pathways of eukaryotic cells it is not evident whether a secretion signal should stem from the expression host or from the foreign gene product. Literature data show that both strategies may be followed. If a foreign proteinis secreted inits homologous host it will probably encode a signal sequence. In the case aof simple presequence this N-terminal extensionwill probably fulfillthe same role in a nonhomologous environment. It is our experience that taking the signal sequenceof the foreign protein is a good starting point. Alternatively, signal sequences derived from yeast proteins may be used. Frequently used yeast presequences are the ones derived from the yeast invertase (SUC2, 19 amino acids), acid phosphatase(PH05,17amino acids), and the yeast a-factor pheromone (MFeZ, 20 amino acids). In yeast, the most commonly employed N-terminal extension with signaling function is the a-factor leader of the e-mating factor precursor. It comprises a 20 amino acid presequence followed by a 61-amino acid prosegment. Thea-factor leader serves, together with the spacer elements, to direct secretion and processing of four units of 13-amino acid mating pheromone peptide (Fig. 3). Despite the frequent of the usea-factor leader sequence, there is no reason to give this signal preference overshort presequences, except for the secretion of smaller peptides (see below). The requirementfor all of the above pre-and prosequences is twofold: they haveto act as secretion signals and they have to be removed efficiently and accurately after doing their job. Unfortunately, predictability for these requirementsis low, although some empirical rules seem to emerge. For size considerationsseems it logicalthat small peptides (e.g., hormones) need additional peptide spacers in order to emerge from the ribosomes before terminationof their synthesis. Since membrane passage occurs cotranslationally, additional amino acids are needed to ensure couplingof synthesis with translocation into the endoplasmic reticulum. Furthermore,

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pre- and prosequence cleavages are enzymatic processes with target sequence as well as steric requirements. Signal sequence removaland precursor processing involvea complicated cascade of enzymatic reactions, starting with the actionof the signal peptidase located in the endoplasmic reticulum followed by endoand exoproteolytic cleavagesat a late Golgi or secretoryvesicle compartment. Of critical importance is endopeptidase F (product of the KEX2 gene) which cuts at the C-terminal sideof a dibasic amino acid motif. Although these enzymatic steps are found in all eukaryotic organisms, subtle differences at the boundaries between homologous and heterologous sequences may explain incomplete or inaccurate processing or signal sequence removal. The first reported caseof foreign protein secretion from yeast cells was the production of human interferon (74). Since then many proteins and peptides have been secreted using a variety of signaland leader sequences(23,47,75-77), some of them already reaching commercial exploitation (78,79). The field of heterologous protein secretion from yeastiscells very complex and reflects the difficulties encounterednot only in synthesizing a protein but also intransporting it via a complex network of membrane structures involving several enzymatic processing steps. More basic knowledge in cell biology and membrane traffic is needed before more rational approaches can be designed. In Section 5 we discuss the production of the leech peptide hirudin by secretion from yeast cells. 2.4.2 PosttranslationalModifications Apart from signal sequence cleavage and other proteolytic maturation steps, additional modifications that are linked to secretion are important. The more prominent ones are the covalent additions ofN- and O-linked sugars (for a review on glycosylation in yeast, see Refs. 80 and 81). Very little is known about the addition of sugars to the hydroxyl groups of serine and threonine; moreinformation is availableabout the process of N-glycosylation. N-Glycosylation in S. cerevisiae follows the same rules as in higher eukaryotic cells(82). A core sugar moiety is transferredto the Namide of asparagine in the sequence context Asn-X-Ser/Thr, with X being As in higher eukaryotic cells this core sugar any amino acid except proline. undergoes additional modification steps in the endoplasmic reticulum and in the Golgi apparatus, the major difference in S. cerevisiae being the absence of the trimming enzymesand modification enzymes adding complex sugars at the Golgi level, which in higher eukaryotes results in the formation of shortened side branches ending with complex sugars (galactose, sialic acid, fucose).S. cerevisiae cells instead'maintain the core

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mannose scaffold, often extended with a large amountof additional mannose residues, which can lead to a substantial increase in the molecular weight ofa protein. Variousmutants are available that block specific glycosylation steps inS. cerevisiae. The most interestingare mnn9, which is responsible for outer-chain additions, and mnnl, which encodes a termnnl minal al,3-mannosyltransferase.Double mutants having defects in and mnn9 have therefore been suggested for use (83).However, sincespecific glycosylation patterns are important features of mammalian proteins and, in addition, wrong sugars may represent immunogenic determinants, S. cerevisiae cells are less suited for the productionof mammalian proteins with complex sugar structures. This is particularlyso with proteins of pharmaceutical interest.

2.4.3 Secretion and Protein Folding The folding of secretory proteins is posttranslational a event taking place in the endoplasmic reticulum, where the environment is particularly favorable for disulfide bridgeformation and the generationof higher-order structures. It is therefore understandable that the secretion mode may represent the only solution to generate a correctly folded foreign protein. This view has been substantiatedby several examples(23,83,84). A number of proteins havebeen identified as being involved in this process,and most of them have also been described for S. cerevisiae. They include protein disulfide isomerase(PDI) (85),the KAR2 gene product (86),prolyl isomerase (87),and immunophilins (88).Very little is known about their role in catalyzingthe folding stepsof foreign proteins. Since proper protein foldingseems to be a prerequisitefor efficient secretion(89), the process of attaining the correct secondary and tertiary structure is crucial. The questionof whether foreign proteins need their homologous counterpart to facilitate an efficient folding process remains to be answered. The role of chaperonins (90) as facilitators or inhibitors of structure formation will most certainly receive moreattention in the field of heterologous protein production. In this context it is worth considering that the prosequence of thea-factor precursor couldwell function as a “folding agent” a neutral extension ofa secretion signal. for foreign proteins and not as just Production of properly folded proteinsis one of the important areas of application ofS. cerevisiae as an expression host.It has obvious technical advantages over mammalian systems and it still outweighs other microbial eukaryotes, due to the extensive knowledge ofthe genetics and cell biology of the system. Since E. coli cells do not readily secreteproteins across their inner and outer membranes that organism does not pre-

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sent a strong alternative.All these reasons makeit attractive to study the S. cerevisiae intensively withthe aim to exploit protein folding process in this feature further. An experimental approach to study the folding path7. way of the human insulin-like growth factor1 in yeast is given in Section

2.5 Stability of Heterologous Proteins Stability of heterologous protein products has received relatively little attention in the past. The reason for this is probably an overestimationof factors that control the early steps of the expression pathway (replication, transcription, translation). This inturn may reflect the fact that parameters controlling these steps are easier to understand and to approach at the molecular and genetic levels. It is, however, quite clear that in many cases failure to obtain adequate levels of heterologous proteins is due to rapid protein turnover. InS. cerevisiae two different proteolytic degradation systems are known. One is the vacuolar degradation system,which contains a numberof unspecific proteases(91); the otheris a nonvacuolar system with a variety of specific peptidases, probably coupled to the ubiquitin system (92).

2.5.7 GeneralPrecautions Many production hosts carry amutation in the gene coding for proteinase yscA, often in the form of the pep43 mutant allele, which, however, exhibits a leaky phenotype.Protease yscA is located in the vacuole and is the major processing protease responsiblefor the maturation of other vacuolar proteasesand other hydrolases (92,93). A defect inyscA therefore affects other potentially harmful proteolytic enzymes,in particular the vacuolar proteinaseyscB. The role of vacuolar proteinases in general protein turnover is still poorly understood but probably involves an as yet unknown import process of proteins destined for degradation (94).

2.5.2 ProteolyticDegradationintheCytoplasm The main degradative system in the cytoplasm seemsto be theATP- and ubiquitin-dependent pathway generally found in all eukaryotes (95). It involves the conjugation of ubiquitin (50 to 76 amino acid peptides)to Part of the degradative machinery proteins that are subsequently degraded. is probably the proteasome particle, a multifunctional enzyme complex which contains among other activities protease yscE (96). The initial sigpathnal that channels a protein to the ubiquitin-dependent degradation way is not yet understood. Interestingly enough, it has been shownthat protein degradation is a function of the amino-terminal residue (N-end rule) (97) and follows the same ubiquitin-dependent pathway(98).

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2.5.3 Protein Degradation in the Secretory Pathway There isno firm evidencefor a general protein degradation system the in secretion pathway. However, thereare a number of well-defined proteolytic activities either residing the in secretory membrane systemor which are transported through this system. Inaddition, all known vacuolar hydrolases travel viathe endoplasmic reticulumand parts of the Golgi apparatus to their final destination. Inaddition to yscA and yscB, two carboxypeptidases, yscY and yscS, are fairly well characterized. Carboxypeptidase yscY is synthesizedas a precursor that is activated in the vacuole only by proteolytic maturation steps, including yscAand yscB (99,100). The enzyme exhibits a broad-specificity spectrum and has been studied extensively (91). The gene codingfor yscS has been cloned and sequenced (101). Surprisingly, yscS does not need activation by yscA or yscB, and thus seems to travel through the secretory pathway asan active enzyme. Other proteases inthe secretory pathway include the two processing pep(91). They are encoded by the genes KEXZ and KEx2, tidases ysca and yscF respectively. These membrane-associated proteases are involved in the ,maturationof the a-factor precursor andof the killer factor (Fig. 3). yscF

STE13

KEXI

Figure 3 The or-factor precursor consists of fouror-factor repeatunitsand hasan Nterminally extended pre- and prosequence. Processing occurs in the endoplasmic reticulum (signal peptidase cleavage) and at a late Golgi or secretory vesicle state. KEX2, endoproteolytic digestion after the dibasic cleavage motif Lys k g ; STE13, dipeptidyl aminopeptidase cleavageto digest N-terminal spacer amino acids; KEX1, carboxypeptidase digestion to remove the C-terminal amino acids Lys and k g .

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is a protease located in thelate Golgi apparatus or the secretory vesicles (102). It cleaves at the C-terminal side ofa pair of basic amino acid residues and as such is a necessarypart of the processing systemfor protein fusions, including the a-factor leader sequence. ysca is a carboxypeptidase witha high specificity for basic amino acids (103). Like yscF, it is membrane associated and localized to later stages of the secretory pathway. Its biological role is the removal of the dibasic aminoafter acids cleavage by yscF. Mutants of KEX1 and KEX2 are perfectly viablebut exhibit a sterile phenotype. Other proteinaseswhich are fully secreted and therefore potential candidatesfor proteolytic degradationof secreted proteins are theBAR1 gene product (104), a pepsin-like proteinasethat inactivates a-factor peptide and aminopeptidases yscll (91). While many of these proteinases serve specific functions and as such exhibit a narrow specificity range, they can become crucial in situations where large amounts of foreign proteinsare being produced (see Section 5). In addition, it is certainly possiblethat high-level production ofproteins may represent a stress signalfor the cell which leadsto the inducttion of a variety of proteolytic activities (92).

3 STABLE YEAST 2-pM VECTORS 3.1

Introduction

As mentioned in Section2.1.2, the native 2-pm plasmid, being stableand is ideal vectorfor present at approximately 60 copies per haploid cell, an the expression of high levels of foreign protein. Thusit is not only surprising but also disappointingthat most engineered 2-pm vectors display such poor stability (Ref. 105 and our unpublished observations). However, mostof these vectors were, with the benefit of hindsight, very badly constructed, having deletions interruptions or in oneor more of the functional regionsof the 2-pm sequence. In recent years, engineered full-length 2-pm vectors having all essential functions intact have been constructed. We and others (106,107) have found that bacterial sequences present on 2-pm-derived plasmidsare bad for plasmid stability, as can be seen by comparing plasmidspFBY 12Rand pFBY 12PR with PFBY8R (Table 2). Plasmid pFBY8R does not contain bacterial sequences when propagated in yeast, whereasthe other plasmids do. Whether the instability is caused by sequences acting as yeast promoters (108-110) or for some other sequence-specific effect isnot clear. It is not simply becausethe inclusion of bacterial sequences makes theplasmids'larger, since plasmids of the same size but lacking bacterialDNA appear to be more stable.

Two-bm-Derived Plasmid St FRT

Plasmid

1

2

pFBY12 pFBY12P pFBY 12R pFBY12PR

-

+

pFBY8R pFBY99 pF~Y96

+

-

+ + + +

-

SnaBI cCDC9 URA3 < CDC9 < URA3

-=

+

U.3> CDC9> U M 3 > CDC9>

+ + +

CDC9> U M 3 >

-

Insert at: BgIII

-

I

-

-

_.



-

ght/left 1.17 3.28 1.17 3.28 2.31 1 .02 1.8

Plasmid-free cells (070) 17 43 4.0 6.5 0.9 1.3 3.2

aThe plasmids listed were all derivatives of A (Fig. 4). Some have a small deletion in either FRTI or FRT2, indicated by a minus sign. Each plasmid also carries a number of genes inserted into either the SnaBI, BgIII, or BamHJ sites. The order of the genes at each site is shown clockwise starting at FRT2 and the direction of transcription of each gene is shown: > is clockwise and < is anticlockwiseas the plasmids are drawn in Fig. 4. GHIR symbolizes an expression cassette for hirudin driven by a truncated GAPDH promoter. Note that those plasmids that have three intact FRT sites will recombine as soon as they are transformed into yeast, thus excising and rapidly losing the bacterial sequences. The ratio of the distance between the Xbal sites in the intact FRTsites present in yeast is indicated. Note that the right segment is that which contains the origin of replication. The percentage of plasmid containing yeast cells after 24 h of growth in rich media was determined by plating aliquots on rich media followed by replica plating onto defined media.

5

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Our results and thoseof others ( 1 11,112) have also shown that the presence of promoter activity affects2-pm plasmid stability.Part of the difference in stability between pFBYSR and either pFBY99 or pFBY% can be accounted for by the strong truncated GAPDH promoter presenton the latter two plasmids (Table2). How transcription mediatesthis reduction of stability is not clear, butit appears to be inpart caused by protein binding at the promoter regions ( 1 12). Avoidance of strong plasmid-borne promoters is, however, not favored if these vectorsare to be used for protein expressionin an industrial context. Even with this increased understanding of the engineered 2-pm plasmids, it is still apparent that these vectors are not as stable as the native 2-pm plasmid and, perhaps more important, are often not stable enoughto be used in large-scale batch or continuous-culture fermentation. Given that the 2-pm plasmid itself is so stable, we felt that it should be possible to improve the stability of engineered 2-pm plasmids.

3.2 Designing Plasmids and Effects of Symmetry on Plasmid Stability Our plasmidscontain all of the 2-pm sequencesso they can be cultured in a cir" host. To put inserts into these plasmids that do not interrupt the known functional regions, others have successfully used the SnaBI site next to the ORI(106,113)or the HpaI site inthe STBlocus (107).We have used the SnaBI in preference to the HpaI site as the latter separates the distal and proximal halves ofthe STB locus.This is not per se deleterious, as STB proximal can stabilize the plasmid independentlyof STB distal unless transcription initiated within the insert runs through STB proximal (114). Other possible sitesfor insertion are between the FLP and REP2 genes or between the RAF and REP1 genes. However, we have not explored these regions betweenthe divergent transcripts, as theyare quite short (1 15) and must containthe binding regionsfor the regulatory complex formed betweenREP1 and REP2, with the possible involvement of RAF ( 1 16,117). Thus a priori one might expectthat inserts in these regions would be deleterious, and its has been shown that inserts into the XmaIII site between the FLP and REP2 genes lead to plasmid instability (107, 113). The other sitesthat we have used were engineeredinto the plasmid to give convenient cloning sites, BamHI and BgIII in Fig. 4, at the 3' ends of the messages of FLPI and REPI. Both of these sites are actually in the 599 bp inverted FRT repeats ( 1 H ) , but they werenot expected to interfere withthe FLP-mediated recombinations,as it is knownthat only

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A

Recombination between FRTl and FRTP in yeast

/

J

Rapidly lost from yeast

Figure 4 A is a physical map of a plasmid that forms the basis of most of the plasmids described in this section. It consists of all of the 2-pm DNA (-) integrated into pTZ18R (=) in such a way as to duplicate the F'T sites (FRT1 DNA. Reand FRT2) as direct repeats separating the bacterial- and yeast-derived

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about 20 bp in the center ofthis region are required for in vitro function (110). We have shownthat inserts in these sites do not affect FRT-mediated recombination or plasmid stability. This The plasmidsthat we have studied also lack all bacterial sequences. can be achieved by cutting and ligating the E. coli-propagated plasmids before transforming them into yeast to remove the bacterial sequences (120), or more elegantly, by flanking the bacterial sequences with FRT direct repeats so that on transformation into yeast, these recombine to yield two plasmids, one containing a single FRTcopy and all the bacterial sequences and the other a 2-pm derived plasmid that is free of bacterial sequences (Fig. 4) (106,121-124). We have used the latter technique,having duplicated a 166-bp FRT-containing site on both sides of the bacterial vector sequences (Fig. 4). The double-rolling-circle model of2-pm plasmid copy-number amplification first proposed by Futcher (125) suggeststhat the ORI should be as close to one of the FRTs as possible, and the other FRT sites should be as distant from thisas possible, to maximize the probabilityof the initiating recombination event occurring after the first FRTsite is replicated and before the other site is replicated. This organization of the FRTs and ORI are conserved in other 2-pm-related plasmids which are found in a variety of other yeasts(126-132) showing almostno sequence homology to one another. It is clear that in all the engineered 2-pm plasmids, this symmetrical positionof the FRT sites in the plasmid is disrupted since all the foreign DNA sequences are integrated into a unique site inthe plasmid. We have shownthat maintaining this symmetrical disposition of the FRT sites in engineered plasmids leads to improved stability, as shown in Table 2. (Compare pFBY12 and pFBYlZP, which differ by 20-bp de-

combination between these two sites, mediated by FLP, general recombination pathways, or in vitro, leads to the plasmids B and C. The latterwill not be maintained in yeast,as it does not contain a yeast origin of replication, consisting merely of the bacterial vector pTZ18Rand a 166-bp fragment containingan FRTsite from 2-pm yeast. However, B will be maintained in yeast in two forms, one as shown and one that is a simple inversion, by recombination between the indirectly repeated FRT sites. The long arrows indicateORFs in this plasmid. The little arrows indicate the orientationof the FRT repeats, and OR1 indicatesthe yeast origin of replication. This basic plasmid has been modified in numerous ways by insertions of different pieces of DNA into some or all of the sites markedor by deletions to make other, more useful plasmids, some of which discussed in this section.

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letions around the XbaI sites presentin the FRT loci and also pFBY 12R and pFBY 12PR.) The more symmetricalpartner of each pair the is more stable. Similarly, when propagated in yeast, pFBY99 and pFBY96 differ only by the position of a 136-bp FRT-containing fragment located either before or after the URA3 gene. In this pair the more symmetrical derivative is also the more stable.

3.3 Use of Stable Plasmids for Heterologous Protein Production We have used these plasmidsas vectors for the expression of both hirudin and insulin-like growth factor 1. In the former case the stable symmetrical plasmids from which bacterial sequences had been deleted resulted ina yield increase of 100to 140% compared with a host-vector system based on plasmid, pDP34. These results from shake flask cultures reflect the good baseline stability of pDP34 carryinga hirudin expression cassette. However, pDP34-derived plasmids carrying IGFl expression cassettes are dramatically unstable with greater than 30% loss inan overnight culture in rich media. Plasmids constructed so as not to contain bacterial sequences and which are also symmetrical show less than a 1.5% loss overnight and over a 200% increase in IGFl titer. 3.4 Conclusion

The 2-pm-derived plasmids are more stable if they: 1. Contain the complete 2-pm sequence and are culture in ciro cells. 2. Have allthe genes and other important regions, FRT, ORI, promoters, and so on, intact and use the SnaBI, BgIII, or BamHI sites at the 3' end of the shown in Fig.4. (One could also engineer a site REP2 gene without deleterious effects,but we have not tried this.) 3. Have no bacterial sequences. (This is achieved most easily by exploiting the FLP recombinase.) 4. Are symmetrical. (We stilldo not know how symmetricalthe plasmids haveto be, butless than 20% difference in the two armsis probably desirable.) 5. Have no inexplicable destabilizing sequences. (The difference in stability between pFBY12and pFBY12R, which differ only by the orientation of the inserted DNA, is large. This instability ofpFBY 12 can be abolished by the deletion of just 4 bp at the end of the insert next to the 5' end of the URA3 gene. There appears to be no consistent explanation for the data concerning this region.)

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It is still apparent that although we have plasmids which are stable enough 1 0 7 0loss inabout 10generafor practical industrial use, exhibiting less than tions on rich media, the rate of loss is still much larger than that seen for native 2-pm plasmid at approximatelyper cellpergeneration.This implies that there is still a lotto be learned aboutthe stability of the 2-pmbased plasmids.

4 CUP1-DIRECTEDHETEROLOGOUSEXPRESSION IN YEAST 4.1

Introduction

The usefulness of yeastas a host for the production of recombinant proteins depends largely on the availability of efficient and versatile promoter systems. In Table 3 we summarize the characteristics of some of the more widely used yeast expression systems (for more detailed information, see Ref. 55 and Section 2.2). According to Table 3, one of the best promoters for use inan expression vector is that of the yeast metallothionein gene, CUPZ. The promoter strength is very high (and can even be adjusted to the requirements of a particular process),the system is genetically well defined, and its application is technically feasible in processes involving large fermentersand complex media. Nevertheless,the CUPZ promoter has so far been used almost exclusively in academic research. In this sectionwe introduce the CUPZ-system and describe its successful application in the production of recombinant hirudin in yeast (see also Section 5). Several comprehensive reviewson metallothioneinsas well as a number of papers dealing withthe construction of vectors for the CUPZ-directed (56,64,133-135). expression of heterologous proteins have been published Therefore, only the essential features of the CUPZ-system are discussed below. Metallothioneins are small, cysteine-rich metal-binding polypeptides widely distributed among eukaryotes(64). S. cerevisiae contains a single metallothionein proteinthat is encoded bythe CUPZ gene (136-138). The CUPZ locus has been shown to confer copper resistanceto yeast cells(139141). Two natural variants of S. cerevisiae with respect to copper resistance have been described: Strains sensitive to 0.3 mMcopper contain a single copy ofthe CUPZ locus andare designated cupZs;strains resistant to 0.3 mM copper are designated CUPP and contain several tandemly repeated copies of the CUPZ locus (136). Copper resistance relies on a combination ofCUPZ amlification and CUPZ translational induction

ie 3 Characteristics of Yeast Expression Systems

Promoter strength ( S ) and tightness

C)

ADH 11

S : One of the strongest promoters currently available. Directs the synthesis of ca. 5% of total protein/mRNA in a homologous situation T: 30-fold induction upon shift from acetatelpyruvate to glucose. Basal level of expression too high to allow the production of toxic proteins. Shift from acetate/ pyruvate to glucose feed.

S: Directs the synthesis of ca. 1% of the soluble protein in a homologous situation. T: mRNA undetectable in the repressed state.

Both S and T are excellent.

Glucose depletion.

Involves release from glucose repression and induction by galactose.

I

Induction mech~ism

GALI, GAL7, CALI0

PGK

PHU5 S: Superior to GAP S: Lower than that of strong constitutive promoter (this promoters (e.g., GAP). study). Can be increased by T: mRNA barely dethe construction of tectable in the rehybrid promoters. pressed state. 30- to Promoter regulation 50-fold induction by is partially lost in hycopper addition. brid promoters. Promoter tightness T: mRNA undetectable regulatable by changunder repressed condiing the number of tions. Ca. 40-fold inCUP1 copies in cells. duction by shift from + P to - P medium. Phosphate depletion. Addition of a copper salt to the medium.

Feasibility/physiological consequences of promoter induction

Manipulations required for promoter activation are simple. Nowever, the cells have to be pregrown on, e.g., acetate or pyruvate. On these carbon sources, growth is relatively slow.

No exact control over time point of inducduction. Promoter induction will also induce proteases.

Promoter regulation

Complex, incompletely understood.

Adjustability of expression levels

No.

Complels (involving catabolite repression), but well defined. No.

Use of complex media

OK.

OK.

No exact control over the time point of induction if cells are grown on glucose. This problem may be overcome by introducing a mutation that results in release from catabolite repression. Galactose is an expensive inducer. See ADHII.

No exact control over anipulations for the time point of inpromoter induction duction. ~ ~ o s p h a ~ e are simple, can occur depletion i n c o ~ p a t ~ ~ at ~ eany time, and have with growth. Increased relatively minor efprotease activity under fects on the host. induced conditions.

Complex, but well defined.

Relatively simple (only two genes involved).

Possible, but difficult since the inducer is also a substrate for growth. OK.

No.

Possible.

Variable phosphate content of media components (e.g., peptone) may create problems.

OK.

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following the addition of exogenous copper (136-138). A &-acting Sequence (UASJ necessary to promote copper-inducible transcription of the CUPZ gene has been identifiedand was mapped to positions -105 to -148 upstream of the transcriptional start site of CUPZ (142-144). Footprint analysis and electrophoretic mobilityshift assays revealed the binding ofa cellular factor to UAS, (143,144). Further analyses showedthat the binding factor is the product of the ACEZ (= CUP2) locus, which is essential for the copper-induced transcription of the CUPZ gene (143145). The ACE1 protein is a transcriptional activator that binds copper (and to a lesser extent, silver) ions in a highly cooperative fashion (143, 146). Metal bindingalters its conformation, thereby activating its DNAbinding domain. The conformational change of ACElp eventually allows the CUPZ gene to be transcribed. If it is to be used as a vehicle for the expression of heterologousproteins, an important feature of the CUPZ-system is its autoregulation. A yeast strain in which the CUPZ locus was deletedfrom the chromosome showed asubstantial level of transcription from a plasmid-borne CUPZ: galKreporter gene even ifno copperwas added to the medium (147). Copper control of CUPZ transcription in a ACUPZ strain was restored by reintroducing a functional CUPZ gene under thecontrol of a constitutive promoter (148). A detailed study by Wright et al. (149) has shown that this autoregulatory behaviorof yeast metallothionein dependson its ability to bind metals. Thus CUPlp appears to repress its own synthesis by complexing free copper ions in the cell, which, in turn, interferes with ACElp activation. This is important for biotechnological applications in that changing the numberof chromosomal copiesof the CUPZ gene may result in altered levels of cloned gene expression (148).

4.2 Use of the CUPI-System in the Expression of Recombinant Hirudin Variant HV1 in Yeast We made use ofthe CUPZ promoter to direct the synthesis and secretion 5 and Ref. 14) in yeast. of recombinant hirudin (variant HV1, see Section The plasmids constructedfor this purpose are shown in Fig.5. They are based on plasmid pDP34, which contains the full 2-pm complement. The pH05 signal sequence fused in frame to expression cassette consists of the the hirudin coding region and a PHO5-derivedterminator (14), all placed under the control of either atruncated version of the strong, constitutive GAP promoter (plasmid pDP34/GAPFL-YHIR) or the CUPZ promoter (Plasmids pPFY56, pPFY58,and pPFY59R). Plasmids pPFY58and

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pPN59R contain, in addition to the hirudin cassette, the ACE1 gene, either under itsown promoter (pPFY58)or fused to the CUPl promoter an appropriate host strain, (pPN59R). The plasmids were transformed into and the transformants were cultivated essentially as described in Section 5. Except for precultures, complex medium was used in all experiments. In a first step, the appropriate time point for the induction of the CUPZ expression cassette was determined in shake flasks. A fixed concentration of copper sulfate (200 CClM) was added to the cultures either (a) immediately after inoculation, (b) during the early exponential phase,(c) late in the exponential phase, (d)at the onset of the diauxic lag-phase, or(e) during the early stationary phase. Irrespective of the type of plasmid used (pPFY56, pPFY58,or pPFY59R), the highest titers of hirudin (as determined by HPLC analysis) were measured in the cultures supplied with copper rightat the time of inoculation.In cells transformed with plasmid pDP34/GAPFL-YHIR, the addition of copper to the medium didnot significantly alter hirudin titers compared with controls without copper. Next, the optimal copper concentrationfor hirudin expressionwas determined. Two different host strains were used, which contained either 3 (strain TR1456) or 10 (strain TR1631) copies of the CUPZ locus. As shown in Fig. 6, approximately 1.5 mM copper (supplied at the time of inoculation) was optimal for hirudin production in strain TR1456. The concentration optimum was the same for all types of plasmid used (pPFY-56, pPFY58, or pPFY59R). Instrain TR1631, the optimal copper concentration was shifted to slightly higher values. However, the maximal hirudin yields were significantly lower when TR1631 was used as the host. This is probably due to the autoregulatory behaviorof the CUPI-system as described above (see also Ref. 148). Strain TR1631 is expected to express the CUPl protein at higher levels than the more sensitive strain, TR1456. This will reduce the concentration of copper ions availablefor ACElp activation, thereby causing the CUPZ-directed expression of hirudin to decrease. Hirudin titers obtained withCUPl the expression plasmids were compared to those measured when using plasmid pPD34/GAPFL/YHIR containing a hirudin cassette under the control of the constitutive GAPFL promoter (dashed line inFig. 6). It is evident that the use of the CUPZ promoter considerably improved hirudin yields. This wastrue not only in shake flasks but also in fed batch fermentationup to the 30-L scale. an optimized versionof the fullNote that the GAPFL variant is already length GAP promoter (14). Thus a regulatable promoter may be superior to a strong constitutiveone even if, as in the case of hirudin, the heterologous protein to be expressed is relative nontoxic to the host cell.

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TR1456IpPFY56

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Figure 6 Hirudin productionby strains TR1456 (three copies ofthe CUPZ locus) and TR1631 (10 copies of the CUP1 locus). Both strains were transformed with the hirudin expression plasmids shownin Fig. 5. The cultures were grown on complex mediumfor 72 h, and hirudinwas determined by HPLC in theculture supernatant. Copper sulfate at the indicated concentrationswas supplied at thetime of inoculation.

Unexpectedly, the presence ofthe ACEI gene on the expression plasmid reduced rather than increased hirudin titers. The lowest titers were obtained with plasmid pPFY58, which contains a copy of the ACE1 gene under its own promoter (Fig. 6). The poor performanceof pPFY58 was, at least inpart, due to the fact that it was lostat a much higher frequency than pPFY56 or pPFY59R, albeit only in the presenceof copper in the medium. In contrast, a plasmid related to pPFY58 but with the hirudin cassette underthe control of the GAPFL instead of the CUP1 promoter was stably maintainedeven inthe presence of copper. Thus the deleterious effect of ACE1 on plasmid stability appeared to be mediated bythe CUPIhirudin expression cassette. We tested whether pPFY58 promoted the synthesis of hirudinat such a high rate that the secretion capacity of cells became limiting. However, no clear indication of a secretion blockwas found. The definite reason for the instabilityof pPFY58therefore remains to be established.

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In contrast to pPFY58, plasmid pPFY59R was maintained almost as to pPFY56 in terms stably as pPFY56. Nevertheless, it was slightly inferior of hirudin production. One possible interpretation of this findingis that the presenceof ACE1 on a multicopy plasmid increasesthe intracellular concentration of CUPlp. This would reduce theamount of free copper ions, thereby causing down-modulation of CUPI-directed gene expression. This hypothesis is currently being testedby reducing the numberof CUPl copies in the host.

4.3 Conclusions The CUPZ-system is one of the most attractive yeast expression systems currently available. The CUPl promoter was used in a pseudoconstitutive way to direct the secretion of recombinant hirudin in yeastand was found to be superior to the strong constitutive GAPFL promoter in both shake flasks and fermentors.

5 YEAST PROTEASES INVOLVED IN DEGRADATION OF RECOMBINANT HIRUDIN 5.1

Introduction

Hirudin, originally isolated from the blood-sucking leechHirudo medicinalis, is the most potent inhibitor of the protease thrombin knownto date (150). Sincethrombin playsan important role not only inthe coagulation cascade but also in the activation of platelets, ultimately resulting in fibrin clotformation and platelet aggregation, inhibition of its activity is believed to provide an important tool for the treatment of various thrombotic disorders. Several isoforms of hirudin have been isolated, all of which contain 65 or 66 amino acid residues, six cysteine residues, and have molecular weights of approximately 7 kDa (15 l , 152). The five N-terminal amino acid residuesare hydrophobic, followedby the core region linked through three disulfide bridges. The C-terminal region is rich in acidic residues, which are important for the binding of hirudins to the recognition exositeon thrombin. Inthe natural molecules, the tyrosine at position 63 is postribosomally modifiedto a tyrosine-O-sulfate.

5.2

Secretion of Recombinant Hirudin into the Medium

To produce sufficient quantities of active inhibitor for clinical evaluation, we decided to express one ofthe variants, HVl, in S. cerevis'iae. A syn-

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thetic gene was constructed which contains the yeast acid phosphatase (PH05) signal sequence fused in frame to the coding region of mature W1 in preferred yeast codon usage. The gene was placed underthe control of a truncated 198-bp-long fragment of the yeast GAP promoter (14), and the construction was completed by addition aofPHOS-derived terminator fragment. The entire expression cassette was either inserted into the multicopy 2-pm-based vector pJDB207 (23) for the transformation of appropriate cir+ yeast strains or pDP34 (23), which contains the full Zpm complement for the transformation of ciP yeast strains. Precultures of the transformed yeast strains were grown under selective conditions in minimal medium; the main cultures were generally grown under nonselective conditions in YPD-based complex mediafor up to 96 h. Hirudin expression was followed with a colorimetric assay measuring thrombin inhibition. A typical fermentation.pattern of a strain transformed with a hirudin expression plasmid is shownFig. in 7. Under the conditions chosen, hirudin is produced ina strictly growth-dependent manner,as one could

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Figure 7 Fermentation of a hirudin-producing yeast strain in YPD-based complex medium. Hirudin was analyzed in a thrombin-inhibition assay.

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expect for the constitutive GAP promoter fragment. Also, the vast majority of the hirudin produced is secreted into the medium, and only a minority, most probably in a transient form, is found inside yeast cells. Using the PH05 signal sequence, large amounts of biologically active hirudin can be secreted into the yeast culture medium. Analyzing the culture broth by high-performance liquid chromatography (HPLC), one major and two minor peaks were observed at retention times where hirudin variants should elute (Fig. 8A). The three proteins were isolated by a combination of adsorption on a macroreticular resin, ion-exchange chromatography, and gel filtration. Fast-atom bombardment/mass spectometry (FAB-MS) gave molecular weights of 6963, 6832,and 6719 for peaks 1, 2, and 3, respectively. Comparison with the molecular weight of native W1 and further protein characterization studies showed that S. cerevisiae is not able to carry out the sulfation reaction at tyrosine 63 which leads to the production of desulfato-HV1 in yeast. Full-length desulfato-HV1 (hirudin 65) corresponds to the major peak, peak 1 on HPLC, whereas peak 2 lackthe two minor peaks represent C-terminally truncated forms, ing the C-terminal glutamine (hirudin64) and peak 3 lacking in addition the penultimate leucine (hirudin 63).

ProteolyticDegradation of Desulfato-Hirudin In Vivo C-terminal truncation of proteins expressed at high level ina recombinant host could bethe result of premature transcription terminationor the result of posttranslational proteolytic degradation by host proteases. Lack of one or two amino acidsat the C-terminal end of hirudin could point to the involvement of yeast proteases, resident in the secretory pathway. We therefore crossed a strain lacking proteinase yscB, carboxypeptidase yscY, and carboxypeptidase yscS (bys232-31-42; Table 4) with a strain lacking carboxypeptidase ysca (96; Table 4). Spores were selected, auxotrophic for leucine and each lacking all but one of the three carboxypeptidases. Also, one spore was isolated lacking all three carboxypeptidases. Four strains were transformed with the pJDB207-based hirudin expression plasmidand analyzed for hirudin production byHPLC. For this experiment a semisynthetic medium containing casamino acids was used (153) which had previously been shownto enhance formationof the byproducts. Figure 9A showsthat thestrain with mutations in yscY, yscS, and ysca is completely devoid of the truncated hirudin 64 and hirudin 63 by-products. Figure9B-D represent the pattern obtained with spores 5.3

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A

Figure 8 HPLC chromatogram of secreted hirudinspecies: (A) yeast strainH449 (KEXZ, PRCZ); p)yeast strain TR1456 (prcZ::ura3. kexZ::ura3). Samples were loaded on a C,, reversed-phase column and eluted with an acetonitrile/trifluoroacetic acid gradient.

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Table 4 Yeast Strainse

Name of %

bys 232-31-42

H449

TR1456 MATa, TR1303 MATa, TR14%

Ref.

MATa, adel, kexl, thrl MATa, prbl-l, prcl-l, cpsl-3,

lys2, leu2, his7 MATa,cpsl, prbl, leu2-3,112, ura3A.5, c i f prbl, cpsl, prcI::ura3AS, kexl::ura3A5, leu2-3,112, ura3A.5, c i f cpsl, prbl, kexl::ura3A.5, pral::ura3A.5, leu2-3,112, ura3A.5, c i f M A Ta, prbl, cpsl, kexl::ura3A5, prcl::ura3A5, pral::ura3A5, leu2-3,112, ura3A.5, c i f

166 166

Our collection

Our collection Our collection

Our collection

'"he gene disruptions in PRCl and KEXI were carried out as described in the text. The disruption in thePRAl gene was generated accordingto Pohlig et al. (167).

that all contain one of the three carboxypeptidases. Figure 9B indicates that yscS has no effect underthe experimental conditions chosen, whereas yscY dramatically degrades hirudin (Fig.9C). Interestingly, the presence of ysca, the gene productof KEXZ, also leads to the formation of hirudin 64 and 63 (Fig. 9D), although to a lesser degree. Based on this experiment, C-terminal truncation of hirudin expressed in S. cerevisiae can be assignedto the activityof two yeast proteases, yscY andpm.yscY resides inthe yeast vacuoleand is known to have arather broad substrate specificity (154). ysca! is an integral membrane protein in a late-Golgi compartment, being involved inthe maturation of the precursors of yeast pheromone a-factQrand the killer protein (103). In both natural protein substrates ysca sequentially removes the dibasic lysine-arginine amino acids exposedat the C-terminus after the initial ysc-F-mediated endoproteolytic cleavage step. The crossing experiment indicatesthat in the case of hirudin, ysca obviously also seems to accept leucineand glutamine as substrates. To gain further insight into the unexpected proteolysis of hirudin by the two proteases,we tried to localize the degradation process in the different carboxypeptidase mutant strains. For this experiment the supernatant medium of the untransformed strains fermentedas described

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flgure 9 HPLC chromatograms of secreted hirudin species in carboxypeptidase mutant strains: (A) (prcl, cpsl, kexl); (B) (prcl, kexl, CPSI); (C) (cpsl, kexl, PRCZ); (D) (cpsl, prcl, KEXI).Samples were analyzed by reversed-phase HPLC as described in Fig. 8.

above was incubated with pure hirudin in vitro and the occurrenceof degradation measured by HPLC. Surprisingly, most of the yscY-assigned hirudin-degrading activity wasfound outside the cell, in a non-cell-associated form. Although active yscY is supposedly found only in the vacuole of the cell, our findings support earlier observations by Stevens et al., pointing to a partial mislocalization of yscY to the cell surface, where the precursor can be processed into an active form (155). YSCa-mediated proteolysis was not found outside the cells, which is consistent with the enzyme being an intracellular, integral membrane protein.

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5.4 Hirudin Degradation in a Strain Disrupted in PRC7 and E X 7 To prove the direct involvement ofthe two carboxypeptidasesyscY and ysca! inthe C-terminal proteolytic degradation of hirudin, we sequentially disrupted their structural genes PRCZ and KEXZ, respectively, in the host strain H449 (Table4). A 2.6-kb CIaI/pvUII fragmentof the genomic PRCZ locus from yeast (156) was subcloned into the NarI/SmaI sites of pUC19. The resulting plasmid was cutat the unique StuI site and a blunt-ended 1.2-kb HindIII fragment containing theURA3 gene wasinserted. pUC19/ prcZ::URA3 was digested withAatII and the fragment usedto transform a ura3- yeast strain. Uracil-independent transformants were tested for correct PRCZ disruption by Southern blotting and again made auxotrophic for uracil by 5-fluoroorotic acid-induced selectionfor ura3- segregants. For disruption of KEXZ a 1.4-kb HindII/BamHI fragment containing the 5’ part of the KEXZ gene (157) was isolated from total genomic yeast DNA and subcloned into pUC19. The blunt-ended 1.2-kb HindIII fragment containingthe URA3 gene was inserted into the unique EcoRV site. pUC19/kexZ::URA3 was digested withHindIII/BamHI and the fragmentused to transform the Ura3-, PRCZdisrupted strain as described above. The 5-fluoroorotic acid-induced selection for Ura- segregants of correctly disrupted Kexl- strains was repeated and the resulting Ura3- strain transformed with a pDP34-based hirudin expression plasmid. The €PLC chromatogram of the strain disrupted in PRCZ both and KEXI is shown in Fig.8B underneath its isogenic parentalstrain shown in Fig. 8A. Incontrast to the parental strain, the disruptedstrain no longer produces the two C-terminal degradation by-products. Intermediate strains, lacking either one of the two carboxypeptidases, contain still measurable quantities of hirudin 64 and hirudin 63 (data not shown). The sequential disruption of the two protease encoding genes PRCZ and KEXZ therefore confirms the result obtained in the crossing experiment and provides proof of the direct involvement ofthe two carboxypeptidasesyscY and ysca! in C-terminal hirudin degradation.

5.5 Influence of Endoproteases on Hirudin Production 5.5.7 Proteinase yscA Expression of heterologous proteins in S. cerevisiae is commonly achieved by useof transformed proteinase yscA-deficient strains (e.g., pep4-3 mutants) (91). Strains lacking yscA accumulate inactive precursors of pro-

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teinase yscBand carboxypeptidaseyscY and show reduced activitiesin a number of other vacuolar hydrolases (99,100,158-162), which consequently results in a significant reduction of overall proteolysis. Since hirudin is C-terminally degradedby carboxypeptidaseyscY, we have comparedthe effects of a disruption in the yscA encoding genePRAZ [PRAZ and PEP4 refer to the same gene (161)] with a direct disruption of the yscY structural gene PRCZ. Figure 10 shows the secretion of hirudin 65, hirudin 64, and hirudin 63 by the isogenic strains TR1456, TR1303,and TR14% (see Table 4)dura complex medium basedon YPD up to 168 h.Strain ing fermentation in TR1456 (PRAZ;prd; kscl) as well as TR1303 (PraZ; PRCZ; kexl) produce exclusively hirudin 65. The lackof C-terminal hirudin degradation is due to the absence of active carboxypeptidase ysca as well as yscY, sinceboth the disruption of PRCZ and the disruption of PRAZ lead to lack of active yscY. However, the total amount of hirudin 65 produced bystrain TR1303 is about 30% lowerthan the amount produced by strain TR1456. This result suggests that a strain containing yscA produces more hirudin than does a yscAdeficient counterpart. This assumption is supported by the observation that additional disruptionof PRAZ in TR1456 leadsto a similar reduction in hirudin titers as observedfor TR1303 (Fig.1OC). The reduction in hirudin titer is not to due growth defects of TR1303 and TR14%, since growth of the yeast strains compared isvery similar in the stationary (Figs. 10 and 11) and logarithmic phases (data not shown). It has been reported that strains lacking yscAare not only severely impaired in their ability to undergo sporulation but alsoshow a dramatically increased loss of viability undernutritional stress conditions such as nitrogen starvation (163). This observation suggests that even though the eliminationof proteinase yscA activityand the consequent eliminationof other proteolytic activities is not lethal to the cell,yscA exerts major functions in the stress

64.

Figure 10 Production of hirudin compounds hirudin 65, hirudin and hirudin 63 by yeast strains:(A) TR1303 (prul::ura3,PRCI,kexI::uru3);(B) TR1456 (PRAI,prcl::ura3, kexl::ura3);(C)TR1496 @ral::urajl,prcl::ura3, kexI::ura3) during fermentation in complex medium. Strains were transformed with plasmid pDP34 containing the hirudin expression cassette. Samples were withdrawn from the cultures after various periods of time, the cells removed by centrifugation, and the supernatants subjected to HPLC. In parallel, the optical density of the cultures was measured at600 nm.

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response. Consequently,we conclude that yscA mightnot be dispensable for the optimal performanceof a yeast strain that is usedfor production of heterologous proteinsat high expression levels. Results confirming thisnotion were obtained by fermenting the three isogenic strains in a semisynthetic medium containing casamino acids, which appears to induce an increase in proteolytic processes (153). Again, the strains lacking yscA activity have a significantly lower titer of total hirudin than that of the strain exhibiting yscA (Fig. 11). However,contrast in to the results obtained in complex medium, we can observe C-terminal hirudin degradation inTR1303. This proteolysis seemsto be due to carboxypeptidase yscY, since it cannot be detected in TR1496, in which in addition to PM1 and KEXI, PRCl is disrupted. Thus surprisingly, uncan be detected in a strain lackder certain growth conditions, active yscY ing yscA. There are two possible explanations for this observation: (a) the precursor of yscY is partially activeor (b) the precursor is activated independently of yscA under certain conditions. Stevenset al. reported PEP4-independent processingof yscY in the periplasmic space of strains overproducingyscY (155)or mislocalizingyscY to the cell surface in vplmutants (164). At this stage we do not know whether our finding can be connected withthe processing protease postulated by Stevens et al. However, since allour strainsused carry a mutationin the PRBl gene (see Table 4), we can rule out proteinase yscB as the potential processing agent of yscY under our conditions. 5.5.2 Proteinase yscB Proteinase yscB has been reported to be a major source of protease problems during biochemical studies ofyeast (93,165). Therefore, we have expressed hirudin mostly in yscBdeficient strains(see Table 4). To examine whether yscB really hasan impact on hirudin degradationor, like yscA, can positively influencetotal hirudin production, we generated an isogenic derivative of TR1456, in which the mutated copy of PRBl, the yscB-encoding gene, was replaced byan intact copy ofthe gene. Preliminary results suggest that yscB does not cause degradationof hirudin. On the other hand,

Figure 11 Production of hirudin compounds hirudin 65, hirudin 6 4 , and hirudin 63 by yeast strains: (A) TR1303 (pral::ura3,PRCI,kexl::ura3);(B) TR1456 (PRAI,prcl::ura3, kexl::ura3); (C)TR14% @ral::ura3,prcl::ura3, kexl::ura3) during fermentation in minimal medium. Strains were transformed and fermentation samples withdrawn and treated as described in the legend to Fig. 10.

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Hlnnen et al.

the presence ofboth yscB and yscA does not amplify the positive effect of yscA alone. No further increase in hirudintiter can be observed. We are currently investigating whether the performance of a yeast production strain that lacks yscA can be improved by the presence of yscB.

6 OVERSECRETION IN YEAST 6.1 Introduction Secretion allows the convenient relocation of proteins outside the cell. The efficiency of the secretion processis an important factor for highlevel expression of secreted proteins. In comparison to filamentous fungi and higher eukaryotic cells,the total amount of protein secreted in yeast is rather low, which raises the question of whether secretion in yeastis rate limiting. Indeed, the issueof secretion capacityis controversial (168). It seems necessary to consider secretionof genuine yeast (homologous) proteins separately from secretion of foreign (heterologous) proteins. The homologous situation clearly involves fewer parameters, which could affect the secretion capacity the of yeast celland therefore seems to be easier to analyze. Secreted yeast proteins such as acid phosphatase or invertase are released into the periplasmic space and are mostly retained there for the benefit of the cell. Nevertheless, some periplasmic enzymes reach the extracellular medium, probably as a result of passive leakage during bud formation (169). There is evidencethat anincreased gene dosage for acid phosphatase and invertase from 2-pm-based multicopy vectors leads to higher enzyme activitiesbut does notsaturate the secretory pathway (170, 171). We describe here, however,a systemthat clearly defines a subpopulation of cells with intracellular accumulation of yeast acid phosphatase. This is associated with a nongrowth phenotype.

6.2 Expression of AcidPhosphataseinYeast Overexpression of acid phosphatasewas achieved by replacingthe inducible promoter of the acid phosphatase gene(PH05) (172) by a strong, (GAP49 = TDH3) (173,174) and cloning the constitutive yeast promoter new expression cassetteinto the multicopy yeast vector pDP34 (23). The new expression plasmid is referred to as pDP34/GAPFL-PHOS (Fig. a poorly expressed 12). Plasmid pDP34 has an URA3 selection marker and dL.EU2 gene (leu24 allele) without itsown promoter (15,175). Yeast strain YB18 (MATa, his3-11, his3-15, ura3A5, leu2::ura3ASEY[cir"])was trans-

Figure 12 Expression plasmid pDP34/GAPFL-PHO5. The pH05 expression cassette was inserted between BamHI and Sal1the inmulticloning site of the yeastE. coli shuttle vector pDP34(23) in an anticlockwise orientation. Arrows indicate length and direction of open reading frames. REPli REP5REP3 (= STB), IR1, IR2: 2p elements (12);AMP, p-lactamase gene;URA3, yeast URA3 gene; dLEU2, defective (promoterless) yeastLEU2 gene (14,15); PH05,acid phosphatase gene without promoter region; GAPFL, 201-bp fragment of the promoter region of the glyceraldehyde-3-phosphate dehydrogenase gene(GAP49 = TDH3; see Refs. 14 and 46). Restriction sites in italics were usedfor cloning, and sites in brackets were lost by cloning steps. 165

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formed with the expression plasmid pDP34/GAPFL-PHO5 or with the control plasmid pDP34. Selectionfor the defective leu2-d marker should result ina higher average plasmid copy number to complement adequately the gene dosage, the expresthe feu2 defect of YBl8. Concomitantly with sion of acid phosphatase should increase.

6.3 Cellular Response to Overexpression of Acid Phosphatase Transformants of YB18 (YBl8/pDP34/GAPFL-PHO5) were cultured under uracil selection. Midlog cells were collected and shifted to a medium with either uracil or leucine selection. Cells under uracil selection grew as well as control cells, and the total acid phosphatase activity (Fig. 13), as well as its cellular distribution (Fig. 14), were as expected. Most of the activity was secreted into the periplasmic spaceand to a small extent into the medium.A small fraction of the activity was detected intracellularly. In contrast, under leucine selection, cell growth stopped (despite slow a increase in optical density). The total acid phosphatase activity per cell was comparably high (Fig. 13)and the cellular distribution changed (Fig. 14). The nongrowth phenotype was not caused by the selection for leucine per se, sincecontrol cells grew wellunder leucine selection(Fig. 13). Further analysisby subfractionation showed that under leucine selection acid phosphatase accumulated to a considerable degree intracellularly (ca.60% of radiolabeled, immunoprecipitated acid phosphatase); a smaller part (ca. 35%) was secreted to the periplasmic space and less than 5% Flgure 13 Growth of YB18 transformants under uracil (-ura)or leucine (-leu) selection and total acidphosphataseactivity. YB18 cells transformed with pDP34 (left) or pDP34/GAPFL-PHO5 (right),respectively,weregrown at 30°C to logarithmic phase in minimal medium supplemented with leucine. were Cellsharvested by centrifugation (2000g), washed, and resuspended(0.4 OD,> in minimal medium. The cell suspension was then dividedand supplemented with either leucine ( m a ) or uracil (-leu). The cultures were grown at 25°C for 24 h at 180 rpm. At different time points aliquots were removed and assayed for optical density (ODm) and acid phosphatase (APase) activity according to Haguenauer-Tsapis and Hinnen (170). Acid phosphatase activitywas determined in the culture supernatant, in the periplasmic space (assay with nonpermeabilized cells), and as cellassociated activity [assay with ethanol/toluol permeabilized cells (170)l. Intracellular acid phosphatase activity was the difference between cell-associatedand periplasmic activity.Total activity was the sum of intracellular, periplasmic,and extracellular activity. Enzyme activity is expressedU, inmol/h/ODm.

acidphosphatase periplasmicspace APase activity [Km01 / h / OD6001

7 65-

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time(h) acidphosphatase intracellular 8

APase activity [ p m 0 1 / h / ODsw]

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reached the medium (Fig. 15). With the same techniquethe distribution of acid phosphatase in cells under uracil selection 30% was (intracellular), 55% (periplasm), and 15% (medium) (Fig. 15). Acid phosphatase as 12sites for N-linked glycosylation (89). The degree of glycosylation an is indicator for intracelluar localizationof a protein. Acid phosphatase synthesized under leucine selection was mostly cell associated and highly glycosylated, if not slightly overglycosylated. This indicates that even the intracellularly accumulated fraction of the acid phosphatase had passed the Golgi, probablyat a slow rate of transport, resulting inan overglycosylated protein. Under uracil selection only mature acid phosphatase was found. The expressionlevel of acidphosphatase increases with the gene dosage in the cell (170). We therefore assume, by way ofextrapolation, that the intracellular accumulationof acid phosphatase under leucine selection is a consequence of high gene dosage. Due to unequal distribution of 2-pm plasmids in a cell population(13), it is rather unlikely that all cells show the same effect,but it could be limitedto a subpopulation of cells only. Immunofluorescence analysis of individual cells is a means to address a subpoputhis question. Acid phosphatase indeed accumulated only in lation of cells. A fraction of cells (ca.10%) exhibited a punctuated (sometimes diffuse) staining, suggesting Golgi localization of the intracellular acid phosphatase. I t is a logical conclusionthat such a subpopulation of cells with a high plasmid copy number also is present in the growing cell culture under uracil selection. Intracellular accumulationof acid phosphatase ina subpopulation of cells resulted inthe aberrant, exaggerated cellular structures seen with the electron microscope. These structures resembled -among others -Berkeley bodies (176) and exaggerated endoplasmic reticular structures. Cytochemical staining of acid phosphatase indicated that acid phosphatase accumulated in those structures. Also, the actin pattern in the cells was changedto diffuse surface staining, and the chitin deposition appeared on the cell surface instead of in the bud region. The higher density of cells with intracellular accumulation of acid phosphatase was used to enrich this subpopulation on a LudoxAM density gradient(176). The majority of cells (9oVo) from the bottom fraction of the gradient had acid phosphatase, which had accumulated intracellularly (immunofluorescent label). Cells Figure 14 Distribution of acid phosphatase (APase) activity in ell cultures grown under uracil (-ura) and leucine (-leu) selection conditions. Acid phosphatase activity was determined in the periplasmic space, within the cells, and in the culture supernatant as described in Fig. 13.

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ngUm 15 Radiolabeling and subfractionation of acid phosphatase. yB18/pDP34/ GAPFL-pH05 transformants were grown under uracil (-uracil) and leucine (-1eucine) selection conditions as described in Fig. 13. After 12 h of fermentation the cells (2 OD,) were harvested and radiolabeled essentiallyaccording to Riederer and Hinnen (89) in 400 p1 of minimal medium without uracil (-uracil) or leucine (-leucine), respectively, for 30 min with 100 pCi [%]methionine (NEN: loo0 Ci/ mmol) and chased for 15 min in the presence of 2 mM methionine. The labeled cells wereseparated from the culture medium (m) by centrifugation. Theells were further incubated with lyticasefor 1 h. After centrifugation the supernatant(p = periplasmic fraction) was collected and the pelleted spheroplasts (i = intracellular fraction) were resuspended incitrate buffer.Acid phosphatase was immunoprecipitated from the subfractions m, p, and i with a polyclonal antiacid phosphatase antibody. The resulting immunoprecipitateswere dissolved, treated with 1 mU of endoglycosidaseH (Boehringer, Mannheim)at 37°C overnight, and analyzed by SDS-polyacrylamidegel electrophoresis. The fluorogram is shown. Subfraction m (lanes 1 and4) represents extracellular acid phosphatase,fraction p (lanes 2 and 5) periplasmic enzyme, andfraction i (lanes3 and 6) intracellular acid phosphatase.

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from the bottom fraction were able to synthesize acid phosphatase under uracil and leucine selection, in contrast to cells from thetop fraction, which showed a reduced ability to synthesize acid phosphatase under leucine selection due to their inefficient leucine complementation. Otherwise, the cells from the top fraction behaved like normal growing cells (e.g., cells under uracil selection) in secreting acid phosphatase into the periplasmic space and the medium, while cells from the bottom fraction did not grow, accumulated acid phosphatase intracellularly, and clearly showed the phenotype of cells grown under leucine selection (Fig. 16). Dense cellsin

bottom -leucine

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Flgure 16 Radiolabeling and subfractionation of acid phosphatase with dense cells enriched in thebottom fraction of a Ludox gradient. YB18/pDP34/GAPFLpH05 transformants were grownunder uracil and leucine selectionconditionsas described in Fig. 13. After 6 h of fermentation the cells were harvested and applied to 55% Ludox-AM (DuPont) gradient solutions. After centrifugation (22.OOOg at 10°Cfor U)min) the cells in the bottom fraction of the gradients were collected. Pulse labeling inthe absence of uracil (-uracil)and leucine (-leucine), respectively, and subfractionation was performed as described in Fig. 15. Subfraction m (lanes 1 and 4) represents extracellular acid phosphatase,fraction p (lanes 2 and 5) periplasmic enzyme, and fraction i (lanes 3 and 6 ) intracellular acid phosphatase.

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the bottom fraction of the gradient had the same properties, whether they originated from a culture under uracil or leucine selection (Fig. 16). 6.4

Discussion

High-level expression of a protein in yeast isoften achieved by using 2pm-based expression vectors. Characteristicfeatures of this type of vector are the high average copy number, but also a rather broad copy number distribution within the cell population due to unequal partitioning (13). We tried to establish a link between gene dosage and the behavior of subpopulations of cells and to make this visible in our system. Under uracil selection,cells with evena low plasmid copy number can grow (efficient complementation). They synthesize and secrete mature acid phosphatase, as expected. Under leucine selection, however, cells with a low plasmid copy number are starved of leucing, dueto the poorly expressed leu2-d allele. Cells with a high copy number produce enough, leucine for growth, but concomitantly, the high gene dosage for acid phosphatase leads to an intracellular buildup of acid phosphatase. This, in turn, results in a block-for reasons not yet understood-of vectorial transport to the bud and an arrest of growth. Changes inthe actin staining pattern indicate changes in the cytoskeleton, which can be correlated to defects in vectorial secretory transport and bud formation (177). Continued synthesis of acid phosphatase leadsto intracellular accumulation of slightly overglycosylated enzymeat a late stage inthe secretory pathway. Part of this overglycosylated acid phosphatase reaches the periplasmic space probably in an undirected, random fashion by fusion of organelles with the plasma membrane. In summary, therefore, under leucine selection, we see two overlapping effects. Leucine starvation for one subpopulation of cells and overproduction of acid phosphatase for another subpopulation of cells resulting in the nongrowth phenotype of the culture. Interestingly, a similar subpopulation of cells with a high plasmid copy number and intracellular accumulationof acid phosphataseis also present under uracil selectionbut is hidden by the majority of normally growing cells in the culture. Dense cells with accumulated acid phosphatase are enriched in thebottom fraction of Ludox density gradients. The dense cells from cultures under uracil or leucine selection behave virtually identically, and the properties of these cellsare very similar to those determinedfor a cell culture under leucine selection as a whole. We have shown that in an overexpression situation for a homologous yeast protein, such as acid phosphatase, a subpopulation of cells has a

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nongrowth phenotype with aberrant intracellular accumulation of the protein. Under leucine selectionthis phenotype dominates and is therefore easily detectable, whereas it is not apparent under uracil selection of acid phosphatase observed conditions. The intracellular accumulation supports the ideathat even for a homologous yeastprotein, the secretion capacity of the cell can be limiting under certain conditions.

7

DISULFIDE-LINKED DIMERIZATION OF RECOMBINANT HUMAN INSULIN-LIKE GROWTH FACTOR-1 RELATED TO ITS FOLDING IN THE YEAST ENDOPLASMIC RETICULUM

7.1 Introduction

Human insulin-like growth factor-l (IGFl) is a single-chain, 70-amino acid polypeptide which shares a high degree of homology withthe twochain insulin molecule (178). Like insulin, IGFl has three intramolecular disulfide bonds.IGFl is of pharmaceutical interest because clinical studies indicate its potential use inthe treatment of someforms of dwarfism and diabetes,and also in hastening the process of wound healing (178). Active monomericIGFl has been produced as product a secreted from the yeast Saccharomyces cerevzkiae.Analysis of the IGF1-like molecules in the yeast culturesupernatant on immune-blots reveals that the secreted molecules are predominantly disulfide-bonded dimers. We have addressed the possibilitythat misfolding of the nascent IGFl polypeptide maybe a reason for dimer formation.

7.2 Protein Folding: The Role of Signal and Leader Peptides Most proteins destinedfor secretion require transient amino-terminal sequences, containing15 to 30 amino acids,for targeting to the endoplasmic reticulum (ER) (179). These sequences, termed signal peptides, mediate translocation of newly synthesized proteins across the membraneof the ER (180). It is believed that the initial foldingof nascent polypeptides is delayed by the presenceof signal sequencesat the N-terminus (181).This delay in foldingis thought to permit a translocation competent state (181, 182). Upontranslocation, the signal peptide is usually cleaved by the enzyme signal peptidase, and then the protein is free to acquire its native folded conformation in the lumen of the ER (183). Secretion of heterologous proteins has been achieved from S. cerevisiae (91) by fusingthe signal sequences of yeast secretory proteins to the coding

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regions of various polypeptides. Proteins have been secreted utilizing the 17-amino acid phosphatase signal sequence encoded by the PH05 .gene (184), the 19-amino acid invertase signal sequence encoded by the SUC2 gene (185), and the 85-amino acid preprosequence of the prepro-a-factor encoded by the MFa gene (186). The preprosequence of the prepro-afactor, usually referred to as the a-factor leader (aFL), is the signal quite often used for the efficient secretion of foreign proteins (187). The aFL is an unusually long secretion signal consisting of a classical 19-amino acid signal or presequence and a 66-amino acid proregion (pro-aFL). The signal sequence segment of the aFL has been used for membrane targeting and has been shown to be cleaved during translocation (79,188). However, the function of the proregion, which is processed only later in the transGolgi by the product of the KEX2 gene (189), in the secretion and folding of heterologous proteins is not clearly understood.

7.3 Secretion of Recombinant Insulin-Like Growth Factor-1 IGFl is secreted from yeast using an a-factor leader-IGF1 gene fusion (153). The IGFl expression cassette consists of a 400-bp fragment of the yeast glyceraldehyde-3-phosphate dehydrogenase (GAPDH) promoter and a 275-bp fragment from the MFa gene which is used for termination of transcription (153,190). It was observed that the signal peptides from the PH0.5, SUC2, and MFa genes (i.e., PHOSss, Invss, and aFss, respectively) do not allow the secretion of recombinant IGFl from yeast (190). It appears that 17- to 19-amino-acid-long classical secretion signals are not sufficient for the translocation of IGFl across the ER. Being the first step in the secretion pathway (191), the translocation event would make possible the secretion of IGFl into the yeast culture medium. Surprisingly, IGFl is secreted only by leader peptides (i.e., PHOSssproaFL, InvssproaFL, and aFL), where each of the three signal peptides, PHOSss, Invss, and aFss, are fused to the 66-amino acid pro-aFL sequence (190). This implies that the proregion of the aFL is essential for the translocation of IGFl . With only signal peptides present at the N-terminus, IGFl probable folds too quickly in the cytoplasm, acquiring a conformation that is translocation incompetent. It is likely that covalent attachment of the pro-aFL to IGFl causes a delay in the folding of IGF1, which confers the optimal conformation crucial for translocation (181,182).

7.4

Disulfide-Linked Dimerization of IGF1 Related to Folding

The majority of the IGFl molecules secreted from yeast using any of the three leader peptides are not active monomers but inactive disulfide-linked

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dimers. It has been suggested that dimer formation could be the result of a specific charged interaction between two IGFl molecules during the process of folding in the yeast cell (192). It can also be argued that after permitting translocation, the covalently linked pro-aFL has a deleterious effect on the folding of IGFl . The proregion is removed in the late Golgi only after folding has occurred. Processing takes place when the membrane-bound endoprotease Kex2p cleaves at a pair of exposed dibasic amino acids linking the N-terminal pro-aFL to the C-terminal IGFl . Therefore, it is possible that the presence of the extraneous pro-aFL sequence during folding in the lumen of the ER could cause a distortion in the folding pathway of IGFl (193), which may result in the formation of dimers.

7.4.I

Mutant Kex2 Enzyme Function in the Endoplasmic Reticulum as a Tool to Study Formation of IGFI Dimers To investigate whether pro-aFL is really involved in IGFl dimnerization, we have examined the effect of a Kex2p variant, soluble Kex2pHDEL (sKex2pHDEL), which could function in the ER (194). This mutant enzyme, if active, should remove the proregion from the precursor protein in the lumen of the ER rather than in the late Golgi. The early processing would diminish the effect of the proregion of the aFL on the folding of IGFl and enable the mature IGFl molecule to fold independently. 7.4.2 C-Terminal His-Asp-Glu-Leu Sequence Allowing Retention of a Soluble Form of the Kex2 Enzyme in the Endoplasmic Reticulum It has been reported that a soluble form of the Kex2 enzyme (sKex2p) (195) is obtained by deleting the C-terminal 200-amino acids from the wtKex2p (196). The deleted region includes the membrane-spanning region of the enzyme. The sKex2p variant has been shown to be secreted in considerable amounts into the yeast culture medium when expressed on a multicopy plasmid (195). No Kex2p-like endoproteolytic activity (102) is observed in the medium when wtKex2p is expressed similarly. Secretion of sKex2p is appreciably lower if the S. cerevisiae ER-retention signal HisAsp-Glu-Leu (HDEL) (197) is attached to the C-terminal end of the soluble form of Kex2p (unpublished observations). To answer the question as to whether the HDEL sequence bestows the property of ER retention on sKex2p, we have used, as in vivo substrates, two precursor IGFl molecules that accumulate in the ER. The enzyme that functions in the ER should generate mature molecules from an ER-accumulated reservoir of intermediates. When the wtaFL sequence is used for secretion, mature IGFl is the major intracellular entity. Through pulse-chase experiments (153) it has

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been shown that maturation of IGFl occurs via intermediatesthat bear Asn-linked core sugars (198). Sincethe proregion of the (YFL,normally removed in the late Golgi, has three sequons for N-glycosylation (AsnXaa-SerIThr), the nature of the oligosaccharides attached to the proaFL can actas convenient tagsto monitor the location of precursor intermediates in the cell (199). The precursors residing in the ER bear only core sugars, which are observed as distinct bands after electrophoresis on polyacrylamide gels. Intermediatesthat have traveled beyondthe ER have a mixture of outer-chain mannosesand are identified by slower gel mobility (i.e., larger molecular mass)and diffuse bandson gels. Two mutated aFL sequences (Mut2 and Dell; Tables 5 and 6 ) fused to IGFl cause intracellular accumulationof precursor IGFl molecules (194). Only a very small amount of the precursors undergoesmaturation. These molecules still have the proregion of the aFL attached to IGF1. Mostly N-linked core sugars are present in these intermediates (Fig. 17), typical of molecules that have not traveled beyond the ER in the secretion pathway (191, 198). If the HDEL tetrapeptide allows retentionof sKex2p in the ER, then among the three Kex2p variants, sKex2pHDEL should be unique in releasing mature IGFl from an intracellular pool of precursors which accumulates in the ER. The three Kex2p variants, wtKex2p, sKex2p, and sKex2pHDEL, have been subcloned on the multicopy plasmid pDP34B S. cerevisiae 2-pm sequence (118). (23,194), which contains the complete The plasmids not only encode one of the Kex2p variants but also carry the IGFl expression cassettes containing the mutated aFL sequences (Table 5). Appropriate plasmids (Table 6) were transformed in a yeast strain, AB1 lOkex2, wherethe genomic copy ofthe KEX2 gene wasdisrupted by gene replacement, and no functional wtKex2p is expressed (194). Yeast transformants were grown in an uracil-selective mediumfor expression

Table 5 Specific Amino Acid Changes in the Mutated aFL Mut2 and a13-AminoAcidDeletionInvolvingthe First Glycosylation Site (Dell) in the aFL (194)

Deletion Mutated aFL sequence Mut2 Dell

Mutation Ala20 to Asp20 and Pro21 to Leu21

-

Pro21 to Ile33

Table 6 Plasmids Used for Expression of IGFP

Expression cassette(s) coding for:

aFL sequence Yeast Plasmid used

strain@)used for transformation ~~

~~

IGFl ABllO and ABllOkex2 WT pBC23 IGFl AB1 10 Mut2 pBCl2 IGFl ABllO pBC13 Del 1 IGFl and wtKex2p pBC17 Mut2 AB1 lOkex2 IGFl and sKex2p ABllOkex2 pBC18 Mut2 IGFl and sKex2pHDEL ABllOkex2 pBC19 Mut2 IGFl and wtKex2p ABllOkexZ pBC20 Del 1 IGFl and sKex2p AB1 lOkex2 pBC211 Del IGFl and sKex2pHDEL ABllOkexZ pBC22 Del 1 IGFl and wtKex2p ABllOkex2 WT pBC24 IGFl and sKex2p AB1 lOkex2 WT pBC25 IGFl and sKexwpHDEL AB1 lOkex2 WT pBC26 Vhe vector pDP34B either bears only the IGFl expression cassette (153) containing one of the aFL secretion signals, or both the IGFl expression cassette and one of the three KEX2 variants. Expressionof IGFl is under the control of the glyceraldehyde-3-phosphate dehydrogenase promoter (153).

Il Figure 17 Endoglycosidase F (Endo F)-digested products (lanes 2,4,6) of intracellular IGF1-likeproteins from AB1 10transformants harboring IGFl expression plasmids pBCl2 and pBC13 (Table 6) bearing the mutated aFL sequences (Table 5). All cell lysates were from yeast cultures (153) grown for 72 h.After completion of digest(l%), 0.5 ml of Laemmli buffer was added. A f t e r separation ona SD%15% polyacrylamide gel, proteins were blotted onto poly(vinylidene diflouride) membrane (Millipore) for Western blot analysis. Transferred proteins were detected with anti-IGF1 polyclonal antiserum (153).Lanes 1 and 2, unglycosylated proteins from Mutl (194), which accumulate inthe cytoplasm (asa control for Endo F digest); lanes 3 and 4, Mut2; lanes5 and 6, Dell; lane 7 , 150 ngof HPLC-purified IGFl monomer. The 17-, 27-, 39-, and 50-kDa bands used as markers belong to the prestained low-range standard proteins (Bio-Rad); M, IGFl monomer.

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Iry

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sKex2p hdei

Figure 18 In vivo comparison of Keep, sKex2p, and sKedpHDEL using as substrates intracellular entities accumulated in the ER (194). IGF1-like proteins from cells grown from ABllOkex2 transformants bearing plasmids (A) pBC17, pBC18, and pBC19; and (B) pBC20, pBC21, and pBC22. All plasmids are listed in Table6. Two transformants from each the of six transformations were used for the Western blot analysis. Proteins from 2 pL cell lysates were reduced with dithiothreitol and were separated by 15% SDS-PAGE. After blotting (as in Fig. 17), IGF1-like proteins were detected with the anti-IGF1 antiserum. The markers used were the same as in Fig. 17.

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of IGFl (153). Harvested cells were lyzed and analyzed by Westernblotting (194). Comparison of the intracellular proteins (Fig. 18) clearly shows that sKexpHDEL is indeed distinct from the other two variants. In contrast to wtKex2p and sKex2p, sKex2pHDEL does form more mature IGFl molecules fromthe intracellular precursor intermediates accumulated in at the C-terminus the ER.This strongly indicatesthat the HDEL sequence of sKex2p has allowed a partial retention of sKex2p in the ER.

7.4.3 Use of sKex2pHDEL to Prevent Intermolecular Disulfide-Linked Dimerization of lGF7 Only 10 to 15% ofthe secreted IGFl molecules are monomeric usingthe wtaFL as a leader peptide (153,190).A large proportion of the total secreted product is dimeric (192,200). Knowing that the C-terminal HDEL tetrapeptide may allow retention of a soluble form of Kex2pthe in endoplasmic reticulum, we have attempted to remove the proregion of the wtcuFL in the lumen of the ER. Plasmids pBC24, pBC25, and pBC26 (Table 6)were transformed in the yeast strain AB1 lOkex2,and supernatants from yeast cultures were compared under nonreducingand reducing conditions (Fig. 19). It appears that dimer formationis scarcely observedon analysis of IGF1-like molecules secretedfrom strains transformed with the plasmid pBC26 and carrying the sKex2pHDEL and IGFl expression cassettes. This indicatesthat the proregionof aFL, after permitting translocation, does influencethe folding of IGFl in a way that facilitates dimer formation (201). 8 CONCLUSION The Kex2 endoprotease can be mislocated from the Golgi to the ER using the S. cerevisiae ER-retention signal HDEL.The proregion of the aFL, which is essentialfor the translocationof IGFl, can be cleaved inthe ER using the ER-retained variant of the Kex2 enzyme. Removal ofthe proregion early inthe secretion pathway hasan impact on the folding of the nascent IGFl polypeptide. It prevents the formation of disulfide-linked IGFl dimers. It is possiblethat sKex2pHDEL could be usedas an in vivo biochemical tool to study the broad role of proregions in the folding and intracellular transport of other precursor proteins. ACKNOWLEDGMENTS Without the helpof many of our colleagues, this chapter would not have been possible. We greatly appreciate theirsupport in providing original

uuu KexPp sKex2p sKex2p

HDEL

Figure 19 Comparison of IGF1-like proteins secreted from ABllOkex2 transformants bearing expression plasmids pBC24, pBC25, and pBC26 (Table 6) on a Western blot (as in Fig. 17). Two transformants from each of the three transformants were usedfor analyses. An AB1 10 transformant of plasmid pBC23was used as a control. Two milliliters of supernatants was concentrated using Centricon-3 filters (Amicon). Supernatants from pBC23 and pBC24 were concentrated twofold and the ones from pBC25 and pBC26 were concentrated fourfold (201). Proteins from 5 pL of concentrated supernatants were analyzed, as in Fig. 17. (A) Lane 1, 150ng of purified IGFl monomer (M); lane 2, pBC23::ABllO; lanes 3 and 4, pBCM:ABllOkex2; lanes 5 and 6, pBC25::ABllOkex2; lanes 7 and 8 , pBC26::ABl lOkex2. Rainbow-colored (Amersham) protein markers (low molecular weight range) were used as standards. (B) Sameas in (A), exceptthat the supernatants were treated with dithiothreitol.

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data and their scientific input. Many thanks to P. Fiirst, H. Grossenbacher, R. Kleene, W. Mtirki, C. Stephan, A. Strauss, K. Takabayashi, and H. J. Treichler.

REFERENCES 1. Tuite MF. Expression of heterologous genes. In: Tuite MF, Oliver SG, eds. Saccharomyces. New York: Plenum Press, 1991:169-212. 2. Barr PJ, Brake AJ, Valemela, P. eds. Yeast genetic engineering. Boston: Butterworth, 1989. 3. Guthrie C,Fink GR, eds. Methods in Enzymology, Vol.194. Guide to Yeast Genetics and Molecular Biology. San Diego: Academic Press, 1991. 4. Strathern JN,Jones EW, BroachJR, eds. The molecular biology ofthe yeast

Saccharomyces. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory, 1981. 5. Rose AH, HarrisonJS, eds. The Yeasts, Vol. 3. San Diego: Academic Press, 1989. 6. Hinnen A, Hicks JB, Fink CR. Transformation of yeast. Proc Natl Acad Sci USA 1978; 75:1929-1933. 7. Rothstein RJ. One-step gene disruption in yeast. Methods Enzymol 1983; 101:202-211. 8. Winston F, Chumley F, Fink

CR. Eviction and transplacement of mutant genes in yeast. Methods Enzymol 1983; 101:211-228. 9. Orr-Weaver TL, Szostak JW, Rothstein RJ. Genetic applications of yeast transformation with linear and gapped plasmids. Methods Enzymol 1983; 101~228-245. 10. Boeke JD, Xu H, Fink GR. A general method for chromosomal amplification of genes in yeast. Science 1988; 239:280-282. 11. Beggs JD. Transformation of yeast by a replicating hybrid plasmid. Nature 1978;275:104-109. 12. Rose AB, Broach JR. Propagation and expression of cloned genes in yeast: 2pm circle-based vectors. Methods Enzymol 1990; 185:234-279. of Saccharomycesmrevisiue. Yeast 1988; 13. Futcher AB. Thecircleplasmid 4:27-40. 14. Janes M, Meyhack B, Zimmermann W, Hinnen A. The influence of GAP

promoter variants on hirudin production, average plasmid copy numberand cell growth in Saccharomyces cerevisiue. Curr Genet 1990; 18:97-103. 15. Erhart E, Hollenberg CP. The presence of a defective LEU2 gene on a 2p DNA recombinant plasmid of Succhuromyces cerevisiue is responsible for curing and high copy number. J Bacteriol 1983; 156:625-635. 16. Unternahrer S, Pridmore D, Hinnen A. A new system to amplify 2p plasmid copy number in Saccharomyces cerevisiae.Mol Microbiol 1991; 5:1539-1548. 17. Hartwell LH, Mortimer RK, Culotti J, Culotti M. Genetic control of the cell division cyclein yeast. V. Genetic analysis ofc& mutants. Genetics 1973; 74~267-286.

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A subfamily of stress proteins facilitates translocation of secretory and mitochondrial precursor proteins. Nature 1988; 332:800-805. 183. Freedman RB. Protein disulfide isomerase: Multiple roles in the modification of nascent secretory proteins. Cell 1989; 57:1069-1072. 184. Meyhack B, Bajwa W, Rudolph H, Hinnen A. Two yeast acid phosphatase structural genes are the result of a tandem duplication and showdifferent degrees of homology in their promoter and coding region. EMBO J 1982; 1:675-680. 185. Taussig R, Carlson M. Nucleotide sequence of the WC2 gene for invertase. Nucleic Acids Res 1983;11:1943-1954. 186. Kurjan J, Herskowitz I. Structure of a yeast pheromone gene (MFa): A

187.

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putative a-factor precursor contains four tandem copies of maturea-factor. Cell 1982;30:933-943. Brake AJ. Secretion of heterologousproteins directed by the yeast a-factor leader. In: Barr PJ, Brake AJ, Valenzuela P, eds. Yeast Genetic Engineering. Boston: Butterworth, 1989:269-280. Waters MG, Evans EA, Blobel G. Preprw-factor has a cleavable sequence. J Biol Chem 1988; 263:6209-6214. Fuller RS, Sterne RE, Thorner J. Enzymes required for yeast prohormone processing. Annu Rev Physiol 1988; 50:345-362. Chaudhuri B, Steube K, Stephan C. The proregion of the yeast prepro-afactor is essential for membrane translocation of human insulin-like growth factor-l in vivo. Eur J Biochem 1992; 206:793-800. Rothman JE, Orci L. Molecular dissection of the secretion pathway. Nature 1992; 355:409-415. Chaudhuri B, Helliwell S, Priestle JP. A L y ~ ~ ~ - t o - Gmutation lu*~ in the human insulin-like growth factor-l prevents disulfide linked dimerisation and allows secretion of BiP when expressed in yeast. FEBS Lett 1991; 294:

213-216. 193. Hober S, Forsberg G, Palm G, Hartmanis M,Nilsson B. Disulfide exchange folding of insulin-like growth factor I. Biochemistry 1992; 31:1749-1756. 194. Chaudhuri B, Latham SE, Helliwell SB, Seeboth PG. A novel Kex2 enzyme

can process the proregion of the yeast a-factor leader in the endoplasmic reticulum instead of in the Golgi. Biochem Biophys Res Commun 1992; 183~212-219. 195. Seeboth PG, Heim J. In-vitro processing of yeast a-factor leader fusion

proteins using a soluble yscF (Kea) variant. Appl Microbiol Biotechnol 1991; 35:771-776. 1%.

Mizuno K, Nakamura T, Ohshima T, Tanaka S, Matsuo H. Yeast KEX2 gene encodes an endopeptidase homologous to subtilisin-like serine proteases. Biochem Biophys Res Commun 1988; 156:246-254.

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197. Pelham HR. Control of protein exit from the endoplasmic reticulum. Annu Rev Cell Biol 1989; 5:l-23. 198. HirschbergCB,SniderMD.Topographyofglycosylation in the rough endoplasmic reticulum and Golgi apparatus.Annu Rev Biochem 1987; 56: 63-87. 199. Graham TR, Emr SD. Compartmental organization of Golgi-specificprotein modification and vacuolar protein sorting events defined in a yeast sed8 ( N W ) mutant. J Cell Biol 1991; 114:207-218. 200. Elliott S, Fagin KD, Narhi LO, Miller JA, Jones M, Koski R, Peters M, Hsieh P, Sachdev R, RosenfeldRD, Rohde MF, Arakawa T. Yeast-derived recombinanthumaninsulin-likegrowth factor I: Production, purification and structural characterization. J Protein Chem 1990; 9:95-104. 201. Chaudhuri B, Stephan C. A modified Kex2 enzyme retained in the endoplasmic reticulum prevents disulfide-linked dimerisation of recombinant human insulin-like growth factor-l secreted from yeast. FEBS Lett 1992; 304:41-45.

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Gene Expression in Methylotrophic Yeasts Gerd Gellissen Rhein Biotech GmbH, Diisseldotf, Germany

Cornelis P. Hollenberg Heinrich-Heine University, Diisseldorf, Germany

Zbigniew A. Janowicz Rhein Biotech GmbH, Dusseldotf, Germany

1 INTRODUCTION The development of several microorganisms and eukaryotic cell lines as hosts for heterologous gene expression has led to an increasing number of options for the production of recombinant proteins. The initial systems, the bacterium Escherichia coli (1,2) and the yeast Saccharomyces cerevisiae (3,4), respectively, are now supplemented by an increasing number of alternatives (5-8), of which some examples are described elsewhere in this volume. For industrial applications the system of choice should produce an optimal amount of authentic bioactive material in a short time in a reproducible and stable production process. Therefore, expression systems have to fullfil several prerequisites to allow such an efficient and economical production of a recombinant protein. The cells should be robust and easy to handle, and they should grow to high cell densities on low-cost media containing cheap energy and carbon sources. Adequate expression vectors must be available and the host must be capable of synthesizing the desired protein either intracellularly or, if certain eukaryotic modifications are required, extracellularly. If it fails to assure a correct fold195

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ing, laborious and cost-intensive in vitro refolding and modification processes might have to be applied. As unicellular eukaryotic organisms, yeasts combine the ease of genetic manipulation with the ability to perform eukaryotic processing steps on the polypeptides expressed. When the traditional S. cerevisiae systemwas used for heterologous gene expression, disadvantages such as strain instability during the production process, low productivity, and an inappropriate glycosylation were encountered in several cases. Therefore, a search was initiatedto find alternative non-Saccharomyces yeasts suitable as hosts for recombinant protein production. Two facultative methylotrophic yeasts, Hansenula polymorpha (9-12) and Pichia pastoris (13, 14), have been developed duringthe recent past. These two ascomycetes are characterized by efficient utilization of methanol as the sole energy and carbon source. The ability to grow on this C, carbon compound is found in a variety of bacteria (15-16) and a limited number of filamentous fungi and yeasts belonging to the genera Candida, Torulopsis, and Hansenula and Pichia, on which we focus (19-22). These yeasts share a general pathwayby which, on the one hand, methanol is metabolized to carbon dioxideto generate energyand is used, on the other hand, for assimilation to produce biomass. In these organisms growth on methanol is accompanied by a strong induction of enzymes involved in methanol metabolism. Genes encoding key enzymes of methanol have been isolated and characterized (23-27). A s a consequence, their strong inducible promoters became availableas components for the expression of foreign genes. Techniques were developedto introduce heterologousDNA into suitable methylotrophic hosts, and expression systems were established for heterologous gene expression whichare characterized by high stability, viability, and productivity. In the following section the expression systems based on these two methylotrophs are described and discussed.

2 METHANOL METABOLISM AND ITS REGULATION IN METHYLOTROPHIC YEASTS The complex methanol utilization pathway shared by the methylotrophic yeasts is highly compartmentalized. The initial reactions take place in specialized microbodies, peroxisomes, followed by subsequent metabolic steps in the cytoplasm (19,27-30). Methanol enters the peroxisomesand is then oxidizedby a specific methanol (alcohol) oxidase (MOX or AOX) to generate formaldehyde and hydrogen peroxide (19,22,28,30-37). Methanol (alcohol) oxidases isolated from different yeast species exhibit similar

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properties. Theenzyme contains eight subunits yieldinga protein of Some 600 kDa (30,34). It is a flavoprotein and contains one FAD moiety per subunit. Theenzymes oxidizeshort-chain (C,to C,) aliphatic primary alcohols. V , , decreases and K,,, increases with increasing chain length. V,, values reported for methanol range between3 and 20 pA4 m i d (mg between 0.2 and 2.0 mM.The K,,, value of the enzyme protein)", the K,,, for formaldehyde, which can serve as a substrate in its hydrated form, value for oxygen of the H. polymorpha is 3 to 10 times higher. The K,,, enzyme lies between0.24 and 0.4 mMmolecular oxygen, dependingon the methanol concentration present in the system (36,37). This low affinity for oxygen forces the enzyme to work at suboptimal oxygen concentrations in the cell (oxygen concentration in air-saturated is water about of the enzyme for its 0.2 mM).This adds to the poor catalytic properties physiological function under in vivo conditions. This deficiency is compensated by a massive production of the protein under conditionsof enzyme requirement (see below). The toxic hydrogen peroxide generated in the oxidase reactionis decomposedto water and molecular oxygen by a peroxisomal catalase (19,35,37,38). The catalase activity is high during growth on methanol. In H. polymorpha, specific activities of 1.45 mM m i d (mg protein)' have been reported, thus being l@ and 104 times in excess of the oxidase (28).MOX and CAT activitieswere found to be coregulated to some degree (19) (see also Section 3). Formaldehyde, generated bythe oxidase reaction, enters either the cytosolic dissimilatory pathway to yield energy or the assimilatory pathway to participate inthe generation of biomass, which starts with a first step of a peroxisomal localization. This first step, a key reaction of the assimilatory pathway,is catalyzed bythe peroxisomal enzyme dihydroxyacetone synthase, yielding dihydroxyacetone and glyceraldehyde-3-phosphate in a transketolase reaction between formaldehyde and xylulose-5-phosphate (39-43). These C, compounds are further assimilated in the cytosol. Dihydroxyacetone is phosphorylated by a dihydroxyacetone kinase.Catalyzed by an aldolase, it reacts subsequently with glyceraldehyde to form fructose-l,6-biphosphate. By the action of a phosphatase, fructose-6phosphate is formed. In several intermediate steps xylulose-5-phosphate is eventually regenerated by transaldolase, transketolase, pentose phosphate isomerase, and epimerase reactions ina cyclic pathway. One-third of the glyceraldehyde-3-phosphate generated is converted into biomass by standard reactions of the central cell metabolism (21,22,28,30). Some of the formaldehyde produced in the methanol oxidase reaction enters the cytosol, where itis catabolized in two subsequent dehydrogenase reactions (44-47). aInfirst step, formaldehydeis oxidizedto formate

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by a formaldehyde dehydrogenase(48-50), formate is subsequently oxidized to carbon dioxide by formate dehydrogenase (49,50). Again, formaldehyde dehydrogenase is similar in all yeasts analyzed so far (30,4850). The enzyme is strictly dependent on glutathione for activity. It does not use free formaldehyde but the thiohemiacetal formed from reduced glutathione and formaldehyde as substrate in a NAD+-dependent reaction to produce S-formylglutathione. S-Formylglutathioneseems to be the substrate of the subsequent formate dehydrogenase reaction in H. polymorpha, since this enzyme has a 40-fold-higher affinity for this compound than for formate (51). The enzyme hydrolyzes S-formylglutathione in the presence of NAD+. Therefore, it is assumedthat a first hydrolysis leads to an enzyme-bound formate which is oxidized in a second step to carbon dioxide. In the methylotrophic yeast speciesKfoeckera sp. 2201 and Candida boidinii, two distinct enzymes have been identified for this process. An initial hydrolysis to glutathione and formate by Sformylglutathione hydrolases (52,53) is followed by formation of carbon dioxide from the formate generated by formate dehydrogenase activity (30). A scheme of this utilization pathway, which has been reviewed comprehensively (30,54), is presented in Fig.1. There might be additional possibilities to yield energy during growth on methanol. Sybirnyet al. (55) isolated H. polyrnorpha mutant strains that are deficient in formaldehyde/formate dehydrogenase. These mutants were found to grow on methanol as a sole energy and carbon source, indicating that enz$nes of direct formaldehyde catabolism are not indispensable for methylotrophic growth. Since inhibition of the tricarboxylic acid cycle results in a growth repression, the authors suggest that dissimilation of methanol and the supply of energy during methylotrophic growth is achieved primarily by oxidation of C, compounds generated by the DHAS transketolase reaction viathe tricarboxylic acidcycle. According to this concept, the direct cytosolic oxidation serves mainly for detoxification of formaldehyde. Growth on different carbon sources results in distinct patterns of intracellular H. polyrnorpha proteins. In methanol-grown cells three prominent polypeptidesare present in large amounts. These abundant proteins are key enzymesof the methanol metabolism described before: methanol oxidase (MOX), formate dehydrogenase (FMDH), and dihydroxyacetone and FMDH canalso be desynthase (DHAS). Significant levels MOX of tected in glycerol-grown cells, but they are absent when glucoseused is as a carbon source (Fig. 2). Thus synthesis of these key enzymes is subject to a repression/derepression/induction mechanism forcedby the carbon

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DHA

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Cytosol Figure 1 Methanol utilization pathway and its compartmentalization in H. polymorphs. Methanol is oxidized within the peroxisome by methanol oxidase (MOX) to generate formaldehyde and hydrogen peroxide (1). The toxic hydrogen peroxide is metabolized to water and oxygen (2). To some extent the formaldehyde generated enters the cytosol to be dissimilated to carbon dioxide by two subsequent dehydrogenase reactions, catalyzed by formaldehyde dehydrogenase (3) and formate dehydrogenase (4) (FMDH). For assimilation, the formaldehyde remaining in the peroxisome reacts with xylulose-5-phosphate (Xu,P) by the action of dihydroxyacetone synthase (DHAS) (5). The resulting C, compounds glyceraldehyde 3-phosphate (GAP)and dihydroxyacetone @HA) enter the cytosol. The DHA is phosphorylated by a dihydroxyacetone kinase (6) to dihydroxyacetone phosphate (DHAP). DHAP and GAP produce fructose l,&biphosphate (FBP) in a reaction catalyzed by fructose 1,6-biphosphate aldolase (7). Subsequently, FBP is dephosphorylatedby fructose 1,5-biphosphatase (8). In further steps X h P is regenerated. One-third of the GAP molecules produced enter the central metabolism of the cell to generate biomass. The H. polymorpha genes isolatedand characterized for methanol oxidase (MOX), formate dehydrogenase (FMD), dihydroxyacetone synthase (DAS), and catalase (CA'I) are indicated in the pathway at the position of the enzymes they are coding for. In Pichia pastoris, two highly homologous alcohol oxidase genes designated AOXl andAOX2 have been isolated and characterized. (From Ref. 42; figure and legend modified according to Refs. 22 and 144).

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A

B

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Figure 2 SDS-PAGE analysis of intracellular H. polymorpha proteins. Crude extract from cells grown in media supplemented withdifferent carbon compounds (lane A, medium containing 2% glucose; lane B, 0.5% glycerol; and lane C, 1% methanol) were separated by SDS-PAGE through 12.5% gels and visualized by Coomassie Blue staining. The synthesis of key enzymes of the methanol utilization pathway is repressed in glucose-containing media, derepressed in glycerolcontaining media, and induced in methanolcontaining media. Key enzymes are dihydroxyacetone synthase@HAS; M, = 78,000), methanol oxidase (MOX; M, = 69,000), and formate dehydrogenase (FMDH; M, = 38,000).

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compounds of the culture medium (56,57). In P. pastoris, substantial amounts are visible only in methanol-grown cells, although low concentrations of alcohol oxidase (2%of the inducedstatus) during growth under glycerol-limited conditions have been reported. Thus expression of these proteins follows a repression/induction mechanism in contrast to the situation in Hansenula (58). Upon addition of methanol, the respective genes are heavily induced (see the next section) and a massive proliferation of the peroxisomes, the compartment for MOX, DHAS, and the catalase (CAT), is promoted (59-61). The methanol oxidaseis an octamer forming a crystalline lattice that constitutes the peroxisomal skeleton (62-64) containing DHAS, CAT, and furtherenzymes within. In recombinantH. polyrnorpha strains overproducing methanol oxidase, formation of irregular giant peroxisomes densily packed with huge enzyme crystalloids were observed. In these transformants, activeMOX can constitute upto 70% of the total cell protein (65). Surprisingly, this crystalline lattice can also be formed in the cytoplasm of peroxisome-deficient (per)mutants of H. polyrnorpha. Thus its formation is independent of a peroxisomal location (66). In methanol-grown continuous cultures of H. pofymorpha,MOX constitutes about 20 to 30% of total cell proteins; FMDHand DHAS, 10 to 20%. Huge peroxisomes can take up to 80% of the cell volume (51; see also Fig. 2). In cells grown ona repressive carbon source such as glucose or ethanol, the typical peroxisomal structures cannot be detected. Instead, a few small microbodies functioning as glycosomes are observed, from which peroxisomes proliferate after transfer into media with inductive conditions (67-70). Yeast peroxisome proliferation and growth in response to the environment are controlled by a variety of different genes (71). First steps ina genetic approach to understanding the complex biogenesis of this organelle have been undertaken in isolating peroxisomedeficient or peroxisome assembly mutants in S. cerevisiae (71,72), H. polyrnorpha (73,74), and P. pastoris (75). Back transfer of induced cells into media witha repressive carbon source results in rapid degradation of peroxisomal enzymes and peroxisomes by an autophagic process(68-70). This process is initiatedby formation of a 2- to 12-layered membrane enclosing the organelle. A vacuole becomes associated with this structure and is eventually incorporatedto release hydrolytic enzymes. Theabundant methanol oxidaseis rapidly degraded (76-79). Since a rapid degradationof the enzymes isnot observed in the cytosolic crystalloids per in mutants, it is believedthat the mechanism triggering the degradative inactivationof methanol metabolism enzymes is not directed against the protein itself but against the organelle

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harboring it(66).Additional inactivation occurs by protein modifications, resulting inthe release of the prosthetic FAD,which is indispensablefor enzyme activity (68). The peroxisomaland cytosolic methanol utilization enzymes are regulated in a coordinated fashion. This is similarly found for all methylotrophic yeasts, but the mechanisms are not identical, as already pointed out. The peroxisomal MOX and cytosolic FMDH are regulated by the catabolite repression/derepression/induction mechanism described earlier (inPichia by the repression/induction mechanism).It is not clear whether methanol itself or another metabolic intermediate acts as the inducing agent. In Hansenula, induction with methanol can also be considered as a strong derepressionby the absence of repressivecarbon sources such as glucose or ethanol. Accordingly, the inductive effect be canoverruled by the high glucose orethanol concentration present inthe system. The derepression effect is concentration dependent. At high concentrations of a derepressive carbon source suchas glycerol (2070) in the culture medium, only a weak synthesis of the methanol-inducible enzymes is observed, which increases at lower concentrations (0.2 to 0.4% glycerol). Consequently, a slight derepression can be detected in low concentrations of a repressive carbon compound such as .glucose(76). The expressionof D M , the peroxisomal assimilatory pathway enzyme, and of CAT seemsto follow closely the same mode. However,the amount of DAS detectable in derepressed cells is relatively low compared to the methanol-induced status.

3 CLONINGANDCHARACTERIZATION OF METHANOL METABOLISM GENES Several genes encoding enzymes of the methanol utilization pathway of the two methylotrophic yeasts have been cloned and characterized. Besides two unidentified open reading frames encoding proteins of 76 and 40 kDa, respectively, a gene encoding an alcohol oxidase (AOX1) was isolated from P. pastoris in 1985 (27). Two years later, a second highly homologous gene(AOX2) was discovered (14). Both genes contain a1992 nucleotide open reading frame encoding enzymes of some 74 kDa (77). A structural comparison revealed two coding sequences92% of and 97% homology at the nucleotide and the deduced amino acid sequence levels, respectively (77). Codon usage exhibited a strong biasto triplets similar to moderately and highly expressed S. cerevisiae genes (78). In contrast to this high homology found within the coding region, no longer sequences of homology were observed in either the 5' or 3' noncoding region. The

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primary transcription site was found 115 nucleotides upstream in both genes. Transcription startswith an adenine which is surrounded by a pyrimidine track. The putative RNA polymerase binding sites (TATA box) are in both cases at position -43 relativeto the primary transcriptioninitiation site. The DNA sequences surrounding the translation initiation

site(CGAAACGATGGCTinAOXl,GAGAAAAGATGGCCinAOX2) were found to follow closely a constrained consensus sequence( A A X X A A A ATG GYT) deduced from highly expressed genes of S. cerevisiae (7981). Within the 3' region of both genes, sequences are present exhibiting to function in3' mRNA some similarities with consensus sequences thought processing, as suggested by various authors (78-80). In H. polyrnorpha, a single methanol oxidase (MOX) gene has been isolated and characterized (23).The gene harbors an open reading frame of 1995 nucleotides encoding a polypeptide of calculated 74,050 Da. The amino acid sequence deduced from this gene exhibits a homology of more than 85% with the Pichia counterparts. However, the codon usage differs greatly in favoringG/C containing triplets,thus following the high G/C content of the H. poljmorpha genome (24).The 5' untranslated regionof mRNA is veryshort and constitutes some20 to 25 nucleotides, as shown -50. The transby S1 mapping. The TATA box is located around position lation initiation sequence ATCA TG AXG exhibits some deviation from the consensus sequence described above. In addition to MOX, the single-copy genesfor formate dehydrogenase ( F M . )(25), dihydroxyacetone synthase(DAS) (M), and catalase (CA7) (26) have been isolated. TheFMD gene harbors an open reading frame of 1085 nucleotides encodinga 35,700-Da protein, DAS a 2109 nucleotide open reading frame for a potential 77,000-Da protein, and CAT a 1521 nucleotide open reading frame encoding a 507-amino acid enzyme. All genes are characterized by the codon usage bias for G/C-containing triplets as reported for MOX. Again, the 5' untranslated regions of the deduced mRNAs are very short, consisting of 15 to 25 nucleotides. Comparative sequence analysis of genes encoding peroxisomal proteins and the analysis of N-terminal amino acidsof the mature protein did not reveal N-terminal leader structures within the deduced amino acid sequence as observed for proteins sortinginto mitochondria orinto the secretory pathway (84,85). However, studies on localization of gene products derived from mutagenized or trucated genes indicated that import into the organelle requires specific targeting signals within the primary sequence of the polypeptide. These signalswere shown to reside in the C-terminus of the protein. A C-terminalS-K-L tripeptide was found to be necessary

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and sufficientfor an organelle import in mammalian cells(86-88). From sequence comparisonof several genesfor peroxisomal proteins a consensus sequenceS-, A-, C-/K-, H-, R/L was deduced (87,88).The targeting signals for the peroxisomal proteins of methylotrophic yeasts reside in the C-terminus aswell. They all show deviations from the S-K-L motif present in mammalian proteins and from the proposed consensus sequence described above. The two alcohol oxidases from Pichia and the methanol oxidase from Hansenula contain a C-terminal A-R-F motif (23,77), the dihydroxyacetone synthaseN-K-L (U), and the catalaseS-K-I (26). This suggests that the targeting motifis less conserved than originally reported (91,92). Furthermore, peroxisomal thiolase was recently shown to contain targeting signals within a cleavable N-terminal leader, reported for the first time for peroxisomal-imported proteins (89,90). The 3’ end regionsof the variousH. pofymorpha genes analyzedso far contain some segments that exhibit a weak similarity characteristic for transcriptional termination and polyadenylation signal sequences. The DAS 3’ region harbors sequences with somestructural similarities to that of the S, cerevisiae ADHl gene (82); the FMD and MOX gene share a TGAA...(A)..ATAGG motif that is repeated three and two times, respectively(93).Analyticalapproachesquantifying transcripts showed that the different methanol pathway genes are controlled primarilyat the transcriptional level (57,58). Detailed studies with MOX promoter variants fused to a bacterial P-lactamasereporter gene revealedthat this region is composed of several positively and negatively cis-acting regions (94,95). A 1.5-kb segment was found to be necessary for full promoter strength. Withgel retardation assays and DNase Ifootprinting, two activating regions have been identified which consist of binding sitesfor yet unknown proteins (94,96). A first element UAS1, defined by footprint and deletion analysis, covers a region between -484 and -471 relative to the ATG start codon of the gene and itwas appointed to a 14-nucleotide 5TCCTTGCACCGCAA3’ motif. Deletion of this region results in20-a fold reductionof reporter gene expression. It could be shown that the unknown factor binds to this element under repressed, derepressed, and induced conditions (95).A sequence highly homologousto this MOXelement with only a single T-to-C exchange is present inCATgene the from position -415 to -401 relative to the start codon.Since this type of element was that it might not detected either in DAS or in FMD the gene, it is assumed be the binding sitefor a protein involved in oxygen-dependent regulation (94). Sequence homologies place UASlinto close relationshipto the welldefined UAS2 of the S. cerevisiae CYCl promoter (95). A second element

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UAS2 was located at position -732 to -718 relative to the MOXstart codon and consists of the sequence5' TCGAGGTCGTGG 3'. This element has no homologyto the first described above and thus probably corresponds to a different regulatory signal. third A element between-921 and -892 is characterized by deletion analysis as an upstream repressing sequence. (95). Since all fourH. polymorpha genes exhibit some degree of coordinated regulation (see the preceding section), a searchfor other sequences with structural similarities as potential regulatory segments within the promoter region of these geneswas tempting. Indeed, a remarkable stretch of homology has been identified in a region about l o o 0 nucleotides upstream of the translation initiation codon. A consecutive segmentof 65 nucleotides of theMOX promoter shows high homologyto a 139-bp region inthe DAS regulon, interspersed by several nonhomologous sequences (93; see also Fig. 3). A similar segment of homology not is found in any other region ofboth genes. When the 0-lactamase reporter gene was fused to a shortened MOX promoter that did not contain this element, only weak expression was observed in methanol-induced cells transformed with this construct (57).

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TAGATATTITCTGCC * TCGTCGTACI'CA-54N-GTGTGATG8N-TCACC-9N-

TCGAAATlTT.TGCCGTCGTCGTAC**A GTGTGATG TCACC m-1052

ATCGCTTCGTACTCGCI'CI'GCAGCIT*CGA

-

ATCGMT GTA AT GAG CI'GCAGCITGCGA -987

Figure 3 Homologous sequences in the DAS and MOX promoters. Sequence comparison of the two methanol-inducible genes revealed an extended stretchof homology in a region about lo00 nucleotides upstream of the translation initiation codon in both genes. (From Ref. 93).

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Further stretches of homology can be deduced from sequence comparisons of the various promoter structures. However, no evidence has been presented so far to attribute a specific functionto these sequences. No sequence has been identified that is common for all methanol-inducible genes despite the fact that they all share a similar induction mode.

3.1 ChromosomalLocalization of MethanolUtilizationGenes In Hamenula, six chromosomes can be separated by pulsed field gel electrophoresis. FMD, MOX, and DAS sequences hybridyze to different chromosomes, I, 11, and VI, respectively, revealinga distinct localization of the methanol utilization pathway genes (97; unpublished results). 4 GENETIC ANALYSIS

The genetic analysis of methylotrophic yeasts is less developed than knowledge of their physiology. The most advanced species with respect to a comprehensive genetic analysis is Pichia pinus. This organism exhibitsa high frequency of sporulation, and the spores are characterized by a good vitality. A wealth of mutants exist and genetic maps couldbe established (98,99). LikeP.pinus, H. polymorpha is consideredto be a homothallic organism, although some evidenceof a bipolarity of mating types exists for both organisms (100,101). A system to perform genetic studies has been developed for both yeasts, but the information published so far is relatively sparse. However,the growing interest in these species has stimulated a rapid increase of knowledge during the past several years. The newly generated mutants for the analysis of peroxisome biogenesisand function described before attest to genetic approachesfor a more comprehensive understanding of these organisms (73-75). Life-cycle stages have been defined. Both organisms have a stable haploid phase under standard culture conditions. Aftertransfer into conditions of restrictive growth, fusion of cells and formation of zygotes occur to some extent. Under further restrictive conditions these zygotes enter meiosis and sporulate. However, after retransfer into a rich medium, the zygotes remain in a stable vegetative diploid phase, which subsequently can be induced under suitable restrictive conditionsto produce asci from which two to four haploid ascospores derive. After germination, haploid coloniesare generated again(101). It appeared to be difficultto obtain some specific types of auxotrophic mutants. Severalauthors observed a strong bias in the types of mutants selected. In H. polymorpha, no useful tryptophane

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pathway mutants could be recovered from a particular strain, whereas ura and ade mutants could be identified at a high frequency.As a possible explanation, the existence of mutationalhot spots and/or a possible aneuploidy has been suggested by various authors (104,105). Nevertheless, mutants suitable to serve as useful auxotrophic hosts for heterologous gene expression have been isolatedfor both organisms (102). The genera Candida and Torulopsis do not have a sexual phaseand thus it is not possible to develop classical genetics.

5 EXPRESSIONSYSTEMSBASEDON METHYLOTROPHIC YEASTS H. polymorpha and P. pastoris have both been developed as hosts for heterologous protein production (4,5). Auxotrophic hostsare transformed with DNAcontaining suitable complementing genes or dominant marker genes for selection. The foreign genes to be expressed are fused to the strong and tightly regulated promoter structures derived fromthe methanol utilizationgenes described before. Vectors can be designed for intracellular production orfor efficient secretion of the recombinant protein (see Section 6). In H. polymorpha, two auxotrophic mutants in either orotidine 5'-phosphate decarboxylase (ura3) (9) or in P-isopropylmalate dehydrogenase (leu2)(10) have been used mostly for transformation. Accordingly, vectors for transformation of these strains have been designed harboring the homologous or the correspondingS. cerevisiae genes for selection (9,lO). In Fig. 4 a representative exampleof an H. polymorpha expression vector designed for transformation of the uracil-deficient strain LR9 is documented (9). In addition to the selection marker, the vector contains further components. Thebla gene and the ori sequence are genetic elements for selection andpropagation in E. coli, and a HARS (Hansenulaautonomously replicating sequence) is a genetic structure for propagation in the yeast host. The presence of this HARS sequence is essential for an efficient transformation frequency of the host cells. However,transformation is possible with plasmide not containing HARS, generating integrants at a low frequency. The expression cassette consists of the tightly regulated FMD (in other constructs, MOX) promoter sequence, which provides a strong and stringently controlled expression. For termination of transcription, a terminator sequence derived from the MOX gene is present. Terminatorand promoter sequencesare separated by a multiple

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Asp718 coR1

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Figure 4 Structure of a H. polymorpha expression vector. For heterologous gene expression,the vector contains an expression cassette with a FMD promoter (FMD-P.) and a MOX terminator (MOX-T.) segment separated by a multiple cloning site for sequence insertion. In addition, the plasmid harbors the following components: the pBR322-derived bla gene and ori sequence for propagation in E. coli. For propagation in the yeast host, it contains a Hansenula autonomously replicating sequence(HAW)for selection ofthe H. polyrnorpha LEU2 and URA3 genes. The plasmid is used to transform leucing- or uracil-deficient hosts. In other constructs, the MOX promoter is used as a control element withinthe expression cassette. Modified vectors contain only a single complementation sequence or harbor a kanamycin resistance gene as a dominant selection marker.

cloning site for insertion of the heterologous geneto be expressed. Variants of this general design are available containing the LEU2 gene or both the URA3 and LEU2 genes for complementation, or a S. cerevisiae ADHl promoter/G418 resistance fusionas a dominant selection marker (103). A similar strategy was applied for the P. pastoris expression system (his4)was generated (13). A mutant deficient in histidinol dehydrogenase from a suitable parental strain. Plasmids containing the homologous HIS4

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gene or that from S. cerevisiae were designedfor transformation to select against the auxotrophic background. Again, the plasmid contains the pBR322-derived components for selection and propagation in E. coli. The expression cassette is composed ofthe AOX1 promoterand AOXl terminator sequences separated byan EcoRI site for insertion ofa sequence of choice. Furthermore, the commonly used plasmids contain an additional copy of the 3'AOXl terminator region positioned distant from the expression cassette. Thisis required for homologous integrationinto the AOXl locus of the P. pastoris host cell (Fig. 5). Transformation of both species follows versions of standard protocols that either require spheroplast formation (104) or employ polyethylene

Flgure 5 Structure of a P. pustorisexpression vector. For expression, the vector contains an AOXl promoter (5'AOXl) and terminator (AOXl-t) separated by an EcoRI cloning site for insertion of a heterologous sequence.The pBR322-derived components are as describedfor the Hamenula expression vector inFig. 4. For selection in a histidine auxotrophic host, the plasmid harbors the P. pastoris HIS4 gene. In addition, the vector contains a second segment of the 3' noncoding region of AOXl (3'AOX1), distant from thatwithin the expression cassette. The plasmid is linearized at the boundary of this segment (BglII digest) for sitedirected integration into the AOXl locus of the host. (Modified after Ref. 145.)

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glycol or Li salt treatment (105), or freeze-thawing methods which do not require protoplast formation (106). In our procedures we stick to a modified versionof the latter which enables long-term storage of competent host cells at -70°C (107). When the expression systems were established, integrative (vectors without the autonomous replicating sequences) and presumptive nonintegrative vectors (vectors harboring an ARS-like element) were used. In the first case, the heterologous DNA integrated readily into the genome. The fate of plasmids containing an ARS-like sequence (108) differed dependingon othersequences present on the plasP.pastoris-derived mid and on the type of ARS-like sequence used. When M S sequences (PARS1and PARS2) were present on vectors, the transformation frequency increasedand the plasmids were maintained as autonomous elements in recombinant P. pastoris cells at least for an exS. cerevisiae-derived ARS tended period of time (13). Using plasmids with sequences or whole S. cerevisiae 2-pm DNA instead, integrationinto the host’s genome was observed, indicating that the 2-pmDNA does not function as ori in P.pastoris (13). Similarly, PARS sequences did not function in transformedS. cerevkiae hosts. Since the Pichiu-specific PARS and the ARS sequences share sequence homologies, it has been argued that the autonomous replication functionis not restricted to this consensus region but might require additional species-specific genetic elements (13). In H. polymorpha, plasmids containing either a homologous HARS or an ARS sequence exhibit a high frequency of integration. It is still not very clear how long plasmids containing such elements are maintained in an episomal state before they eventually integrate into the genome. Tihomorova observed spontaneous formation of multimeric structures a plasmid encompassingan “imin recombinant strains transformed with perfect HARS element”,but this element evoked some kind of “autonomous behavior” (L. Tihomorova, personal communication).In our standard proceduresfor the generationof recombinant H. polymorpha strains containing integrated DNA, cells transformed with HARScontaining (40-60 genvectors first undergo several passages in a selective medium erations) and a subsequent stabilization on a nonselective medium. The resulting strains bear the heterologous DNA integratedinto the genome, and no free plasmid canbe detected sincethe episomal DNA is selected out during the growth on nonselective media. Strains can be identified that contain up to 100 copies of the foreign DNA, exhibiting a head-totail arrangement. In the example presented in Fig. 6, single-copy integrants and recombinants with up to eight copies have been identified. A

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a b c d e f g h i k l

094 kb 083 kb-

Figure 6 Copy number determination of integrated heterologous DNA in H. polymorpha. The genomic DNA of an untransformed host strain and different recombinant isolates of a particular construct were digested with the restriction enzyme XhoI. The restrictedDNA was separated on 0.8% agarose gels, transferred to nitrocellulose and hybridized to a 32P-labeled fragment of the cloned FMD promoter sequence. The hybridization pattern reveals two signals in similar electrophoretic positions, one for the genuine single-copyFMD gene, the other for the slightly larger heterologous fusion.In dilutions of the various DNA samples, the signal intensity of the foreign DNA can be compared to the intrinsic singlecopy control. In the example presented in this figure, recombinant strains with one to eight copies of the integrated DNA have been identified. Left lane, size marker; lane 1, untransformed host strain; lanes a to k, different recombinant isolates. In the arrow-marked lane (b) a four-copy integrant could be identified; in (c), a twocopy integrant; and in (h), an eight-copy integrant.

gene dosage effect of gene expression was clearly apparent in recombinant H. polyrnorpha strains. For instance, product yields obtained from a hirudin-secreting strain with 20 copies werefound to be much higherthan those from strains harboring 5 and 10 copies of the expression cassette (12,109). In glucoamlyase-secretingstrains, however, maximal yields of 1.4 g/l were secreted from a four-copy strain and no further increase in productivity couldbe obtained by strains with higher copy numbers. The reason for this exceptionally weak gene dosage effect in this particular case is unknown (1 10). The vectors commonly used to generate recombinantP.pastoris strains are integrative plasmids. They contain a further 3’140x1 segmentin

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addition to that within the expression cassette. The plasmids are linearized at the boundary of this sequence, targetingthe linearized plasmidto the host’s AOXl gene via homologous recombination (see Fig. 4). In other cases, the heterologous DNA is targeted to the P. pastoris HIS4 gene (1 11). HIS4 integrants were found to be the less stable type of HIS4-selected transformants, since recombination can result in the excision of the expression cassette, leaving behind theHIS4 gene. In contrast to the situation in Hansenula, homologous recombination usually results in the integration of a single copy of the heterologous DNA. To increase the yield ofa productthat appears to be gene dose-dependent in this species(1 11,112),protocols have been worked out to obtain multicopy integrants similarto those in recombinantH. polymorpha strains. They are obtained either using vectors containing multiple expression cassettes or by screening for multiple integration events (see also the next section). In both species, Hansenulaand Pichia, the transformants bearing the multicopy integrantswere found to be mitotically stable. Stable multimeric integrants have been identified for a P. pastoris strain expressing Bordatella pertussis pertactin (1 13) or a tetanus toxin fragment C producing strain (110),which will be discussedin more detail inthe next H. polymorpha strains producing different section. Several recombinant secretory or intracellularly expressed proteins were tested for mitotic stability by isolating and analyzing the heterologous DNA before and after growth for an extended period of time. Tests after 40, 100,and 800 generations confirmed the high stability. Molecular analysis revealing the genetic stabilityof a particularH. polymorpha strain expressing hepatitis S antigen (HBsAg) is documented in Fig. 7. In P.pastoris, the modeof integration might influence the level of foreign gene expression.In particular, this has been shown for strains with disrupted genomicAOXl generated by the transplacement event during transformation. During induction theseaoxl transformants do not simultaneously produce high levelsof the AOX enzymeand the heterologous product. They are characterizedby a slower growthrate exhibiting a much lower 0, demand comparedto wild-type strains. In a comparative study, expression of the Bordatella pertussis pertactin was analyzed usingstrains of slow growth (methanol utilization slow; MutS)and wild-type strains (Mut+)as hosts. The MutSintegrant expressed the recombinant protein Mut+ strain at 10% of total cell protein in a fermenter cultivation, while the reached onlya 5% level (1 IO). Fermentation protocols have been worked types of recombinant out to match the specific demands of these two basic P. pastoris strains (see Section7).

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Figure 7 Mitotic stability of aH. polymorphs recombinant strain H415 expressing HBsAg during growth on a nonselective medium. The medium (YEP) was supplemented with glycerolas carbon source. Rounds of inoculations into new media were carried out with cells transferred from late log-phase cultures. Twelve individual colonieswere isolated beforeand after a 40-generation growth period.The respective DNA was isolated and characterized by Southern analysis. Sufi/SspIdigested samples were hydrized to a 32Plabeled probe derived from the plasmid used for transformation. The hydribization pattern confirms that the foreign DNA is integrated into the genome and reveals its stabilityafter growth. Left lane, undigested DNAfrom strain H415;I, restricted DNA isolated from colonies before growth and 11. after growth; pRBS 269, plasmid DNA used for transformation (from Ref. 114) as control.

6 RECOMBINANTPROTEINSEXPRESSED IN METHYLOTROPHIC YEASTS Both systems have been applied successfully for the productionof heterologous proteins duringthe recent past and are used commercially in ongoing developments. An increasing number of intracellularly produced and secreted proteins attest to the quality of methylotrophic hosts for a summary is preheterologous gene expression. In the following section sented focusing ona few selected. representative product examples obtained

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from the two species;further aspects and examples are discussed in more detail in the forthcoming sections. 6.1 RecombinantProteinsIntracellularlyExpressed in Methylotrophic Hosts The possibility of a stable multimeric integration makes H. polymorpha an ideal host for the expression of complex composite structures suchas polyvalent vaccines. This was demonstrated convincingly by recombinant P.polymorpha strains coexpressing and L S hepatitis B envelope proteins in a balanced predeterminedratio, thus mimicking the compositionof the natural viral coat (1 14,115). The envelope of the hepatitisB virus (HBV) is composed of the S-antigen as the major viral protein and two minor related proteins, the M- and L-antigens (116). S-antigen was expressed successfully inS. cerevisiae inthe early 1980s (117). Vaccines formulated with S. cerevisiae-derived hepatitisB surface antigens have been available commercially for several years(1 18,119). The recombinant vaccines consist of the S-antigen alone, which is assembled in vivo in the yeast host into highly immunogenic 22-nm particles similar to those found in the serum of hepatitis B patients. S-antigen-expressing strains have also been obtained for P.pastoris (120) and H . polymorpha (114,115) which were found to produce up to 0.4 and 0.5 to 0.6 g/L, respectively. Since additional protective epitopes are present in the L-protein which are absent in presently available recombinant vaccines (121), strains were constructed simultaneously expressing both theS- and L-antigens(114,115). The composite particles derived from such strains might provide the base for an improved vaccineor diagnostic probe. To construct such strains, the uracil-deficient H. polymorpha hostLR9 was transformed with a plasmid harboringan expression cassettefor the 3. Mitotically stable L-antigen analogousto the example presented in Fig. strains were obtained containing one or several copies integrated into the genome. Selected recombinant strains characterized by different L-antigen expression levels were transformed again with an S-antigen expression vector. This vector contained as a dominant selection marker the Tn5derived kanamycin resistance gene expressed from S.thecerevisiaeADHl promoter (130). Different strains were identified as producingboth proteins, with fixed L/S ratios ranging from 1 :8 to 1 :15, which correlates with the dose of integrated genes. The proteins producedwere assembled into particles, now containing both antigens in the ratios noted above (Fig. 8). During fermentation, the ratio remained constant due to the stable

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Figure 8 Analysisofrecombinant HBsAg particles (small hepatitis B virus surfaceantigen)producedin H. polymorpha. (A)Quantification of theexpressed antigens by Western blot analysis. Different recombinant strains were constructed with either a FMD- or a MOX-promoter controlled expression. Crude extracts from the various isolates were assayed from HBsAg content with specificmonoclonal antibodies comparing the immunological signal of the different samples with those from purified HBsAgstandards. Lanes 1 to 5, HBSAg standards containing 0.03, 0.07, 0.15, 0.3, and 0.6 pg of protein, respectively; lanes to 6 to 8, 3-, 6-, and 12-pg samples (total soluble protein) from strain H415 crude extracts (MOX-promotercontrolled expression of HBsAg); lanes9 to 11, 3-, 6 ,and 12-pg samples from strain H352 crude extracts (FMD-promoter-controlledexpression of HBsAg). (B)Identification of HBsAg particles by sucrose gradient centrifugation. Crude extract from strain H415 was centrifuged in a linearto20 50% sucrose gradient. After centrifugation the gradient fractions were assayed for antigen content by Western blot analysisas described above and by a particle-specific Auszyme assay. The peak indicatesthe efficient formation of 22-nm particles. (C)Electron micrograph of 22-nm HBsAg particles. HBsAg particles preparedfrom H. polymorpha strain H415 crude extract were depositedon a colloidon gridand stained with uranyl acetate. Magnification x 180,000. (From Reg. 114.)

integration of the heterologous genes within the host’s genome and the well-balanced functional promoters in both expression cassettes (1 14, 115). A potential subunit vaccine against tetanus, the tetanus toxin fragment C, was produced intracellularly inP.pastoris (1 11). Tetanus fragmentC is a 50-kDa polypeptide derived by papain cleavage from Clostridium the

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tetani toxin (122). The development of a recombinant production system might providea safe nontoxic alternative production process. The current vaccine consistsof formaldehyde-inactivated tetanus toxin, which has to be produced in a potentially hazardous process(123). For expression in P.pastoris, a plasmid was designed analogous to the example shown in Fig. 4. A synthetic sequence encoding the fragment C was insertedinto the cloning site ofthe expression cassette.The resulting vectorwas linearized for targeting into specific loci of the host’s genome. Using different strategies, plasmids were either targeted to transplace theAOXl or targeted to other chromosomal loci. By the first procedurethe genuine gene is disrupted and the original gene function is lost, resulting in slow methanol utilizing mutants (MutS). Alternatively,the DNA was linearized in such a way that the intactness of the AOXl function was not affected by the crossover event, leading to recombinant strains of the Mut+ type. In a third approach, the foreign gene was targeted to the HIS4 locus of the host (see also Section5). The expression of the proteinwas found to be independent of the site and mode of integration, contrastingthe finding for recombinant pertactin-producing strains described before. As in the H. polymorpha example above,an increase in the expression level correlates with the gene dosage. Multicopy integrants canbe obtained either by constructing vectors with several expression cassettesbyorscreening for multicopy integrants. They could be distinguished from transformations using DNA fragments targeted for single-copy transplacement. It is assumed that they arose by circularization of the transforming DNA followed by multiple integration into the chromosome via repeated singlecrossover recombination events(1 11). In scale-up experiments, yields of several grams per literof biologically active material could be obtained.

6.2 Recombinant Proteins Secreted from Methylotrophic Hosts Eukaryotic secretory proteinsare targeted to the secretory apparatus by N-terminal leader sequencesthat are removed from a precursor protein on entry into the endoplasmic retriculum(ER). For many proteins, passage through the secretory pathway is a prerequisite for proper folding by formation of S-S bridges and appropiate protein modifications such as glycosylation. For secretion of a recombinant protein, it is necessary to fuse the polypeptide to an N-terminal leader sequence that directs it to the secretory apparatus of the host cell. A variety of different leader sequences has been usedto drive secretion of heterologous proteins in yeasts. The most common elements in the traditional S. cerevisiae host are derived

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from genuine genes encoding secretory proteins: that for invertase (SUC2) (124), acid phosphatase (PH05)(125), and the a mating factor (MFal) (126). Leader sequences usually consist of some 20 amino acids and are composed of a hydrophobic core 6ofto 15 amino acids framed by hydrophilic amino acid residues. A theoretical model deduced from a compilation of protein sequences allows prediction of the processing(127). site In the case ofMFal, a two-step maturationof a prepro-polypeptide takes on entry into the ER (128) as stated place. First,a leader sequence is cleaved above. The remaining propeptideis further processed by the proteolytic activity of the KEX2 protease within the Golgi apparatus (129). In most constructs using this leader, the heterologous sequence is fused to this KEX2 recognition site. In the two methylotrophic hosts, fusions with these leader sequences and those from other eukaryotic sourceswere found to be faithfully processed, indicating a compatible mode of secretion and of cleavage atthe Lys-Arg ( K E X 2 ) processing. As a single exception, lack processing siteand inappropiate N-terminal extensions ofthe mature secreted protein have been reported for a prepro-MFdaprotinin fusion expressed in a P. pastoris host. It is assumed that structural properties of the aprotinin force the inaccurate removal of the preprosequence(130). In glycoproteins, oligosaccharidesare either N-linkedto an asparagine residue or 0-linked to a threonine or serine residue of the polypeptide chain. In N-glycosylation,a core consisting of nine mannose, two N-acetylglucosamine, and three glucose residuesis added to an asparagine within the ER. After subsequent removal of a mannose andthe glucose residues, further processing take place within the Golgi apparatus, resulting indifferent typesof oligosaccharide side chains. S. cerevisiae lacks Golgi mannosidases, by whichfurther mannose residues are removed in many other eukaryotic cells. This removal is necessaryto generate high-mannose or complex-type side chains typically found in animal cells. Instead, further mannose are added, resulting in heterogeneous side chains with up to 75 mannose residues (131). As a consequence, many heterologous proteins are overglycosylated (>40 mannose residues within a side chain) when secreted from a S. cerevisiae host (132). In methylotrophs, overglycosylation was not observed in many secreted recombinant proteins (see below). As exceptions, overglycosylation is reportedfor a recombinant aglactosidase secretedfrom a H. polymorpha host (133) and HIV gp 120 secreted from a P.pastoris host (C.A. Scorer, cited in Ref. 8). In the first example describing the production aofsecretory protein in H. polymorpha, the glucoamylase(GAM) gene from the amylolytic yeast Schwanniomyces occidentah (134) was expressed usingthe genuine leader

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for export. Forthis purpose a plasmid was constructed by inserting a fragment withthe entire coding region, including the leader sequenceinto the H. polymorpha expression vector. The heterologous gene was expressed from a FMD promoter. Again, transformation with the final construct resulted in several recombinant strains harboring different copy numbers of the expression cassette stably integrated into the genome. Strains with one to eight copies couldbe identified. A particular strain with four copies was analyzed in more detail.It was found to secrete 1.4g of biologically active enzymeat a cell mass of130 g dry weight per liter (see also Section 7). When the recombinant enzymewas compared with its S. occidentalis counterpart, identical N-termini were determined demonstrating a correct processing ofthe signal sequence. Comparative analysis of the sugar moieties revealedan identical extent ofN- and O-glycosylation of the two polypeptides, as shown in Fig.9 (1 10). No overglycosylationW& observed for invertase (see below) and L-antigens from the hepatitisB virus which are typically overglycosylatedin S. cerevisiae. Obviously, the expressed Lantigens enter the secretory pathwayto some extent. High-level secretion of 2.5 g/L was observed in recombinant P.pas(135). The invertase expression vector was toris strains secreting invertase constructed by ligating an S. cerevisiae invertase (SUC2) gene (124) sequence into the cloning site of the expression vector. The sequencewas composed of a suitable gene fragement and a synthetic oligonucleotide encompassing the N-terminal leader sequence designed accordingto the genuine gene. The resulting vector was targeted for transplacement into a Suc+ phenotype. the AOXl locus. The resulting transformants exhibited Again, an appropiate signal peptide cleavage was observed. Comparative electrophoretic analysis revealed differences between the S. cerevisiaederived enzymeand the recombinant product. Invertase from S. cerevisiae exhibited a highly variable ektent of glycosylation, yielding proteins ranging in size between1 0 0 and 1 4 0 kDa (136). In contrast, the P.pastoris-derived product appeared to be a more homogeneous protein with a molecular weight of some85 kDa. Deglycosylation ofboth species in both cases resulted in proteins with a molecular weight58ofkDa. A detailed analysis of the size distribution and the general structural features of the sugar moieties revealed that the invertase produced inPichia contained more than 85% of the N-linked oligosaccharides as structures in the size range of Maq,,GlcNAc+ Thus glycosylation of the recombinant invertase resembles closely the pattern observed in high-mannose oligosaccharides of glycoproteins in higher eukaryotes (137). When invertase was expressed in a Hansenufa host, a comparable type of glycosylation was observed (138).

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Figure 9 Glycosylation of recombinant glycoamylase expressed in H. polymorph. Glucoamylase (GAM) was isolated from the culture medium ofa recombinant H. polymorpha strain expressing the GAM gene from Schwanniomyces occidentalis. The isolated protein was deglycosylated withEndo H. Samples of the undigested and the deglycosylated enzyme wereseparated through 7.5% gels and visualized by staining with periodic acid-Schiff reagent for glycopeptide detection (153). The electrophortic mobility ofthe proteins was compared to thatof isolates from S. occidentalis. An identical extent of N-and O-glycosylation was found for both enzymes. Lane a, undigested heterologous GAM; lane b, deglycosylated recombinant GAM; lane c,deglycosylated GAM from S. occidentaliq lane d, undigested GAM from S. occidentalis. (from Ref. 110.)

Correct foldingand formation of S-S bridges in the appropriate position was clearly demonstrated for heterologous products secreted from both hosts. Examplesare recombinant aprotinin secreted from a P. pastoris host (130) or hirudin and miniproinsulin synthesized by recombinant H. polymorpha strains (U. Weydemann, Rhein Biotech GmbH, personal communication). Many proteins have been produced at high levels in the two methylotrophs. These include the foregoing examples of intracellular expression and the described secretory proteins. When the yields of specific heterologous proteins produced in different expression systems were compared, methylotrophs werefound to be at least as efficient as other yeasts or proved to be the superior hosts (6). The increasing number of

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heterologous compounds expressed in the two methylotrophic yeasts comprise a variety of proteins, including enzymes, hormones, anticlotting factors, and vaccines ranging in size from 7 to 150 kDa. A compilation of the different polypeptides produced the in two species is given in Table 1.

7 FERMENTATION An advantage of the exploitationof the two methylotrophsfor heterologous protein productionis the possibility to combine the control of gene Table 1 Production of Foreign Proteins in MethvlotroDhic Yeasts

Yeast

Protein

Promoter

Refs.

H. polymorpha

P-Lactamase" HBsAg Pre-S2-HBsAga Pre-SlS2-HBsAga cr-Galactosidaseb Glucoamylaseb Hirudinb Human serum albuminb Invertaseb Mini-proinsulinb

MOX FMD MOX FMD MOX MOX FMD MOX FMD FMD MOX MOX FMD

P.pastoris

P-Balactosidase HBsAg Tetanus toxin fragment C Pertactina

AOX AOX AOX AOX AOX AOX AOX AOX

138 114,115 153 114,115 132,152 110 12 151 138 U. Weydemann, personal communication 146 120 111 133 147 148 149 C. A. Scorer, cited in Ref. 8 135 143 112 150 130 Cited in Ref. 8

TNFe Streptokinase

sow

HIV g p 1208 Invertaseb Bovine lysozymeb Murine EGF6 Human EGI? Aprotininb Human serum albuminb aIntracellular expression. bSecretory Proteins.

AOX AOX AOX AOX AOX AOX

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expression by strong and stringent promoter elements with the favorable fermentation properties of these yeast strains at an industrialscale. These yeasts can growto extremely high cell densities(130 to 150 g dryweight per liter culture medium), excel by fast growth rates, and feed on cheap carbon sources such as glycerol and methanol. The useof glycerol is advantageous for a fermentation process since it results in a very limited level of ethanol formation. Ethanol negatively influences the production process of heterologous compounds in scale-up fermentations. This is found to be a problem in recombinantS. cerevisiae strains, which cannot use glycerol efficientlyand which often produce higher levels ofethanol in glucose-supplemented media(139). The commonly used culture media for methylotrophs (Table2) are based on simple synthetic components. They contain trace metal ions and adequate nitrogen sources which are required for efficient expression and cell yield (140,141) and do not contain proteins. Since thesecell strains normally do not secrete many proteins that may contaminate or even degrade heterologous products, they can easily be purified. High stability and lackof degradation was shownfor several proteins secreted fromH.a polymorpha host when stored in culture supernatants for a long period of time. In addition, no other major proteins or substances interfering Table 2 StandardMedium for H. polymorph# m4)ZS04

G&)PO, CaCl, MgSO, 7&0 NaCl FeCI, 6&0 CuSO, 5&0

ZnS04-7H,0 MnSO,.H,O Ni SO, 6H,O CoCb 6H,O

K1 %BO, mg/L N%MoO, d-Biotin Thiamine HCl

-

3.45 g/L 1.33 g/L 0.03 g/L 0.24 g/L 0.04 g/L 1.2 mg/L 1.2 mg/L 4.05 mg/L 0.5 mg/L 0.15 mg/L 0.15 mg/L 0.15 mg/L 0.15 0.15 mg/L 0.02 mg/L 2.7 mg/L

%Suitableamounts of a carbon source haveto be added.

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(12,110).

The stable integration of the foreign DNA adds further to the superior fermentation characteristics of the two yeasts. The production process is not impaired by plasmid instability or plasmid rearrangements observed in transformants with episomal heterologous DNA (142). Any nonselective medium can be used, and a medium can be designed or optimized to match the special requirements and demands of an economic production and downstream process fora product of choice. In the case of recombinant H. polymorpha strains, different fermentation modes can be distinguished. The mode selection depends on the promoter sequence used for heterologous gene expressionand the inherent control mechanism imposed by this structure (see previous sections)and may vary with the type of proteinto be expressed. Ina twocarbon-source mode, the recombinantstrain is first grown on high glycerol or glucose concentrations to a certain cell density, in some casesup to 80 to 100 g dry weight perliter. At the endof this growth phase, as the concentration of the first carbon source is depleted, methanolis added as1 to 1.5% (v/v) to the medium. Fifteento twenty hoursafter methanol addition, a strong induction of the methanol-inducible promoter is observed resulting in a massive expression of the heterologous compound. Maximal productivity is reached after 20 to 30 h. The total fermentation time for both phases depends on the strain, the culture medium,and on propertiesof the protein produced and varies between 70 and 120 h. This two-phase fermentation mode assures a high per cell productivity to duean optimal induction of the heterologous expression cassette. Both promoters,the MOX and F M . promoters, respectively, can serve as control elements and proas a repressive carbon source vide an efficient expression. The use of glucose during the first step of fermentation offers the possibility to express compounds that might interfere withcell growth orthat are potentially hazardous or toxic to the cell. The disadvantageof this two-step mode isthe necessity to change the carbon source during the production process. This requires careful controlof the carbon compound levels inthe culturemedium, but a highly reproducible process can be established. A possible mode of fermentation would beto perform the entirefermentation process on methanol. However, since the growth rate with methanol is low, it is advisableto grow the cells initially on another carbon source and to switch later to methanol for the production phase as described above. The second fermentation mode is based on the high activity level of the FMD promoter under derepressed growth conditions. In this process, glycerol is used as a sole energyand carbon source. This typeof

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fermentation startswith a feed of 1to 2% glycerol, resulting in rapid growth of the culture to high cell densities. Upon almost complete depletion of glycerol at an anticipated cell mass level, glycerol is maintainedat concentrations between 0.1 and 0.3% (w/v). Under these conditions both promoters are derepressed (see Section2). The advantage of this mode is the easycontrol of the process,and for some applications glycerol-grown cells were found to perform better than those grown on methanol. For instance, the cellsare bigger and the cell wallsare less rigid. Although the per cell productivityis 20 to 50% less than that observed inthe two-step fermentation mode, the economy of the process can be superior due to a shortened fermentation time and the possibility of reaching very high cell densities. This mode is especially advantageous in a FMD-promoterdriven production process. The activity of the FMD promoter reaches50 to 90% of the induced status under these conditions. maximal yields were found to be correlated with cell mass over a wide range when glucoamylase was expressed from a FMD promoter (110; see Fig. 10).

0.75

dry weight ( m

Figure 10 Correlationbetweenmaximalyields of secretedglucoamylaseand cell mass. Cells were grown in 3% glycerol (w/v) to different densities before being transferred to induction conditions inmethol. Growth continuedfor at least 24 h before yield determination. Filled circles represent yields routinely obtained in fermentations of recombinant strain harboring four copies of the heterologous expression cassette. (from Ref.110.)

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As an example, a one-step fermentation of a hirudin-secreting H. polymorpha strain is documented in Fig. 11 exhibiting some of the features stressed before. For fermentation studies, a recombinant strain was constructed harboring 20 copies of an expression cassette, stably integrated into the chromosome (12). Secretion was achieved by fusing the hirudin sequence to the S. cerevisiae MFal preprosequence. A productivity of to 110g dry weight. The HPCL 1.8 g/L was observedat a cell density of 80 analysis indicated perfect folding and proper processing of the signal sequence. Recombinant hirudinis especially susceptibleto proteolytic activity, by which the C-terminal amino acidsare removed to yield a shortened protein. This shortened form is typically recovered in high amounts when S. cerevisiae is used as an expression system. The highamount of Hansenula is due intact hirudin (more than 80% of the expressed protein) in to some extent to intrinsic properties of the methylotrophic host, but it was further improvedby a suitable medium composition (data not shown). In a modified version the of one-step fermentation mode, a feed becan initiated usinga mixture of glycerol and methanol as a carbonsource during the entire fermentation process. This type of process results in a superior per cell productivity, but the fermentation time is usually longer. Both promoters, MOXand FMD perform perfectlywell under these conditions. In Pichia pastoris, the control mechanism of the methanol metabolism pathway genes differs,as pointed out earlier. The AOXl promoter, which is homologous to the MOX promoter in Hansenula, is the only component commonly used for control of heterologous gene expression. Since strong derepression is not observed, fermentation and scale-up processes are established which closely resemble the two-step fermentation mode described for H. polymorpha. A convincing example is represented by recombinant P. pastoris strains expressing and efficiently assembling the

Figure 11 Fermentation of H. pobmorpha strain H-22 secreting a hirudin (F” promoter controlled expression). Strain H-22 was grown in a batch-fed 10-L fermentor on a semisynthetic nonselective medium supplemented with glycerol as carbon source. In the first growth phase, cells are cultured to high cell densities with a high-level supplementation of glycerol; the subsequent production phase is characterized by low levels of glycerol. The insert documentsHPLC analysis of the medium sampled at the end of fermentation. The undegraded 65-amino acid form of hirudin (peakH) constitutes some 80% of the secreted hirudin (back filled peaks inthe chromatogram) indicatinga low amount of products degraded at the C-terminus. For further details, see the text.

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hepatatis B surface antigen (HBsAg)(120). First the strains were grown in a defined minimal medium with glycerol as a carbon source. Upon depletion, methanol was added. Cells were harvested200 h after a shift to inductive conditions, and yields of 0.4 g/L were obtained at a cell density of 60 g dry weight per liter. This productivity was similarlyfound in scaleup fermentations at a 240-L scale when a similar mode of cultivation was applied (120). In another example, the fermentation conditions were optimized for recombinant P. pastoris strains expressing bovine lysozyme (143). The fermentation developmentwas carried out for strains of the MutS (disruption of the host’s AOXl gene by the recombination event)and the Mut+ (recombinants withan intact AOXl gene) type.A first approach employed a fermentation protocol comparable to that recombinant Muts strains. In described abovefor the HBsAg-producing strain, an induction phase required 175 h to yield maximal lysozyme levels of 250 mg/L at a cell density of60 g dryweight per liter. Foran attempt at improving productivity, the compositionof carbon compounds were altered duringthe induction phase. In a mixed feed of glycerol and methanol in a 4:l ratio (3.6g of glycerol per 0.9 g of MeOH per hour) a maximalproduct concentration of 180 mg/L was obtained within39 h at a cell density of82 g dryweight per liter. When the composition was altered to a 2:1 ratio, 290 mg/L was 85 g dry weight. In a modified version of yielded in43 h at a cell mass of the 2:l protocol, the MeOH feed was increased to a point where excess (nonlimiting) methanol beganto accumulate. This type of fermentation yielded an amount of 375 mg/L within 45 h at a cell density of 103 g dry weight per liter. Thus an up to fourfold increase in volumetric productivity (mg/L-h) over the original methanol feedwas observed, but the per cell yield (5.2 mg/g dry weight in the methanol feed) decreased, ranging from 4.0 mg/g dry weight (modified2:1 version) to 2.3 mg/g dry weight (4:1). In the second approach to shorten thetotal fermentation time, recombinant strainsof the Mut+ type were analyzed. Mut+ strains are sensitive to changes in the residual methanol level. Therefore, a three-step mode of fermentation was applied. First, the cultures were grown in glycerol excess. In a second step, a feed was initiated with lowamounts of glycerol. This provides afurther increase in cell massand allows that a weak derethe AOXl propression of the AOXl promoter (under these conditions, 2% of the induced status, as mentioned earlier moter activity reaches some in Section2). In a third phase, production is induced aby ramped methanol 450 mg of lysozyme per literat a feed. This fermentation protocol yielded

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cell mass of84 g/L. In scale-up fermentationsto a 10-Lvolume 560 mg/L was obtained within 30 h. Thus this type of optimization forced a 6.5fold increase in productivity relative to the original Mutsstrain. In this protocol, a smooth transition from the growth to the production phase is provided by the intermediate feed on limited glycerol concentrations. In this fermentation mode, a stringent control of medium components has to be obeyed. In contrast, this smooth transitionis readily obtained in Hansenula. Due to the high activity levels of methanol pathway genes under derepressed conditions,the simple one-step fermentation modeor modified versions of it can be applied as described before.

8 DIFFERENCES BETWEEN THE H. POLYMORPHA AND THE P. PASTORlS EXPRESSION SYSTEMS In the previous sections we documented the most promising performances of the two methylotrophic yeast speciesas hosts for heterologous gene expression. In both casesthe cells are shownto grow to very high densities (100 to 130 g dryweight per liter). Generally, these high densities are obtained within a short fermentation time(see Sections 5 and 7 ) , although some HBsAg-producing P. pastoris strains are characterized by a very long induction/production phase of 80 to 100 h (135). Integration of the heterologous sequences resulted in mitotically stable recombinant strains (see Section 5 andFig. 6).Both organisms followa similar control mechanism for the promoters of methanol utilization genes, which are exploited as control elements for the expressionof foreign genes (see Sections2, 3, and 5). Despite the obvious similarities, the two systems were shown to differ in the following aspects. Transformation inP.pastoris usually resultsin the integration of a single copy of the heterologous sequence. It is only recentlythat protocols have been workedout to obtain strains with multiple copies comparable to the situation in Hamenula, as described before for the tetanus toxin fragment C-producing strains. For expression in Pichia, foreign genes are fused to the AOXl promoter. In Hansenula, two strong methanol-inducible promoter structures are applied for heterologous gene expression: the MOX and FMD promoters. In Pichia, methanol is the only carbon compound that elicits high levels of methanol-inducible proteins. HanIn senula, expression of these proteins is also observed in glycerol-grown derepressed cells. This allows the elimination of a methanol induction step. FMD-promoter-driven production especially can be obtained in strains grown on a mixture of methanol and glycerol or on glycerol alone, en-

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abling a one-step fermentation and resulting in a high production rate. The differences betweenthe two systems are summarized in Table 3.

9 CONCLUDING REMARKS The capabilitiesof the methylotrophs presented in this chapterattest to the favorable characteristicsof these yeasts as expression systems for heterologous proteins. Their performance as hosts for recombinant protein production make them highly competitive in comparisonto other commonly used bacterial, yeast,and eukaryotic cell line systems.To a large extent they meetthe characteristics generally desiredfor an efficient industrial host, including rapid growth on low-cost media, stringent control of a reproducible production process, high expression levels ofthe desired product using both an intracellular and a secretory expression mode, proper folding, and appropriate modifications. Limitation may be encountered in cases where a complex mammalian type of N-linked glycosylation is indispensable.An appropriate glycosylationwas observed in several recombinant proteinsthat were found to be overglycosylated when secretedfrom a S. cerevisiae host. This indicatesthat the methylotrophs are able to express structures similarto the mammalian high-mannose type of N-linked glycosylation. The low amounts of genuine contaminating proteinsand other compounds provide for easy purification of secreted recombinant products. Since virus particles, pyrogens, pathogens, or cellderived components with an oncogenic potentialare not observed in these yeast species, reliaTable 3 Differences Among the Methylotrophic Yeasts H. polymorpha

P. pastoris

Mostly nonhomologous recombination Highcopy integration single-copy Mainly integration (up to 100 copies) MOX and FMD promoters as components for expressionplasmids FMD/MOX follow a repression/ derepression/induction mechanism mechanism One-step fermentationon glycerol or on glycerol/methanol possible glycerol Uraciland leucine-deficient

hosts

Homologous recombination AOX promoter as component for expressionplasmids AOX follows a repression/induction Two-step fermentation: growth on followed by induction with methanol Histidine-deficient host

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ble expression systemsare at hand that assure a high safetystandard for products considered for administration to humans. Ongoing risk assessment studies confirm the ecological safety of recombinants derived from H. polymorphs. In summary, the methylotrophs represent useful advanced alternatives for heterologous protein production and in many instances may provide the system of choice for the expression of a recombinant product.

ACKNOWLEDGMENTS We thank our co-workers J. Diirkop, H. Ervens, and P. Mackscheidt and our colleagues U. Weydemann, U. Dahlems, K. Melber, M. Piontek, P. Keup, F. Muller, and A. W.M. Strasser at Rhein Biotech GmbH, Diisseldorf for their valuable help and contributions to part of the experimental work reviewed in this chapter, and for their suggestions and support in finishing the manuscript.

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116. Heermann K, Goldmann U, Schwartz W, Seyffarth T, Baumgarten H, Gerlich W. Large surface proteins of hepatitis B virus containing the pre-S sequence. J Virol 1984; 52:396-402. 117. Valenzuela P, Medina A, Rutter WJ, Ammerer G, Hall BD. Synthesis and assembly of hepatitis B surface antigen particles in yeast. Nature 1982; 298: 347-350. 118. Emini EA, Ellis RW, Miller WJ, McAleer WJ, Scolnick EM, Gerety RJ. Production and analysis of recombinant hepatitis B vaccine.J Infect 1986; 13A:3-9. 119. Petre J, Wijnendaele van J, de Neys B, Conrath K, Opstal van 0, Hauser P, Rutgers T, Cabezon T, Capiau C, Harford N, de Wilde M, Stephenne J, Carr S, Hemling H, Swadesh J. Development of a hepatitis B vaccine from transformed yeast cells. Postgrad Med J 1987; 63(suppl2):73-81. 120. Cregg JM, Tschopp JF, Stillman C, Siege1 R, Akong M, Craig WS, Buckholz RC, Madden KR, KellarisPA, Davis GR, Smiley BL, CruzeJ, Torregossa R, VelicelebiG, Thill GP.High-level expression andefficient assembly of hepatitis B surface antigen in the methylotrophic yeast, Pichia pastoris. Bio/Technology 1987; 5:479-485. 121. Neurath A R , Jameson BA, Huima T. Hepatitis B virus proteins eliciting protective immunity. Microbiol Sci 1987; 4:45-51. 122. Helting TB, Zwisler0. Structure of tetanus toxin I. Breakdownof the molecule and descrimination between polypeptide fragments. J Biol Chem 1977; 252:187-193. 123. Helting TB, NauHH. Analysis ofthe immune responseto papain digestion products of tetanus toxin. Acta Pathol Microbiol Scand Sect C 1984; 92: 59-63. 124. Taussig R, Carlson M. Nucleotide sequence of the yeast SUC2 gene for invertase. Nucleic Acids Res 1983; 11:1943-1954. 125. Mayhack B, Bajwa W, Rudolph H, Hinnen A. Two yeast acid phosphatase structural genes are the result of a tandem duplication and show different degrees of homology in their promoter and coding sequences. EMBO J 1982; 1~675-680. 126. Brake AJ, Merryweather JP, Coit DC, Heberlein UA, Masiarz FR, Mullenbach GT, Urdea MS, ValenzuelaP, Barr PJ. a-factor-directed synthesis and secretion of mature foreign proteins inSaccharomyces cerevisiae.Proc Natl Acad Sci USA 1984; 81:4642-4646. 127. Heijne von G. A new method for predicting signal sequence cleavage sites. Nucleic Acids Res 1986; 14:4683-4690. 128. Waters MC, Evans EA, Blobel G. Prepro-a-factor has a cleavable signal sequence. J Biol Chem 1988; 263:6209-6214. 129. Julius D, Brake A, Blair L, KunisawaR, Thorner J. Isolation of the putative structural gene for thelysine-arginine-cleaving endopeptidase required for processing of yeast prepro-a-factor. Cell 1984; 37:1075-1083.

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130. Vedvick T, Buckholz RG, Engel M, Urcan M, Kinney J, Provow S, Siegel RS, Thill GP. High-level secretion of biologically active aprotinin from the yeast Pichiapastoris. J Ind Microbiol 1991; 7:197-202. 131. Kornfeld R, Kornfeld S. Assembly of asparagine-linked oligosaccharides. Annu Rev Biochem 1985; 54:631-664. 132. Innis MA. Glycosylation of heterologous proteins in Saccharomyces cerevisiae. In: Barr PJ, Brake AJ, Valenzuela P, eds. Yeast Genetic Engineering. Boston: Butterworth, 1989:233-246. 133. Fellinger AJ, Verbakel JA, Veale R A , Sudbery PE, Bom IJ, Overbeke N, Verrips CT. Expression of the a-galctosidase from Cyamopsk tetragonoIoba (guar) by Hansenulapolymorpha. Yeast 1991; 7:463-473. 134. Dohmen RJ, Strasser AWM, Dahlems U, Roggenkamp RO, Hollenberg CP. Cloning ofthe Schwanniomyces occidentalk gucoamylase gene( G A M ) and its expression in Saccharomyces cerevisiae. Gene 1990; 95:lll-121. 135. Tschopp JF, Sverlow G, Kosson R, Craig W, Grinna L. High-level secretion of glycosylated invertase inthe methylotrophic yeast, Pichia pastoris. Bio/Technology 1987; 5:1305-1308. 136. Ballou CE. Yeast cell wall and cell surface. In: Strathern JN, Jones EW, Broach JR, eds. Molecular Biology ofthe Yeast Saccharomyces:Metabolism and Gene Expression. Cold Spring Harbor Laboratory, 198:335-360. 137. Grinna LS, Tschopp JF. Size distribution and general structural features of N-liked oligosaccharides from the methylotrophic yeast, Pichiapastork. Yeast 1989; 5:107-115. 138. Janowicz ZA, Merckelbach A, Eckart M, Weydemann U, Roggenkamp RO, Hollenberg CP. Expression system basedon the methylotrophic yeast Hansenulapolymorpha. Yeast 1988; 4S:155. 139. Fiechter A, Fuhrmann GF, Kappeli C. Regulation of glucose metabolism in growing yeast cells. Adv Microbiol Physiol 1981; 22:123-183. 140. Egli T,Fiechter A. Theoretical analysis of media used in the growth of yeasts on methanol. J Gen Microbiol 1981; 111:137-144. 141. Egli T, Quayle JR. Influence of the carbon:nitrogen ratio of the growth medium on cellular compositionand the ability ofthe methylotrophic yeast Hansenula polymorpha to utilize mixed carbon sources. J Gen Microbiol 1986;132:1779-1788. 142. Da Silva NA, BaileyE.Influence of dilution rate and inductionof cloned gene expression in continuous fermentations of recombinant yeast. Biotechno1 Bioeng 1991; 37:318-324. 143. Brierley RA, Bussinau C, Kosson R, Melton A, Siegel RS. Fermentation development of recombinant Pichia pastoris expressing the heterologous gene: Bovine lysozyme. Ann NY Acad Sci 1990; 589:350-362. 144. Ciellissen G. Heterologous gene expression in C-l utilizing yeast. In: Murooka Y, Imanaka T, eds. Recombinant Microbesfor Industrial and Agricultural Applications. New York: Marcel Dekker, 1994:787-796.

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145. Sreekrishna K, Potenz RHB, Cruze JA, McCombie W R , Parker KA, Nelles

L, Mazzaferro PK, HoldenKA, Harrison RG, Wood PJ, Phelps DA, Hibbard C E , Fuke M. High level expression of heterologous proteins in methylotrophic yeast Pichiapastork. J Basic Microbiol 1988; 28:265-278. 146. Tschopp JF, Brust PF, Stillman CA, Gingeras TR. Expressionof the lacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucleic Acids Res 1987; 153859-3876. 147. Sreekrishna K, Nelles L, Potenz R, Cruze J, Mazzaferro P, Fish W, Motohiro F, Holden K, Phelps D, Wood P, Parker K. High-level expression, purification, and characterisation of recombinant human tumournecrosis factor synthesised in the methylotrophic yeastPichiapastoris. Biochemistry 1989; 28:4117-4125. 148. Hagenson MJ, Holden

KA, Parker KA, Wood PJ, Cruze JA, Fuke M, Hopkins TR, StromanDW. Expression of streptokinase in Pichiapastoris yeast. Enzyme Microb Techno1 1989; 11:650-656. 149. Thill GP,Davis GR, Stillman C, Holtz G , Brierley R, Engel M, Buckholz R, Kinney J, Provow S, Vedvick T, Siegel RS. Positiveand negative effects of multi-copy integrated expression vectorson protein expression in Pichia pastoris. In: Heslot H, Davies J, Florent J, Bobichon L, Durand G , Penasse L, eds. Proceedings of the 6th International Symposium on Genetics of Microorganisms, Vol. 11. Paris SocietC FrancaisedeMicrobiologie, 1990:477-490. 150. Siegel RS, Buckholz R, Thill GP. Production of epidermal growth factor

in Pichiapastorisyeast cells. European patent applicationEPA W090/106!37,

1989. 151. Hodgkins MA, Sudbery PE, Kerry-Williams S, Goodey A. Secretion of human serum albumin from Hansenula polymorpha. Yeast 1990; 63:435. 152. Sierkstra LN, Verbakel JMA, Verrips CT. Optimisation of a host/vector

system for heterologous gene expressionby Hansenula polymorpha. Curr Genet 1991;19:81-87. 153. Shen HS, Bastien L, Nguyen T, Fung M, Sliiaty SN. Synthesis and secretion of hepatitis middle surface antigen by the methylotrophic yeastHansenula polymorpha. Gene 1989,84:303-309. 154. Zacharius R M , Zell TE, Morrison JH, Woodlock JJ. Glycoprotein staining following electrophoresis on acrylamide gels. Anal Biochem 1969; 30: 148-152.

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5 Gene Expression in Filamentous Fungi Expression of Pectinases and Glucose Oxidase in Aspergillus niger Jaap Visser, Henk-Jan Bussink, and Cor Witteveen Wageningen Agricultural University, Wageningen, The Netherlands

1 INTRODUCTION Filamentous fungi are a diverse group of lower eukaryotes that can be isolated from a wide range ofhabitats. In nature many are found as soilborne saprophytes,which play an important role in biodegradationand elemental recycling. However, filamentous fungi have also found a variety of industrial applicationsas producers of fermented foods suchas cheese, soy sauce, tempeh, and beverages such as.sakC (l), of primary metabolites such as organic acids and vitamins (2) and secondary metabolites (antibiotics, alkaloids, gibberellins; see Refs. 3-9, and as excellent producers of a broad spectrum of extracellular enzymes(6-9). Furthermore, hyphal fungi are exploited in biotransformation processes that involve (stereo)specific modificationsof steroids or antibiotics (10,ll). Due to the high secretory capacityfor homologous enzymes, filamentous fungi may have a potential as hoststo produce foreign proteins. Amongthe fungi of industrial interestare various Aspergillus spp., such as A . niger, A . oryzae, A . sojae, A . awamori,Trichodermareesei,Penicilliumchrysogenum,

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and Acremonium chrysogenum(= Cephalosporium acromenium); Rhizomucor spp. such as R. miehhei; and Rhizopus spp. In agriculture hyphal fungi have an important negative impactas plant pathogens; at the same time there is increasing interest in this sectorto apply filamentous fungi for the biological control of plant pathogenic fungal or insect pests. Those speciesthat synthesize mycotoxins such as aflatoxins are also a potential danger for human healthby infecting food crops. Last but not least, as a resultof early genetic studies(12,13) some filamentous fungi (Neurospora crassa, Aspergillus nidulans)were found to be attractive models to investigate metabolic pathways, genetic control of metabolism, and fungal development and differentiation. The wide variety of carbon and nitrogen sources utilizedand relatively simpleadditional nutritional requirements, their growth underwide a range of external conditions, suchas pH, temperature, and oxygenation level, a short life cycle, and haploid genomesare important features of these systems. The speciesN. crassa and A . nidulans were also the first two hyphal fungi for which transformation was reported and for which manipulation of the genome was realized. In the case of N. crmsa, transformation was based on complementationof mutants by the cloned qa-2 gene (14), whereas for A . nidulans this was realized usingthe heterologous N. crassa pyr-4 gene (15) as well as homologous genes: amdS (16), trpC (17), and argB (18). However, this technology has progressed far beyond application to laboratory species and now includes a number of industriallyimportant fungi. Heterologous and homologous transformation systems have been developed for Aspergillus niger(see Section3. l), Penicillium chrysogenum (19-23), and for example, Trichoderma reesei(24-26). In the future, industrial strain breedingwill rely stronglyon detailed gene expression studies and genetic manipulationbut ideally should be combined with traditional approaches such as mutagenesis and recombination. A number of reviews covering molecular manipulation and gene expression in filamentous fungi, including heterologous gene expression, have appeared (27-42). These are complemented by some useful compilations on industrial fungal enzymes from Aspergillus (43) and those involved in lignocellulosedegradation (44)and in the Oriental food and beverage industries(45). We therefore limit ourselves and give only a brief and generaloutline of the current knowledge on fungal gene expression. Rather than becoming repetitive, thiscontribution will focus on two specific systems inA . niger that are biotechnologically relevantand illustrative. The first case deals with pectinase gene expression and its relevance

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for the production of this class of food-grade enzymes. The second case treats the production of a primary metabolite, gluconate, and includes an analysis of glucose oxidase gene expression. The production of glucose oxidase as such is also interesting, as this enzyme is used commercially for various applications,the most common onethat of a diagnostictool to determine glucose concentrations.

2 METABOLIC REGULATION AND FUNGAL GENE EXPRESSION Both in natureand in fermentation practice, hyphal fungi usually encounter complex substratesand there is preference inthe order by which the various nutrients are consumed. This requires optimaladaptation as reflected by inductionof appropriate uptake systems and catabolic enzyme activities as well as proper enzyme levels of anabolic and central pathnidulans, have ways. Biochemical and genetic studies, in particular A. with shown pathway-specific regulatory systems whichcontrol a set of genes This has been that have to be expressedto catabolize particular substrates. described, for example, for genes involved in the utilization nitrate of or the utilization of particular carbon sources, such as acetate, alcohol, or quinate, amino acids such as proline, purines, and in w-aminoacid, acetamide, and lactam catabolism, as reviewed byArst and Scazzacchio(46). Clustering of genes operating in one catabolic pathway not is uncommon in filamentous fungi but certainly is not a rule. Besides pathwayspecific regulation, broader forms of regulation exist that affect a large number of individual genes in different pathways. These wide-domain regulatory mechanisms mediate carbon catabolite and nitrogen metabolite repression and sulfur and phosporus control but also enablethe fungus to respondto changes in externalpH and level of oxygenation. Many genes are foundto be subject to more than one regulatory system. Loss of function mutations leading to noninducibility of all genes under control of the same regulator have beenfound for most of these regulatory genes, indicating that positive control mechanisms prevail. The isolation of mutations both in regulatorygenes and in some target genes, including mutations in the upstream regions of the target, has allowed the construction of detailed fine-structure maps by recombination and physical mapping. As molecular characterizationof these systems progresses, such mutations become valuableto analyze the functional domains inthe regulator protein encoded and to establish target sites in the promoter region of the genes regulated.

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The review by Arst and Scazzacchio (46)gives a very useful summary of the studies on the control of Aspergillus gene expression in a premolecular era. Detailed molecular studies onan increasing number of control functions are in progress. These involve, for example, the positive-acting regulatory gene areA mediating nitrogen metabolite repression (47-50), the negative-acting regulatory gene creA mediating carbon catabolite repression (51-53), and pathway-specific regulatory functions such as alcR (54-59), amdR (60-63),facB (64),prnA (65), nirA (66,67), and qutA (68) involved in inductionof enzymes in acetate, proline,and quinate catabolism. Also, some informative reviewson this topic have appeared (69-71). As these control systems operate in a very similar way in related fungi, this will certainly stimulate and expand gene expression to studies in fungi of industrial interest. Sequences or regionsin fungal promotersthat control the level of transcription by activation or repression can be identified in different ways. Among the approaches used are (a) computer homology searches as illustrated for the alcA/aldA genes of A. nidulans (72); (b) deletion analysis, often in combination with a reporter gene to monitor expression in vivo as used, for example, to identify functional elements in the promoter of the constitutive, highly expressedgpd gene of A. nidulans (73) or the inducible glaA gene of A. niger (74);and (c) DNAfobtprinting studies as used, for example, to identify the targetsof the qa-1F activator protein in theN. crassa qa gene cluster (75,76). The sequence requirementsfunin gal promoters that determine the transcriptional start point (tsp)and lead to basal levels of transcription (core promoter elements) arenot clearly established. Often more than one tsp is observed. Although CAATand found in many fungal TATA motifsas well as CT-rich regions have been genes, systematic functional analysis is largely lacking. The functional significance of TATA elements for core promoter function is not altogether clear, as these sites can be found at a variable distancefrom the tsp. Moreover, TATA boxesare also lacking in some fungal promoters: for example, in the A. nodulans argB gene (77), in the highly expressed triosephosphate isomerase(tpi)gene (78), and in the pyrivate kinase (pkiA) gene (79). CT box motifs have been described for a large number offungal genes, as summarized by Gurr et al.(80) and more recentlyby Unkles (41), and are often found immediately before the major tsp. Deletion analysis of the A. nodulans trpC promoter, also lacking atrue TATA element (81), and of the gpdA gene (73)and oliC gene (82) ofA. nidulans, has shownthe importance of the CT boxesto position transcription initiation. The T-rich region, approximately 30 bp of the tsp, which occurs in both the pkiA and tpi genes of A. nidulans, have been implicatedto

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serve this role(79). For more detailedinformation about this topic, some useful compilations shouldbe consulted (41,80,83).

3

DNA-MEDIATED TRANSFORMATION OF A. NIGER

3.1 SelectableMarkers in (Co)transformation Transformation in fungi has been reviewed several times (40,84-87), and therefore we focus hereon A. niger transformation. Several transformation systemsare available for this fungus, including systems based on homologousgenessuch as the orotidine-5’-phosphate-decarboxylasegene, designated pyrA (88) and pyrG (89) by different groups, and the nitrate reductase-encoding niaD gene (90).In these casesmutants that can serve as the transformation host can be isolated by resistance againstS-flUOrOorotic acid and chlorate, respectively. Transformation systems that do not require prior strain selection include those based on the semidominant A. niger oliC3 gene that encodes an oligomycin-resistant variant of the mitochondrial ATP synthase subunit9 (91), the heterologousA. nidulans acetamidase-encoding amdS gene (92), and an E. coli hygromycin B resistance gene (93). Genes to be introduced inthe fungal genome can be inserted ainvector that contains the selectable marker of choice. Physical linkage to the marker gene is, however, not required, as cotransformation of Aspergillus has been well established. When A. niger was cotransformed withapproximately equimolar mixtures of a plasmid containing the marker gene and a plasmid containing an unselected gene,rather low (80%). In our laboratory it is preferredto add the cotransforming plasmid inat least 20-fold excess of the plasmid containing thepyrA gene. In this way high cotransformation frequencies (>70%) are routinely achieved, while a significant fraction of the cotransformants show the high copy numbers ofthe unselected gene(95,96). In none of the pyrA/pkiA contransformants analyzed had integration occurred at the pkiA locus, whereas only a small fraction of the pyrA/pgaII contransformantsshowed integration at either thepyrA (approx. 10%) or pgaZZ (approx. 10%) loci. These results contrast with those obtained transby formation with a single plasmid containing the pyrA or pyrG gene, where integration occurs primarilyat the resident locus, frequently resulting in gene replacement (88,89). Analysis of niaD transformants alsoshowed that

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most integration events werea result of homologous recombination(97). Although the factors that determine the type of integration arenot fully understood, they may include the composition of the vector, the nature of the selectable marker, the genetic backgroundof the acceptor strain, and other factors, such as the amount of transforming DNA used (86).

3.2 Stability of TransformedStrains

As transforming DNA is integrated in theA. niger genome, transformants are generally considered to be mitotically stable. When they are grown in the absence of selection pressureand subsequently testedfor the transformed phenotype, there are usually no indications of instability (89,98). Transformants sometimes show tandem integration of multiple copiesof the plasmid withthe gene of interest, and this is of course often desired to increase the gene dosage effectively. However, because of the tandem duplication of sequences, this type of integration is potentially unstable (40,99). Plasmid DNA can be lost by intrachromosomal recombination, which is probably the reason why transforming DNA can be recovered from transformants in the absence of prior restriction of chromosomal DNA (88,100). Recently, selection systems that allow positive selection of reverted transformants have been usedto estimate the stabilityof A. niger niaD (90) and amdS (101) transformants and pyrA cotransformants (95). In all three cases the highest reversion frequencies observed were approximatelyThelatterstudyshowed that in a multicopy cotransformant, generally only part of the array of tandemly integrated plasmids was excised, suggestingthat reversion frequencies may underestimate actual instability in those multicopy transformants where each plasmidcarries the selectable marker. In a niaD transformant carrying a single plasmid copy integrated at the homologous locus,about 0.5% of the conidia (asexual spores) had lost the marker (102). These results stress the importance of proper strain maintenance to avoid degeneration of recombinant strains (103). It appears that multiple plasmidsoften integrate at a single chromosomal site in individual A. niger multiple-copy transformants. Tandem integration of several copies of a plasmid is usually detected as a strongly hybridizing band of the size of the plasmid on Southern blots of chromosomal DNA restricted withan enzyme that cuts the plasmid once. On such size cannot be predictedand which blots other bands are usually Seen whose may represent junction fragments that border the inserted DNA. When several of such bandsare observed, they are sometimes interpreted asto

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reflect plasmid integrationat multiple sites in the genome. However, they can also resultfrom rearrangements of the transforming DNA (104). Rearrangements were indeed observed in plasmids reclonedfrom an A . niger amdS transformant, and only one plasmid was recovered that contained A. niger chromosomal DNA, supporting the assumption that integration of multiple copies occurredat a single genomic site (105). Moreover, in multiple-copy amdS transformants the amdS insert behaved as a single genetic marker that in each individualtransformant could beassigned to a single linkage group (101). The inserts mapped in seven of the eight linkage groups known in A . niger. These transformants were also used to assign specific chromosomal bands an in electrophoretic karyotype of A . niger to linkage groups (106). Finkelstein et al. (107) also concluded that tandemly integrated glucoamylase gene clusters can behave as single genetic loci. After introduction of additional markers, different transformants were crossed usingthe parasexual cycle,and a class of progenywasobtainedthathadinheritedbothparentalgeneclusters.The glucoamylase yield in this recombinant class was significantly elevated relative to the two parental progeny classes. 4

EXPRESSION OF PECTINASES

4.1

PectinStructureandPectolyticEnzymes

Pectolytic enzymes are involved in degradation of pectin (Fig. l), a heteropolysaccharide with a backbone consisting mainly of (1 “4)-cr-D-gdaCturonan which is partially esterified with methanol. These enzymes have been reviewed several times (108-110). Pectin esteraseor pectin methylesterase (EC 3.1.1.11) hydrolyzes pectin, producing pectic acid (polygalacturonic acid) and methanol. The depolymerases split the glycosidic bonds of the D-galacturonan chain of their preferred substrates, pectate or pectin, either by hydrolysis (hydrolases) or by P-elimination (lyases) and are further classified on the basis of their modeof attack [i.e., random (endo) or terminal (exo) splitting ofthe glycosidic bonds]. They include endopolygalacturonase(EC 3.2.1.15), exopolygalacturonase @C 3.2.1.67), endopectate lyase(EC 4.2.2.2), exopectate lyase(EC4.2.2.9), and endopectin lyase (EC 4.2.2.10). Although the main chain of pectin consists mainly of homogalacturonan, it also contains some 1,Zlinked L-rhamnose residues. A novel enzyme, rhamnogalacturonase, which can split galactopyranosyluronic-rhamnopyranosyl linkages has been reported (1 11).

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* H'

H0

CH30H

pectin esterase

F-

0

PG polygalacturonase

H oH+0i i

0'

/

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-

pectin lyase

O

)

H0 H0

+ o

I1

-

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0

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H%H 0 0

i Figure 1 Chemical structureof a homogalacturonan segmentof pectin and mode of action of the three major fungal pectinolytic activities. This figure does not reflect the precise substrate requirements of each enzyme in terms of number of subsites involved or the precise pattern of esterification.

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The parent compound of pectin in intact immature plant tissueprois topectin, an insoluble substance located primarily inthe middle lamella that serves as the glue to hold cells togetherand in the cell walls. Pectic are heterosubstances, as they are extracted from various plant sources, geneous with respect to composition and molecular mass, dependingon the plant source and method of isolation. For example, more or less of the galacturonate residues may be acetylatedat the C-2 and C-3 positions. Apple pectin contains small xylose side chains and highly branched arabinogalactan side chains that are linked to relatively short segments of the rhamnogalacturonan backbone rich in L-rhamnose, the so-called hairy regions (112,113). These hairy regions are also found in other pectins, such as sugar beet pectin, in which a phenolic compound, ferulic acid, is ester-linked to the neutral sugar side chains(114). Degradation of pectin is thus a quite complex process, also involving activities other than those mentioned above.It has, however, been well establishedthat purifiedpectin-depolymerizing enzymes of the endo-splitting type can cause maceration (cell separation) of plant tissues (115). Also, treatment of isolated plant cell walls with endo-polygalacturonase increases the susceptibility to other plant cell wall-degrading enzymes(116). With regardto applications in plant biomass conversion, a synergistic action has been demonstrated for technical cellulolytic and pectolytic enzyme preparations derived from Trichoderma virideand A . niger, respectively (117). Pectolysis isan important phenomenon associated with many biological processes in which plant material is involved, and pectolytic enzymesare produced by plants, bacteria, fungi, yeasts,protozoa, insects, and nematodes. Microbial pectolytic enzymes have been studied extensively in relation to plant pathogenesis (1 18). The molecular biology of the bacterial enzymes, exploiting the expression of the correspondinggenes in Escherichia coli, which does not normally produce pectolytic enzymes, is well developed comparedto that of fungal pathogens.The cloned genes have been used to mutate chromosomal genes inorder to study the importance of the encoded proteins in pathogenesis(119). For example, it has been shown that with Agrobacterium tumefaciens,biovar 3 production of polygalacturonase is required for root decay in grapes, and that polygalacturonase contributesto, but is not essential for, tumor formation by this bacterium on grapes (120).

4.2 Application of Pectolytic Enzymes Only a few microorganisms are allowed for use in the food industry,which is probably the reasonwhy most of the commerical "pectinases" (indus-

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trial pectolytic enzyme preparations)are derived form A. niger. The applications of pectinases infruit and vegetable processing have been developed further from the original useof pectolytic enzymes for treatment of soft fruit to ensure high levels of juice and pigments upon pressing and for the clarificationof raw press juices and now also include juice recovery from apples, manufacture of pulpy nectars,and isolation of essential oils 109, 121, and 122). There isalso inand pigments (for reviews, see Refs. terest in the use of specific pectolytic enzymes to modify commercial pectins, which are used in food gels and beverages (123). Specific applications make different demands on the enzyme formulations to be used. For example, preparations with both polygalacturonase and pectin methylesterase may be usedfor clarification and depectinizationof juices, which enables concentrationand avoids gelling ofthe concentrates, whereas preparations free of pectin methylesterase and containing predominantly endopolygalacturonase are used for the production of pulpy nectars, which requires only limited pectin degradation, particularly in the middle lamella (maceration). However, commercial enzymepreparations are generally crude and are oftenjust precipitates ofthe fermentation liquor; thus there is a need for novel techniques to obtain more specific technical enzyme preparations and facilitate the development of new applications (121).

4.3 FermentationMedia The composition of the culture medium is a crucial factor in pectinase production (for reviews see Refs. 109 and 124). Pectic substances are genpecerally a constitutent of culture media. In industrial processes, crude tin-containing substancesare used for reasons of economy; media based on sugar beet, citrus peel, and apple pomace have been reported. With the use of complex media, a variety of enzymes other than pectolytic enzymes are produced, some of which may adversely affect the quality of the fruit product. For example, the presence of arabinofuranosidasein commercial enzymepreparations can cause hazeformation in juices such as appleand pear (125). The concentrationof the carbon source is an important factorfor pectinase production.Its optimization for a sugar beetbased medium has been described by Zetelaki (126). Readily metabolizable sugars such as sucrose and glucose may repress enzyme production (127that high enzyme yields can be 129). Laboratory experiments have shown obtainedusingwell-balancedconcentrationsofmixedcarbonsources, such as a combinationof glucose and pectin, the enzymes being produced when the sugar concentration inthe medium drops rapidly (109). Using a defined medium containing glucose, saccharose, and pectin, itwas found

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that production of polygalacturonaseby A. niger VTT-D-86267 was most efficient at 18°C and insignificantat 30°C (130). However, on a technical (beet extraction waste) medium containing less readily available nutrients, production of polygalacturonasewas considerably more efficientat 30°C than at 18°C. Other important factorsthat have been investigated include inoculum size,pH control, and aeration and agitation regimes (131-133). Culturing conditions promoting compact mycelial structure have been correlated with increased polygalacturonase production (134). In the fermentation industry pectolytic enzymes are also still frequently produced by surface culture processes. With regard to downstream processing of industrial polysaccharide-convertingenzymes byaffinity chromatography (135), A. niger endopolygalacturonase has been purified from an industrial pectolytic enzyme preparation using alginate as an affinity adsorbent in a fluidized-bed reactor (136). The substrate analog alginate, which is not degradedby pectolytic enzymes,was transformed into beads byCa2+ complexation.

4.4 Multiple Molecular Forms of PG and PL Many fungi areknown to produce multiple molecular forms or isoenzymes of endopolygalacturonase(1 10,137,138), but in general there is little information on thestructural differences between these enzymes. Different polygalacturonases from a single organism sometimes appear to be structurally very similar (139) and in this regard the most abundant A. niger polygalacturonase (PGII) isolated from a commercial pectinase was found to be heterogeneous with respect to its isoelectric point (140), although no other significant differences were detected betweenthe two most abundant forms of PGII, which are clearly the products of the same gene (our unpublished results). However, the second most abundant A. niger enzyme, PGI, is quite different fromPG11 inits physicochemical properties. It has a much lower specific activity and a different mode of action on oligomeric substrates. In addition to these polygalacturonases, Kester and Visser (140) purified three other polygalacturonases that resembled a very small PG1 in their physicochemical properties and represented only fraction of the total polygalacturonase activity of the pectinase. From PLI and the commercial A. niger pectinase Ultrazym, two pectin lyases, PLII, together responsiblefor 95% of the total pectin lyase activity, have been purified (141). Biochemically, PLI and PLII show marked differences: the K,,, value of PLI is much higher than the value found for PLII, and PLI has 9 or 10 subsites, whereas PLII has only 8. These enzymes are, however, similar with respect to molecular mass, isoelectric point,

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and amino acid composition,and they also show immunological crossreactivity.

4.5 CloningPectolyticEnzyme-EncodingGenes Many pectolytic enzyme-encoding genes have recently been cloned from Aspergillus. Dean and Timberlake (142) used antisera raised againstA. nidulans pectate lyaseto identify pectate lyase clones in a cDNA expression library. Several of these clones expressed pectate lyase activity E. in coli. The single-copy pectate lyase gene, designated pelA, was subsequently isolated from a cosmid bank. Targeted mutation ofthe gene in A. nidulans resulted in complete loss of pectate lyase activity, while growth on polygalacturonic acidand polygalacturonase activitywas unaffected by the pelA disruption. With A. niger, many of the pectolytic enzyme-encoding cDNAor genomic clones have been isolated using enzyme-specific oligonucleotide probe mixtures designed on the basis of known amino acid sequences. This approach was used to isolate the pectin lyase D-encoding pelD gene ofA. niger (143), a polygalacturonase gene of A. niger RH5344 (144,145),the PGII-encodingpgallgene of A. nigerN400 (146), the PGI-encodingpgal gene of A. niger N400 (147), and the pectin methyl esterase-encoding gene (pmeA) of A. niger RH5344 (148,149). Another five putative pectin lyase genes (pelA-C, pelE, and p e l 0 were isolated using the pelD gene as probe (150). Hybridization conditions of moderate stringency were employed: hybridization at 60°C in a standard buffer and with washes of two times standard saline citrate at 60°C.The product of thepelD gene, PLD, corresponds to PLI; thepelA gene, which hybridizes strongest withthepelD gene, encodesPLA that corresponds to the other major pectin lyase from Ultrazym, PLII. Similarly, five more polygalacturonase genes (pguA-E) were isolated from A. niger N400 using thepgallgene as probe (151). Furthermore, ithas been suggested that there are at least two distinct pectin methylesterase genes in A. niger RH5344, one of which has been clonedand characterized (149). Thus from molecular analysis the pectolytic enzyme system of A. niger appears to be even summary of the cloned more complexthan anticipated from other results. A genes is given in Table 1.

4.6 pel Genes and Aspergillus Taxonomy: An Intermezzo Different groups of investigators often use different wild-type A. niger strains and pectolytic-enzyme overproducing or other mutants thereof.

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As it was observed that two "A. niger" strains showed, for example, a somewhat different polygalacturonase isoenzyme pattern, we became interested in A . niger classification. The black aspergilli[Aspergillus sect. Nigri ( M ) ] have been studied frequently from a taxonomic viewpoint. Species inthis section appear to be closely related, and identification of individual species is difficult. In the species concept of the black aspergilli according to Al-Musallam (156), seven speciesare accepted, A . niger representing an aggregate ofsix varieties and two formae (e.g., A . niger var. awarnorl). Several blackAspergillus isolates were investigatedfurther by biochemical and molecular methods (157). Pectin lyase production was analyzed using Western blots, while Southern blots probed with thepeZD gene were usedto look for homology in these isolates with respect to their pectin lyase gene(s). In this way, Aall. niger isolates could easilybe identified, although their classificationwas different from the previous classification onthe basis of morphological characteristics. The other species of sect. Nigri (e.g., A . japonicus) differed in one or more aspects from the A . niger aggregate, except A . foetidus. The A . niger aggregate was then studied further by analyzing restriction fragment length polymorphisms, as detected in ribosomal bandingpatterns in ethidium bromidestained gels of chromosomal digests aswell as on Southern blots probed with several pectin lyase genes (158). The 23 A . niger isolates investigated were dividedinto two distinct groups, and wasit proposedthat these groups represent two different species,A . niger and A . tubigensis. With regard to biotechnology, the consequences of the divergence between A . niger and A . tubigensis depend on the system considered. In the case of the xylanase-encodingxInA gene isolated from an A . tubigensis strain that was previously designatedA . niger, a highly similar gene wasnot detected in A . niger N400, which is itselfa xylanolyticstrain (159). The polygalacturonase gene of A . niger RH5344 isolatedby Ruttkowski etal. (144) is as, the pgall almost identicalto, and encodes the same polygalacturonase gene ofA . tubigensis NW756 (160). This polygalacturonase shows, however, only 94% identical amino acids the in mature protein comparedto A . niger RH5344 polygalacPG11 of A . niger N400. The sequence of the turonase corresponds to that of a polygalacturonase isolatedfrom pectinase ROHAPECT 5DL (Rohm, Darmstadt, Germany) and that of A . niger N400 PG11corresponds to the sequence of a polygalacturonase isolated from Pectinase K2B 078 (Rapidase, Gist-brocades Seclin, France). Therefore, bothA . niger and A. tubigemis have been usedfor production of pectinases that are used in the food industry, and thus both fungi appear to have obtained GRAS status before they were recognized as two distinct species.

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4.7 Overproduction in Multicopy Transformants Several of the cloned A . niger pectolytic-enzyme genes have been used successfully to obtain overproduction of the corresponding enzymesby A. niger and have also been expressed in other Aspergillus species. Using the polygalacturonase geneof A. niger RH5344 situated on plasmid pPG A . niger RH5344 101, Ruttkowski et al. (161) achieved the best results with (untransformed strain: ca 20 to 30 PG units/mL; transformant number 7: ca. 240 to 270 PG units/mL). These authors also used this plasmid to transformA . awamori NRRL 31 12(untransformed strain:< 10 PG units/ mL;transformant number 12: ca. 120 to 150 PG units mL) andA. oryzae ATTC 11601(untransformed strain:c 10 PG units mL; transformant number 4:ca. 20 to 30 PG units/mL) to produce theA . niger polygalacturonase. In the cases of A . niger NRLL3 and A . awamori DSM5574, transformants constructed with theA . niger RH5344 polygalacturonase gene on plasmid pPG67/H24, a two- to sixfold-enhanced polygalacturonase activity relativeto the wild types wasreported, and the polygalacturonase appeared to be the major protein secreted by an A . awamori transformant subclonedpgallgene (145). A . niger N W strains cotransformed with the produced up to 50-fold-higher polygalacturonase activity than the untransformed strain (95). A somewhat earlier PG11 production was observed when it was expressedfrom a clone that contained only799 bp of upstream sequence, in comparison to the original clone, which contained 1356 bp of upstream sequence. A high overproduction of pectin lyase PLA by A. nigerpelA transformants has also been reported (150,162). A . niger Furthermore, productionof pectin methylesterase activity by two NRRL3 transformants was &fold higher than that by the wild-type strain in the presenceof pectin inthe medium (149). Thesetransformants were obtained using the A. niger RH5344 pmeA gene with only 276 nucleotides of 5' flanking region, containing typical CAAT box, TATAA box, and CT motifs. Maximum production was obtained in strains that had the highestpmeA gene copy numberand mRNA levels.It is thus evident that the main pectolytic enzymes can be overproduced in a simple way, by increasing the copy numbers of the corresponding genes. The A.niger pectin lyasesand polygalacturonases are encoded by gene families and it is therefore important to obtain information regarding synthesis of the individual enzymes. Several relevant factors have already been identified. Pectin lyase PLBproduction by A. nigerpelB transformants was observed following only a shift to fresh medium (163). PLB detectable at 8 h after the shift disappearedfrom the medium thereafter. When the concentration of monobasic potassium phosphate in the culture

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medium was increased 10-fold (from 1.5 g/L to 15 g/L), the shift was no longer requiredto detect PLB,and its concentration inthe medium now did not decrease after 8 h following the shift. The important point to be made here isthat the levels ofthe other pectin lyase, PLA, were not greatly influenced by the phosphate concentration, as opposed to the situation with PLB. These investigators suggested that the higher PLB production seen in the medium with the higher phosphate concentration may be due to reduced activityof proteases that degrade PLB, as relatedto a higher pH observed in the medium of higher buffer strength. Also, Archer et al. (164) have shown improved hen egg-white lysozyme secretion A.in niger when the medium is supplemented with 50 to 100 mMsodium phosphate buffer, allowing a drop in pH to about 4.0, compared to 2.0 for unbuffered medium. However, it was unclear whether this was due to a pH effect, since similar results were obtained with sodium chloride, suggesting that itwas more likelyto be due to a “salting” or osmotic effect (165). The nitrogen source usedcan also have different effects on the levels of the individual enzymes. PGI-overproducingA. nigerpgaI transformants were also obtained, but like the untransformed strain, these produced very little PG1on a medium optimizedfor the productionof PGII, (i.e., with ammonium chloride instead of urea as the nitrogen source, as used for PG1 production, and pectin and sugar beet pulp as the carbon source) (Fig. 2). As one might expect,the complex carbon source constitutes another factor that influences the isoenzyme spectrum. For example,PG11 the was found as the major activity when sugar beet pulp was used ascarbon source, whereasa brown-band pectin favored PG1 production (160). It is noted that isoelectric focusing followed by polygalacturonase activity staining can be quite useful to visualize the distinct activities (166). In the literature concerning process development, however, there is unfortunately little information regarding the abundancy of individual polygalacturonases and pectin lyases.

4.8 ExpressionwithReducedContaminating Pectolytic Activities The roleof the individual pectin lyases and polygalacturonases inthe degradation of plant cell walls isnot understood and has been difficult to address, as this requiressubstantial amounts of enzyme free of contaminating pectolytic activities. To obtain such enzymes, two strategies are being used inour laboratory. The first takes advantage of a strong glycolytic A. niger promoter, that of the pyruvate kinase-encodingpkiA gene

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3

Figure 2 Western blot analysisof the effect of the nitrogen source on PG1 levels produced by A. niger using a polyclonal antibody raised against PGI. Culture media were obtained from A. niger N402 (wild type, lane 1) and from A. niger N593-pGW1900 (apgal multicopy transformant, lanes 2 and3) which were grown for 48 h on 1070 pectin and 1070 sugar beet pulp as carbon source and with urea (4.0 g/L) (lanes 1 and 2) or ammonium chloride (4.0 g/L) (lane 3) as nitrogen source. A medium lowin potassium phosphate(1.5 g&) was used. (From Ref. 147.)

(96). Pyruvate kinasewas reported to constitute at least 30% of the total protein contentof an A. nigerpkiA transformant. Another usefulproperty of this promoteris that full promoter activity can be obtained using, for example, glucose asthe carbon source, on which A. niger N400 does not produce significant pectolytic activity. The construction of a pkiApelB fusion geneand its usefor PLB production has been reported(163). The second strategy, expression of A. niger pectolytic-enzyme genes in A. nidulans under the control of their own promoters, has also proved successful, although the reasonsfor this are not fully understood.In the cases of A. nidulanspelA and pgaZZ multicopy transformants, production of the corresponding enzymes at similar levels was observed, whether these strainswere grownon a medium with glucoseor on an inducing medium with pectinhugar beet pulp as thecarbon source (151,162). This is a general characteristicof these A. nidulans transformants, although it was not seen with A. niger, in the acceptor strain as well as in transformants. In the latter fungus, expression of thepelA andpgaZIgenes is strongly regulated bythe carbon sourceat the level of transcription. A. nidulans is itself a pectolytic organism in whichthe regulation of the production

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of pectolytic activities in response to the carbon source used (142,167) appears, by and large, to be quite similar to that of pectolytic A . niger enzymes. The A . niger pelA and pgaZZ genes inA . nidulans transformants must have escaped,at least inpart, from theA . nidulans regulatory mechanism involved inthe expression of its own pectolytic-enzyme genes. The pelB andpgazgenes have also been expressed successfully A.in nidulans, although in these cases significantproduction of PLB and PG1 was observed onlyon pectinhgar beet pulp-containing growth media. Expression of the pgal gene in A . nidulans remains quite attractive, however. Transformants were obtained that secreted PG1 asthe major protein in the culture medium (approximately 1 g/L in batch culture), producing about 700-fold-higher polygalacturonase activity than untransformed A . nidulans itself and also much higher activities than observed for A . niger transformants (Fig. 3). Increasing the phosphate concentration of the culture medium had a major positive effect on heterologous polygalacturonase production by A . nidulans. A . nidulans was also used to identify the polygalacturonases encoded by those A . niger polygalacturonase genes isolated by heterologous hybridization (151). This funguswas used as the host since it was expected that detection of these polygalacturonases, following the introduction of the genesinto the A . niger genome, wouldnot be straightforward. With regard to the pgaA, pgaB, pgaD,and pgaE genes, cotransformants were constructed using phage DNA without prior subcloningof these genes. as These A . nidulans transformants produced novel polygalacturonases, indicated by Western-blot analysis using a polyclonal antibody raised against PG1and in most cases alsoby activity measurements. One may expect that these lessabundant A . niger polygalacturonases are produced at very high levels byA . nidulans following the subcloning of the genes in plasmid vectorsand the construction of multicopy transformants. This is indicated by the caseof the pgaC gene in whichstrains producing about 1 g/L of PGC in batch culture have already been obtained (Fig. 3). To the best of our knowledge, this polygalacturonase had not been described previously. 4.9 HomologyinPolygalacturonaseGeneStructure

To date three polygalacturonasegenes have been sequenced from a single organism: the pga1, pgaZZ, and pgaC genes ofA . niger N400.These genes have diverged significantly in that, for example, PGI, PGII, and PGC A similar show 56 to 61% amino acid identity between the mature proteins.

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-

Pi

L

I L

I H

-

c

H

H

259

Figure 3 High-level expression of the A. niger pguZ and pguC genes in A. niduluns proteins in the culture medium A. of nidulunstransformants grown for46 h on 1% pectin and 1% sugar beet pulp medium with a low (L: 1.5 g/L) or a high(H: 15 g/L) phosphateconcentrationwereseparatedona 10% SDS-PAGE geland stainedwithCoomassieBrilliantBlue.Ammoniumchloride (4.0 g/L)was used as nitrogen source. Shown from left to right are: lane 1, A. niduluns pyrA transformant (control); lane 2, pguZ transformant number G191 (pGW1900)6; lane 3, as lane 2; lane 4, as lane 1; lane 5, pguC transformant numberG191 (pGW1910)3. (From Ref. 151.)

degree of homology is observed when these sequencesare compared to the polygalacturonase of C. carbonurn (see Fig. 4). The PG1 sequence is in an intermediate position with respect to the PG11 and PGC sequences. The N-terminal amino acid sequencesthe of A. niger polygalacturonases display characteristic amino acid insertions or deletions that arealso observed in polygalacturonases of phytopathogenic fungi; in this respect the most abundant polygalacturonase of Sclerotinia sclerotiorurn (168) resembles PGC and that of Colletotrichurn lindernuthianurn (168), like the C. carbonurn polygalacturonase, resemblesPGII. The threeA. niger polygalacturonases contain small N-linked glycans, as indicated by enzymic deglycosylation. The conserved N-glycosylation site is thus considered a functional site, but isit not present in the C. carbonurn polygalacturonase, which is itself a glycoprotein. Endopolygalacturonase is thought to act by general acid catalysis. The effect of pH and temperatureon polygalacturonase activity suggested the protonated imidazole groupa histidine of (169). The presence of an essential histidine to be the general acid catalyst

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PGC PG1 PG1 PG

PGC PG PG1 PG

41 32 I28 28

91

I 79 I74 74

C

ATTCTFSGSEGASKASKSKTSCSTIYLSDVAVPSGTTLDLSDLNDGTHVI ASTCTFT---SASEASESISSCSDWLSSIEVPAGETLDLSDAADGSTIT DS-CTFT---TAAAAKAGKAKCSTITLNNIEVPAGTTLDLTGLTSGTKVI DG-CTFT---DAATAIKNKASCSNIVISGMPAGTTLDLTGLKSGATVT

..

................

***.

................

FQGETTFGYEEWEGPLVRVSGTDIWEGESDAVLNGDGSRWWDGEGGNGG FEGTTSFGYKEWKGPLIRFGGKDLTVTMADGAVIDGDGSRWWDSKGTNGG FEGTTTFQYEEWAGPLISMSGEHITVTGASGHLINCDGARWWDGKGTSGFQGTTTFGYKEWEGPLISVSGTNIKVVGASGHTIDAAGQKWWDGKGSNGG

...........

***.. .*....* . . . . . . * . * * * . . * . . *

PGC PG1 PG11 PG

14 128 12 124

PGC PG1 PG11 PG

191 178 172 174

DTDDLAANTDGFDIGESTYITITGAEIYNQDDCVAINSGENIYFSAVCS DDNG-GHNTDGFDISESTGVYISGATVKNQDDCIAINSGESISFTGGTCS DTQG-GHNTDAFDVGNSVGVNIIKPWVHNQDDCLAVNSGENIWFTGGTCI DSAG-AHNTDAFDIGSSSGITISNANIKNQDDCVAINSGSDIHVTNCQCS

PGC PG1 PG1I PG

241 227 221 223

GGHGLSIGSVGGRDDNTVKNVTFKSQQAIRIKTIYGDTGSVSEV

PGC PG1 PG11 PG

291 277 271 273

TYHEIAFSDATDYGIVIEQNYDDTSKT--PTTGVPITDFVLENIVGTCED TYSNIQLSGITDYGIVIEQDYENGSPTGTPSTGIPITDVTVDGVTGTLED

PGC PG1 PG11 PG

339 327 321 323

DDCTEVYIACGDGSCSDWThTGVSVTGGSVSDDCLNVPSGISCDL DA-TQVYILCGDGSCSDWTWSGVDLSGGKTSDKCENVPSGASC-GA-TEIYLLCGSGSCSDWTWDDVKVTGGKKSTACKNFPSVAC--

1 KTKPKFFYAHDLTSSTIKSIYIENSPVQVFSIDGSTDLTMTDITVDNTDG

KTKPKFMYIHDVEDSTFKGINIKNTPVQAISVQA-TNVHLNDFTIDNSDG 3 KKKPKFFYAHGLDSSSITGLNIKNTPLMAFSVQA-NDITFTDVTINNADG KTKPKFFYAHSLTTSSISGLNIKNTPVQAFSINGVTGLTLDRITIDNSAG

...................

*.*.*.

v

.................. VTC

............ .

*... .

*

v

140 128 122 123

190 177 171 173

C

.. *

*****.*.***"*

240 226 220 222

#

GGHGLSIGSVGGRDDNTVKNVTISDSTVSNSANGVRIKTIYKETGDVSEI GGHGLSIGSVGDRSNIEHSTVSNSENAVRIKTISGATGSVSEI GGHGVSIGSVGGRKSGTTIANSDNGVRIKTISGATGSVSDI

......................

..

.

.*....*****

.**.*+..

TYSNIVMSGISDYGVVIQQDYEDGKPTGKP~GVTIQDVKLESVTGSVDS

TYENITLKNIAKYGIVIEQDYLNGGPTGKPTTGVPITGVTLKNVAGSVTG

** .*

90 78 73 73

. . . . .**.**.*.* .. c

c

*..*..* . . . . . . . . . . C

* * * * * * * . * . * * * * * * * * **** * * **

338 326 320 322

C

SG-TEIYVLCGKGSCSGWNWSGVSITGGKKSSSCLNVPSGSC--

.. *"*.

290 276 270 272

**

383 368 362 364

Figure 4 Comparison of the deduced amino acid sequences of PGC, PGI, PGII, and the PG of Cochliobolus carbonum(PG). Where alignment has been improved by the introductionof insertions, thisis marked bydashes, conserved amino acids by asterisks, and conservative substitutions by points. Possible catalytic residues (V), conserves cysteines(c), and the conserved N-glycosylation site in the A. niger PGs (#) are indicated.

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is further indicated by chemical modification studies(170,171). The pH effect on polygalacturonase activityand, more recently, chemical modification data (169) also indicatedan essential (ionized) carboxylate group. Upon multiple sequence alignment of fungal,plant, and bacterial polygalacturonases, the regionof highest similarity is only short (seven conserved amino acids)and contains a histidine, possibly the catalytic residue (160,173). On the basis of such sequence alignments, several acidic amino acids that could correspondto a catalytic residue have also been suggested. As expected for secreted proteins,PGII, PGI, and PGC are synthesized as precursors withan N-terminal extension.The start of the mature proteins is, however, not immediately preceded by a likely signal peptidase cleavage site(174), and therefore it is likelythat these polygalacturonases are synthesized as prepro-proteins which are first cleaved bya signal peptidase and subsequently by another processing enzyme (see below). Another striking feature of the three sequenced A. niger polygalacturonase genes is the diverged intron/exon organization, which can be explained bythe gain or loss of two single introns (Fig. 5). An intron corC.carbonurn polygalacturonase gene responding to the only intron of the is not found in any of the threeA. niger genes. From comparison of polygalacturonase genes,A. niger pectin lyase genes(163), and several homologous A. nidulans and Neurospora crassa genes (175) it appearsthat in the evolution of the filamentous Ascomycetes and related fungi imperfecti, introns have been acquired or lost rather freely. 4.10 StrainBreeding

Considerable progress has thus been made in cloning of the genes that encode pectolytic activities, and their expression to obtain overproduction of pectolytic enzymes,or production by other hosts, or production under novel favorable fermentation conditions. Strain breeding by molecular means holdsthe promise of rather straightforward manipulation of the pectolytic enzyme spectrum, in comparison to current breeding strategies employing multistep mutagenesis and extensive screening for desired strains (176). As a prerequisitefor future applications, much more needs to be knownabout the activities of the distinct minor pectin lyases and polygalacturonases. Moreover, this should providefurther clues regarding the importance of the A. niger pectin lyaseand polygalacturonase gene families as relatedto the saprophytic lifestyleof the fungus. It can be expected that polygalacturonase-encoding gene familieswill be found in other fungi(177), although the maize pathogen,Cochliobolus carbonurn,

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-

similarity 56 61 % 21 + 6

PGll R

I

*

I

*

18+13

PGI KK

I

I

*

I

Figure 5 Schematic representationof the PGII, PGI, and PGC proteins of A. niger, indicating

the putative processing sites for the signal peptide and the monobasic and dibasic processing sites for the propeptide. The position of the introns in the corresponding genes has been indicated by a bar. probably has a single endopolygalacturonase-encodinggene (173,178). In this connection it may be of relevancethat monocotyledonous plants (e.g., maize) contain less pectin that dicotyledons, which appears to be reflected by the differences in the spectra of cell-wall-degrading enzymes produced by the pathogens of the respective host plants (179). Furthermore, there is still little information on the regulation of A . niger pectolytic-enzyme genes,due to the lackof specific and well-characterized mutants which can now be made by gene disruption. The cloned genes clearly provide the opportunity to study their own regulation in Aspergillus. Another matter of interest is the structure of these pectolytic-enzyme genes in its own right as well as regarding the possible use of sequences derived from these genes in expression or secretion vectors. We therefore extend the discussion brieflyto gene regulation and proteolytic processing aspects.

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5 GENE REGULATION When compared to the situation in A. nidulans (see Section 1) there is still little known about gene regulation in industrial fungi. As there are important industrial applications of Aspergillus glucoamylases and aamylases in starch solubilizationand saccharification inthe brewing and food-processing industries, it not is surprising that their genesare among those best characterized(45). Furthermore, these highly expressed genes have been used to develop efficient expression systemsfor foreign proteins, including bovine chymosin production using the A. niger glucoamylase gene (180). The fungal Rhizomucor miehei aspartic proteinase was secreted in excess of3 g/L by A. oryzae when expressedfrom an A. oryzae a-amylase promoter(1 81). Aspergillus glucoamylase and a-amylase genes are strongly regulated in response to the carbon source used (182,183). Malto-oligosaccharides derivedfrom starch, such as maltose, induce the formationof a-amylase inA. oryzae, and it has been suggested that this is dueto inducers of the a-glucosyl glucose configuration.Isomaltose (6-O-a-D-glucopyranosyl-D-glucose) is a stronger inducer than itself, and is produced by maltose (4-O-a-~-glucopyranosyl-~-glucose) an A. oryzae transglucosidase activity(184). Xylose represses glucoamylase production in A. niger when starch is used to induce glucoamylase synthesis, but if maltose is used, theaddition of xylose has little effect on glucoamylase production(185).As a possible explanation, was it suggested that xylose may repressthe production of an enzyme that releases a product from starch that induces high-level glucoamylase synthesis. Deletion analyses of the glucoamylase-encodingglaA gene of A. niger (185), the a-amylase (Taka-amylase A) encoding amyl? gene of A. oryzue (186), and the glucoamylase-encodingglaA gene of A. oryzae (187)have been reported. In the case of the A. niger glaAgene, truncated versions of the glaA gene with deletions extending into the 5’ sequences were reintroduced ina glddeleted host, whereas thetruncated A. oryzae genes were analyzed using a &glucuronidase (GUS) reporter gene.A number oftransformants of each construct, differing with respect to both the sites of integration and copy numbers, were assayed for glucoamylase/P-glucuronidase production. With all three genes, a large reduction inenzyme pro-562 duction was observed upon deleting specific upstream regions [from to -318 in A. niger glA, from -377to -290in A. oryzae amyl?, from -247 to -348in A. oryzae glaA (positions referto the start codon)]. These regions are required for high level expression (and starch induction), whereas sequences further downstream appearto be sufficientfor low-level tran-

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scription. Furthermore,a comparison of the nucleotide sequences of these promoters showed two homologous regions, ofone which isfound in the regions required for high-level expression as defined by the unidirectional three deletion analyses(187). The other occurs farther downstream, in all genes at a similar position,starting at 229 to 201 before thestart codon. Specific deletion of these regions from the A. oryzae glaA gene indicated that both are essential for high-level expression on maltose. Using an approach similar to that described above, a putative regulatory element in A. niger polygalacturonase genes has been identified. Deletion analysis ofthepgaII gene showedthat a sequence between positions -799 and -576 is required for high-level gene expression(95). A search for DNA sequences inthis region revealed a the presence of yeast regulatory sequence of high homology with the well-characterized upstream activation site UAS2 of the S. cerevisiae CYCI gene (Fig. 6). Homologous sequences are present in the pgaI and pgaC genes, in the reverse orientation and closerto the putativeTATA boxes. A similar sequencewas, however, not observed inA . niger pectin lyaseencoding genes. The consensus sequence derived from the A. niger polygalacturonase genesthat includes a CCAAT sequence onthe opposite strand is an extended versionof the consensus sequence derivedfrom yeast genes that are under the control of the HAP2/3/4 system [see Forsburg and Guarente (188) for a review

(UAS2UP1) CYCl pgaII (N400)

-639

G A G C G T T G A T T G G T G G A T C A A G C + * + * * * + + + + * + + + * t * + * G A C C G T T C A T T G G T G G A A C T A G C

pgaII (NW756)

-632

A A T C A T T C A T T G G T G G A A T T T G C

-610

-206

C C T C C A T T A T T G G T G G A A A G A C C

-228

-359

A G G T A A T C A T T G G T G G A G A T A A C

-381

pga

consensus

yeast consensus

-617

T Y A T T G G T G G A T N A T T G G T

Figure 6 Conserved sequencein the promoter of the A.niger polygalacturonase genes p g d , pgaII, and pgaC and its similarity to a yeast upstream activation site in the CYCl gene, which is activated bythe HAP2/3/4 regulatory complex. The asterisks indicatethe matches between the yeastUAS2 with the UP1 point mutation and theA. niger N4OOpgaII gene. The secondpgaII sequence is fromA. tubigensis NW756. The positions of the 5' start (on the left) and the 3' end (on the right) of the sequences are given with respectto the translation initiation codon and also indicate the orientation of the sequences. (From Ref. 151.)

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and Bowman et al. (189) for a recent compilation]. The heteromeric activation complex bound at UAS2 ofthe CYCZ gene consists ofat least three proteins, HAP2, HAP3, and HAP4. This regulatory complexis similar to the mammalian heteromeric CCAAT-bindingtranscription factors in that the DNA-binding properties ofa factor from HeLa cells, CP1, and of the yeast complexare virtually identical. Subunits of CPl and HAPY HAP3 are also functionally interchangeable in virtro (190). The HAP2/3/4 system activates the genes under its control when cells are shifted from glucose to a nonfermentable carbon source and, interestingly, levels of HAP4 RNA are also regulated by carbon catabolite repression (191). At first sight the similarity of the putative regulatory region in the A . niger polygalacturonase genes and upstream activation sites in yeast genes is somewhat puzzling, sincethe latter (nuclear) genes often encode (mitochondrial) proteins involved in respiratory chain functioning, or a subunit of a citric acidcycle enzyme. However, thecarbon source glycerol is known to promote HAP2/3/4mediated gene expression in yeast (189), and it is also considered an intermediate of D-galacturonate catabolism in A . nidulans (192). It is therefore quite conceivable that polygalacturonase synthesis inA . niger is under the control of a HAP2/3/4-like regulatory system. 6 PROTEOLYTIC PROCESSING AT BASIC SITES

An increasing number of secreted fungal proteins have been found to be synthesized as prepro-proteins, processing of which often involves cleavage after one or two basicamino acids [previously reviewed in Calmels et al. (193)l. From Table2 it canbe seen that the pair Lys-Arg isthe most frequently observed dibasic cleavage site, and in those cases it is generally assumed that the cleavage is catalyzed by a processing enzyme related to the S. cerevisiae Kex2 protease, a membrane-bound Ca2+-dependent serine endoprotease that is present in a late Golgi compartment (194,195). In that the protein the caseof Aspergillus glucoamylase it was indeed shown isolated from a S. cerevisiae kex;! mutant had the signal peptide removed but still contained the short propeptide (196). Processing of the lignin peroxidase Lip2 precursor of the basidiomycete Phanerochaete chrysosporium was studied by in vitrotranslation of the corresponding RNA in a rabbit reticulocyte lysate in the presence of canine pancreatic microsomes, which resulted in cleavageof the signal peptidebut not of the seven-amino-acid propeptide (197). More extensive processing has been described for the KP6 preprotoxinof Ustilago maydis, a fungal pathogenof maize

~ o ~ ~ ~of iFungal s o nr e ~ r o - ~ e ~ tSequences ide an Their (~utative)Processing Sites Dibasic sitesa (24

(28) (28)

(1071 (138) (10% (40)

(31)

S G L V G T G L A L S L T A A N A A L S L S A A N A A A V I E K R R H R D D P P P T A S D I G K R S S G Y G G G Y G Y W K N K R P M V T E A P D V N L V E K R S A A P A P S R V S E F A K K

* A T L D

S

T C A N

* A T C S N * T I Q D S * G K la P R * Q S E E Y * A T T C T * A S T C T

G~uco~~lase A . awa~ori Lip2 LiPconsensus P. chrysosporium KP6 (a C-terminus) U.~aydis ~P6~N-ter~in U.~m) aydis A . niger var. macrosporus ProteinaseA A . nigerN400 PGC PGI A . niger N400

196 193 197 198 198 200 151 147

Monobasic

(221 (27) (41) (27) (27) (271

S L V V S L A A A L P H Y I R

* * * * * * *

S N G I E

Glucoseoxidase A . niger 203 198 KP6 (a N-ter~~nus)U maydis I P E G S L Q F L S L R P I Galactose oxidase L). dendroides 214 XylanaseA A . tubigensis 159 I N A A P A P E P D L V S R A , giganteus 215 T A L A V P S P L E A R T a-Sarcin A . restrictus 216 S V L A A P S P L D A R T C Restrictocin Cellobiohydrolase 11 T. reesei 217 (24 A T L A A S V P L E E R S S C.carbonu~ 173 T F Polygalacturonase (27) L V A A A P S G L D A R A T F A S A S P I E A R T F PGII A . niger N 146 (27) S A T L A S A S P I E A R T F Polyg~ac~uronase A . niger RH5344 144 (27) PGII A . tubigmsis NW75 160 initiationsite to the putativeprocessing site,which is indicated by an asterisk. aThefigures indicatethe number of mino acid residuesfromthe trans~ati~n

L G T T T

L T A A A A T A G V A A

A G F V V L A G

G A S A L P N G L S P R

N A S A A

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F C

~

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(198). From this precursor, two secreted KP6 toxin polypeptides are derived, KP6 alpha and beta. Proteolytic cleavagewas considered to occur (b) at the at four sites: (a) at the predicted signal peptidase cleavage site, monobasic cleavage site precedingthe N-terminus of KP6 alpha, (c) at the predicted dibasic cleavage siteat the C,terminus of KP6 alpha, and (d) at the dibasic cleavage site preceding the N-terminus of KP6 beta. The A. niger var. macrosponrs proteinase A precursor is also processed in a complex way.The mature proteinase consistsof two noncovalentlyassociated polypeptide chains (199), a light (L) and a heavy (H) chain, which to involve four are encoded by a single gene (200). Processing is thought proteolytic steps: (a) cleavage at a signal peptidase cleavage site, (b) after the asparagine residue preceding the N-terminus of the L-chain, (c) after the tyrosine at the C-terminus of the L-chain, and (d) after the dibasic site preceding the N-terminus of the H-chain. The C-terminus of the Lchain, the N-terminus of the Hchain, and the small intervening amino acid sequenceare shown in Table2. In addition, the glutamineat the Nterminus of the H-chainwas found to be convertedto a pyroglutamic acid residue, a modificationalso found, for example, at the N-termini of T. reesei endoglucanase I11 (201)and an A. acufeatuscellulase (202). In several precursors of secreted fungal proteins the start of the mature protein is preceded by a putative monobasic cleavage site (Table 2),and for some of these precursors processing according to the pre(signa1)pro format has been suggested. In the case the of glucose oxidase precursor this has been evidenced by sequence analysis of partially processed protein, which was obtained from S. cerevisiae as a minor component (203). Processing at monobasic sites is alsooften found for peptide precursors in higher eukaryotes, and a series ofand rules tendencies that govern monobasic cleavages has been proposed (218). With the exception of the glucose oxidase precursor, the other monobasic sites listed in Table 2 conflict with one or two of Devi’s four rules. In particular regarding the most stringent (positively formulated) rule, the presence of a second basic amino acid ( k g , Lys, His)at either position- 3, - 5, or - 7 with respectto the Arg at the cleavage site. Devi found that his rules were also applicable to a large number of dibasic sites. In those fungal precursors where there is another basic amino acid inthe region preceding the dibasic cleavage site (LiP2, KP6alpha, proteinase A,and PGI), it is indeed found at one of the positions predicted. At position - 3 with respectto the basic amino acid at the cleavage site thereis a preferencefor a hydrophobic aliphatic amino acidas noted by Tao et al. (198). Interestingly, the sequences found in the ribonucleolytic toxins cy-sarcin and restrictocin, cellobiohydrolase

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11, and somepolygalacturonasesshowconsiderablesimilarityin the six amino acids precedingthe start of the mature proteins; the sequence of a-sarcin in this region can be taken as consensus sequence. These precursors also show a conserved alanine at position - 9 with respectto the N-terminus of the mature proteins.This might, perpaps, relateto the abundancy of this amino acid at the - 1 and - 3 positions with respectto signal peptidase cleavage sites(174). Unfortunately, nothingis knownabout the specificity of the processing enzymes that cleave protein precursors in filamentous fungi. It is thus not clear if processing occurs by an enzyme of relaxed substrate specificity, or by twoor more different enzymes, similar to the situation seen with mammalian subtilisin-like prohormone convertases (204-206). Recently, evidence has been obtainedfor the existence of a distinctS. cerevisiae monobasic-specific protease whose activity is independentof the K, killer preKex2 enzyme(207). Cleavage at a monobasic site in the yeast protoxin, however, is dependent on K e d protease (208). To produce foreign geneproducts, there is also interest inthe use of prbcessing enzymesfor site-specific cleavageof recombinant fusionproteins (209,210). Improved chymosinproduction was obtained when it was expressed from a glucoamylase/chymosin fusion gene, in which thycase mosin was probably autocatalytically released from the fusion protein after secretion (211). Contreras et al. (212) havereported 250-fold-higher production of human interleukin-6 when it was initially synthesized as glucoamylase/interleukin-6 fusion protein by A. nidulans, in comparison to previous constructions containing only the glucoamylase preor preprosecretion signals. The mature interleukin-6 was fused through a short spacer peptide (kg-Met-Asp-Lys-Arg) to the C-terminus of the entire A. niger glucoamylase sequence,and this fusion protein was efficiently cleaved byan A. nidulans Kea-like activity. This study alsoprovided evidencefor the existence of dipeptidylaminopeptidaseactivity (213) in a filamentous fungus.

7 EXPRESSION OF GLUCOSE OXIDASE IN ASPERGILLUS NlGER

7.1 Glucose Oxidase: A Brief Introduction and Its Applications Glucose oxidase has several commercially interesting applications, which

can be roughly divided into the productionof gluconic acid, in which case

the intact fungus containing enzyme the is generally used,and applications

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for which isolated enzyme is required. Different approaches for strain improvement are required for these two applications. Strains used for the production of gluconic acid have to be more resistantto the conditions used inthe fermentation process, suchas high oxygen levels, mechanical shear, high sugar concentrations, and high levelsof hydrogen peroxide, whereas this isnot necessary or is less necessaryfor strains used to produce the enzyme. The availability of the structural gene for glucose oxidase allows for separate production of enzyme in other organisms. The reaction catalyzed by glucose oxidase (/3-D-ghcose:oxygen l-oxidoreductase; EC 1.1.3.4) is the oxidationof glucoseto glucono-&lactone with concomitant reductionof oxygento hydrogen peroxide.In A. niger the glucono-&lactone canbe hydrolyzed by a lactonase(EC 3.1.1.17) or it hydrolyzes spontaneously, whereasthe hydrogen peroxide is degraded by catalases (EC 1.11. l .6)(Fig. 7). Glucose oxidase inA. niger was first describedby Miiller in 1928 (219). Franke and Lorenz (220) showed the formation of H202 in the reaction and demonstratedthat other hydrogen acceptors can replace oxygen. They partially purified glucose oxidase and obtained evidence that glucose oxidase is a flavoprotein (221). In Penicillium glucose oxidase was initially described asan antibiotic: penicillinA, but was soon recognized as being a glucose oxidase and renamed as notatin, an enzyme similarto that already described for A. niger (222,223). Keilin and Hartree (224) characterized a highly purified glucose oxidase from P. notatum. In addition to A. niger and severalPenicillium species, glucose oxidase can be found in a number of other filamentous fungi such asPhaneroa function in the degradation chaete chrysosporium,where it might have

tactonase L 7

oxidase gluconic glucono-&lactone 0-Dglucose

COOH H-&OH HO-~H

~ 4 - 0 ~

~-6-0~ h20H

add

Figure 7 Enzymatic reactions in the oxidation of p-D-glucose to gluconic acid by A. niger.

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of lignin by lignin peroxidases(225,226)and Tahromycesj7avu.s. In this fungus the hydrogen peroxide formed by glucose oxidase is supposedto be involved in the biocontrol of another fungus, Verticillium dahliae(227). The physiological function of glucose oxidase isnot always clear, since the organismcannot utilize the energy released the in oxidation reaction. The most likely function the is contribution to the competitiveness of the organism, by the removal of the easily degradable glucose,by the concomitant acidification of the immediate environment,or by the formation of the toxic HzOz,to which A. niger is highly resistant. In someorganisms the provision of H202as a substrate for a peroxidase might be an important function, as is suggested for P. chrysosporium. Commercial preparations of glucose oxidase usuallycontain other enzymes, especially catalase. For some applicationsof glucose oxidase the presence of catalase is necessary to neutralize the toxic H,O,. In other cases (e.g., for analytical purposes)the catalase hasto be removed(228). A. niger catalase is also commercially available. Applications of glucose oxidase are all based on the capacity ofthe enzyme to form gluconate and hydrogen peroxide and to remove glucose and oxygen. All four functions have commercial applications (229).The use of A. niger for the formation of gluconate is discussed later in this chapter. Glucose removal by glucose oxidase was used for “desugaring eggs” to prevent the Maillard reaction (the reaction of an aldehyde and an amino group)(229).Oxygen removal by glucose oxidase is used to prevent oxidation and thusto prevent color and flavor changes. For this function itis sometimes used insoft drinks, beer,wine, dried foods, and foods (229,230).A relacontaining oil emulsions in water, such as mayonnaise tively new application is the use intoothpaste together with amyloglucoH,O,,which is used byan antibacterial sidase. The glucose oxidase forms system in the saliva based on lactoperoxidase. The high specificity for glucose in combination withthe formation of the easily detectableH,O, and the high stability of the enzyme makes it a very attractive system for (231). the determinationof glucose in biological samples Finally, glucose oxidase is probablythe most frequentlyused enzyme in biosensors. The stability, high specificity,and several possibilitiesfor detecting the activity of the enzyme makes it an ideal enzyme for application in biosensors. Determination of glucose with high specificity has many uses, including biomedical applications. For reviews on the application of glucose oxidase in biosensors,we refer to Schmid and Karube (232)and Janata (233)and references therein.

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7.2 Production of GluconicAcid A. niger has been used for the production of gluconate since the 1930s.

In that period efficient processesfor the productionof gluconic acid using submerged A. niger cultures were developed. Elevated air pressure and neutralization with calcium carbonate were applied in these processes. In 1952, Blom et al. (234) developed a process for the production of sodium gluconate using soldium hydroxide for the neutralizationof the gluconic acid formed. Fora more detailed descriptionof the history of the gluconate fermentation process,we refer to Miall (235) and Rohr et al. (236).

The high Michaelis-Menten constant of glucose oxidase with respect to oxygen [K, for oxygen = 0.48 mMat 27°C (237)] makes the dissolved oxygen levela key variable in the process kinetics of gluconate formation. Increasing the oxygen concentration under production conditions by elevation of the air pressure is the easiestand cheapest way to increase the formation rate of gluconate (238). A mathematical model describingthe kinetics of growthand production coupled with gadliquid oxygen transfer rates has been developed by Reuss etal. (239). Their model takesinto account the effects of pH on growth and production rate and the influence of sugar and gluconate concentrations on the solubility of oxygen and the k,a (the mass transfer coefficient). The main parameters and their effects included inthe model are: 1. The product formation rate is proportional to the dissolved oxygen

concentration at normal production conditions because of the high K, value for oxygen. 2. The optimal pH for growth is lower than than the optimal pH for enzyme activity. 3. An increase in the glucose and/or gluconate concentration results in a lower solubility andk,a of oxygen. From experimental data of product formation they calculated a theoretical K, value for oxygen. This value (0.36 mM)is comparable to the value of the isolated enzyme. The model predicts an increased product formation when glucose is added continuously to the culture compared to a culture where the glucose is added batchwise. This is because of the better oxygen solubility and higher k,a at lower sugar concentrations. Adapting thepH at the beginning ofthe fermentation to values resulting in higher biomassformation does not give a relevant increase inthe pro-

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duct formation under normal conditions (using 1 bar air for aeration). This is because the increase in biomass and thus in glucose oxidase is counterbalanced by a decrease in the oxygen level due to the higher amount of enzyme, resulting in lower turnover per unit of enzyme. However, when the air pressure is increased to 4 bar, the higher biomass does result an in increased product formation rate. Glucose oxidase overproducing strains become, of course, interesting as well under these high-pressure conditions. They have the advantage that lower amounts of biomass are necessary to obtain the same even or higher activities. This meansthat a loweramount of glucose is usedfor the formation and maintenance of biomass and also that the amount of other organic acids, such as oxalic acid, that areformed under these fermentation conditions will decrease. Another advantage is the lower viscosity of the culturefluid, which will leadto higher k,a values. Two approaches are available for constructing strains with higher glucose oxidase levels: mutagenesis and subsequent selectionof overproducers or the use of molecular biological techniques to increase the production of glucose oxidase. The advantage of mutant selectionthat is mutations with a broader effect can be isolated. For instance, the goxB mutant (see Section 7.5) has, in addition to an increased level of glucose oxidase, a higher level of two catalases, which might beimportant for the protection of the cell during gluconic acid fermentation. 7.3

Enzyme Properties

7.3.7 Glucose Oxidase Glucose oxidasefrom A. niger is a glycoprotein with a molecular weight of approximately 150 kDa (240-242). Estimations of the carbohydrate content varyfrom 10 to 25% (240,243-245). The native enzyme consists of two identical subunits(203,246,247) and two tightlybut noncovalently bound FADS (242,248). The holoenzyme contains two free SH groups and two disulfide groups(244,246,249). O’Malley and Weaver (246) reported an intersubunit disulfide bridge, whereas Tsuge et al. (250), Jones et al.(251), and Ye and Combes(252) found that the subunitswere bound noncovalently. (241,253-255). The Both 0-and N-linked carbohydrates were reported exact composition ofthe carbohydrates boundis variable and dependent on the production lots of manufacturers (256). When separated on isoelectric point, several bands of glucose oxidaseare usually found which differ only in carbohydrate composition (257). The carbohydrates are

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mainly mannoseand glucosamine (6: 1)and a smallamount of galactose. Several studies in which the function of the carbohydrates of glucose oxidase is investigated have been published(240,243,245,247,253,258,259). Summarizing, it can be said that at pH 5 to 6, where glucose oxidase is most stable, the carbohydrates do not contribute to the stability,but under less favorable pH conditions they contribute to the stability of the protein. Using deglycosylated protein Kalisz et al. (259) were able to obtain crystals of A . niger glucose oxidase. Kusaiet al. (260) described the crystallization of native Penicillium amagasakienseglucose oxidase. According to Hendle et al.(261) these crystalswere not suitable,so they used deglycosylated glucose oxidasefrom this organismfor crystallization. The availability of the crystal structure [as recently published(262)] and the structural gene for glucose oxidase opennew possibilities for research by studying enzymesthat are modified using molecular genetic techniques. The specificity of glucose oxidase was the subject of several reports (242,263-266). Both the Aspergillus and Penicillium enzymes are highly specific for the &form of D-glucose. D-galactose, D-mannose, andD-xylose are oxidized with less than 1% of the rate of oxidation of glucose. Both 2-deoxyglucoseand 6deoxyglucose are oxidized by glucose oxidase, although at lower rates than glucose. Kleppe (267) and Greenfield et al.(268) showed that glucose oxidaseof A . niger is inactivated by H,O, and the enzyme is much more sensitive when the flavin groupsare in the reduced state. The mechanism of inactivation isnot clear, but the most likely mechanism seems to be the oxidation of methionine residuesto methionine sulfoxide.

7.3.2 Catalases Catalase activity is necessary for detoxification of the hydrogen peroxide generated during the production of glucose oxidase. This is essential for the viability of the cells and the stability of the glucose oxidase. Witteveen et al. (269) showed that A. niger forms at least four different catalases, two of which are induced parallel with glucose oxidase (CATIII and CATIV) (see Fig. 9). Several publications described the purificationand characterization of an A . niger catalase. Since these catalasesare glycosylated and are purified from commercial preparations, which very likely originate from glucose oxidase fermentations, they probably correspond with CATIV, the cell wall-bound catalase which is induced parallel with glucose oxidase. This catalase is a tetramer with a reported molecular weight that varies strongly: 323 kDa (270), 338 kDa (271), 385 kDa (272), and 460 kDa (273). This is probably caused by differences the in carbohydrate

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content (10 to 33%). The amino acid composition has been determined by Gruft et al. (270) and Mosavi-Movahedi et al. (271). The enzyme is much morestable than beef liver catalase, especially with respect to H,O, resistance, proteolytic degradation, and temperature and pH stability, especially at low pH (271,274), and is relatively insensitiveto inhibition by HCN, HN,, and HF (270,272). However, the A. niger has a specific activity that is approximately 10-fold, lowerthan that of bovine liver catalase.

7.3.3 Lactonase The physiological importanceof this enzyme is not clear since the hydrolysis of glucono-&lactone also occurs spontaneously (half-time approximately 0.5 h, strongly dependent on the pH). Gluconob-lactone is known to be a strong inhibitor of @-glucosidase; therefore, lactonase is supposed to contribute to cellulose degradationby hydrolysis of the lactones(275, 276). The induction of the enzyme is coordinately regulated with glucose oxidase (see Section 7.6). Lactonase was partially purified from a commercial A. niger enzyme preparation by Bruchmann et al.(276). They concluded that there are several lactonase isoenzymes. The molecular weight is approximately 70 kDa.

7.4 Localization of the Glucose Oxidase System For a long time itwas assumed that glucose oxidase ofA. niger was located intracellularly(277). This was further supported by ultrastructural studies by van Dijken and Veenhuis, who concluded that the enzymewas located in peroxisomes(278). Mischak et al.(279) showed that under manganese-deficient growth conditions glucose oxidase is found almost quantitatively in theculture fluid. They explained this by a cell-wall-localized glucose oxidase which enters the culture fluid because ofan altered cellwall composition resultingfrom manganese deficiency. Using immunocytochemical methods Witteveenet al. (269) demonstrated that glucose oxidase was localized in the cell wall (Fig. 8). An extracellular localization of glucose oxidase is confirmed by data obtained from the sequence of the glucose oxidase gene. The amino acid sequence derived from this DNA sequence shows a secretion signal peptide typical for secretory proteins (203). The observationof Van Dijken and Veenhuis (278) that glucose oxidase is localized the in peroxisomes might be the result of a misinterpretation of their data. Their cytochemical data may be explained as an artifact of the method used. Within this view the observed positive staining of microbodies, caused by catalase, may result from H,O, gen-

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Figure 8 Immunocytochemical staining of an A. niger cell after glucose oxidase induction. Specific antibodies against deglycosylated glucose oxidase and protein A-gold were used. Labeling is confinedto the cell wall. Peroxisomes are not labeled. N, nucleus; P, peroxisome; bar, 0.5 pm. (From Ref. 269.)

erated by glucose oxidase locatedthe in cell wallafter the addition of glucose to the incubation sample, which subsequently diffuses inside the cell. The high glucose oxidase activities in the cell wall might produce sufficient 30,for the peroxisomal staining observed(269). In Penicillium amagusakiense glucose oxidase is secreted as well (260). An extracellular localizationof glucose oxidase activity impliesan extracellular production of hydrogen peroxide. The sequestration of this toxic process will prevent damage to the cell. However, there remains the necessity for the efficient removal of H,O, since this compound is known to inactivate glucose oxidase (267) and can easily diffuse over the cell membrane. Mycelium containing high levelsof glucose oxidase can produce 50 to 100 mmol of hydrogen peroxide per hour per gramof myceliumdry weight without large-scale cell lysis or inactivation of glucose oxidase; thus an effective protective system must exist. Witteveen et(269) al. demonstrated the presence of four catalases inA. niger using nativegel elecrophoresis and staining of the gels for catalase activity. Twoof these catalases are more or less constitutive.By determining the activityof the catalases in both mycelia and protoplasts, they were able to demonstrate that one of these catalases is localized intracellularly (CATI), whereas the other oneis cell-wall bound (CATII). Under glucose oxidase-inducing

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conditions twoextra catalases are synthesized, one of them extracellular (CATIV) and one intracellular (CATIII) (Fig.9). Witteveen et al. (269) reported that 50% of the lactonase activitywas present in the culture fluid and 50% was mycelium bound. Considering the location of glucose oxidase and two of the catalases, it seems very likely that lactonase is secreted,too. The cell wall isnot a separate compartment, so there must bea mechanism by which the proteins are retained. The proteins might be bound to cell-wall components, or they might be retained because the pore size is too small to let them diffuseout of the cell wall. Approximately20% of glucose oxidase,less than 5% of catalase, and 40 to 50% of lactonase is found in the medium. This might be explained by the difference in size of the proteins. Catalase, which has a very high molecular weight (at (70 kDa) least 350kDa), is retained almost completely, whereas lactonase (150 kDa) takesan interpasses the cell wallmore easily. Glucose oxidase mediate position in this.In yeast it was shown that the passage of proteins throughthe cell wall isnot just determined bythe molecular weight of the proteinsbut is influencedby multiple factors (280). However, the distribution of catalase, glucose oxidase, and lactonase do suggest that molecular size is an important factor. Expression of glucose oxidase in S. cerevisiae resulted in a strongly overglycosylated protein, which apwas obparently could easily pass the cell (203). wall A comparable result

A

B

C

D

E

F

Figure 9 Catalase in mycelial and protoplast extracts of glucose oxidase-induced and noninduced A. niger wild-type mycelia visualized on nondenaturing polyacrylamide gels. Lanes: A and B, extracts of mycelium and protoplasts of noninduced mycelium; C and D, extracts of mycelium and protoplasts of induced mycelium; E, same as described for lane C; F, material released from induced mycelium when lowconcentrations of Novozyme 234 were used. (From Ref. 269.)

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tained when the cellobiohydrolase from Trichoderma reeseiwas synthesized by S. cerevisiue (281).

7.5 Isolation of GlucoseOxidaseOverproducing and Negative Mutants Regulatory and structural mutants for glucose oxidase are very useful for both strain improvement and a better understanding of the regulation of the glucose oxidase system. Several groups reported the isolation of glucose oxidase-overproducingstrains obtained by mutagenization (282285). They all usedplate screening methods basedon either acidification of H202 (285). of the medium around a colony (282-284) or the production The glucose oxidase levels of the overproducing mutants described in different articles by different groups cannot be compared because they all used different cultivation conditions. On the basis of genomic size and the assumption that only a few genes will bedirectly involved inthe regulation of glucose oxidase gene expression, one would expectto find useful mutants onlyinafrequencyof about to As enrichment techniques cannot be applied, this implies that a large numberof colonies have to be screened. Fiedurek etal. (283) and Kundu andDas (282)screened only low numbers of mutatedstrains, so it is not very likely that mutations in genes directly involved in glucose oxidase regulation were isolated. Markwell et al. (284) chose mutant selection conditions that reThis was under suboptimal sulted in a mutation frequency of 6.6 x acidifying conditions (relatively low glucose,100mM). Witteveen et al. (285) used a much more sensitive method for glucose oxidase activity detection, which could infact only be usedfor the selection of mutants producing under noninducing conditions and for the selection of negative mutants. For selection under noninducing conditions, carbon sources other than glucose were usedor glucose in combination with low oxygen levels. A mutationfrequency ofwas found when theselection was done on fructose, whereason glycerol and gluconate a frequency of 10-5 was found, which is comparable to the frequency of mutation found by Markwell et al. (284). The mutants could be classified in nine complementation groups which were assigned to the eight different linkage groups of A. niger (286). Mutants of three of these complementation groups (goxB, goxCJgoxE) showed phenotypesthat were sufficiently pronounced to be used for a further analysis of the regulation of glucose oxidase, catalase, and lactonase, thethree enzymes relevantfor glucose oxidation in A. niger (see Section 7.6). These three mutations are recessive and are glucose oxidase negative(goxC) or glucose oxidase overproducing (goxB

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and goxE). The goxB strains showa more orless constitutive phenotype and are less dependent on the oxygen level for glucose oxidase expression (one of the goxB mutants was selected for glucose oxidase expression under conditionsof low oxygen levels). The goxE mutants produce glucose oxidase on several carbon sources and thus seem to have lost the dependency on the presence of glucosefor glucose oxidase induction.In the goxC complementation group (linkage group 11) only one mutant (NWlO1) wasfound. In this mutant no glucose oxidase protein or mRNA was detected. Transformation of this strain with the structural gene of glucose oxidase restored wild-type expression of glucose oxidase. This was taken as evidence that the goxC mutation defines thestructural gene for glucose oxidase. Several glucose oxidase-overproducing mutants were isolated which belongedto the goxB and goxE complementation groups located on linkage groupsI1 and VII, respectively. The characteristicsof these mutants with respectto glucose oxidase, catalase,and lactonase expression is described below (see Section 7.6). The level of glucos eoxidase in the goxB mutants, which showed the strongest effecton glucose oxidase expression,was increased about twofold when compared to wild-type mutants grown under optimal inducing conditions. The knowledge of the chromosomal location and the availability of strains with markers on most of the chromosomes allowsfor the recombinationof different gox mutations. In this way a strain was constructed that combined different gox mutations together with a gcaAZ mutation. The latter mutation results in the inability to use gluconate as a carbon source (Witteveen et al., unpublished results). Someof the recombinantstrains showed just a small increase in production of glucose oxidase with respect to the original parent strains. 7.6 Induction of the Glucose Oxidase System Factors important for optimal induction of glucose oxidaseare (a) high glucose concentrations (287),(b) a pH around 5.5, and (c) a high level of dissolved oxygen (288). Control of the pH can be achieved by titration or by adding CaCO, to the medium. The presence of manganese has been claimed to be important for glucose oxidase induction (289), but Mischak et al. (279) showedthat this is not true. Nitrogen limitationis not necessary to obtain efficient inductionof glucose oxidase (285), although this was previously reportedto stimulate theformation of glucose oxidase in both Penicillium (290) and Aspergillus (291). The latter publication relates to surface culturesof A. niger and is therefore not directly comparable. Induction of glucose oxidase requires the presence of glucose or of

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2-deoxyglucose (279,285). The effect of the pH is more complicated. There is no doubt that the optimalpH for induction isaround 5.5, but at pH values as low as 2.5, biosynthesis of glucose oxidase could still be' demonstrated (285,292). At a pH lower than 2.5, glucose oxidase is no longer stable. Raising the pH from 2 to 5.5, Mischak et al. (279) could demonstrate de novo synthesisof glucose oxidaseand Wirsel et al. (293) demonstrated the inductionof glucose oxidase mRNA. Considering these facts, it is noteworthy that optimal conditions for glucose oxidase induction are optimal conditions for the enzymatic activity. Unraveling the molecular mechanism of induction is in progress (294). Witteveen etal. used the goxB, goxC, and goxE mutants to study the induction of glucose oxidaseas well as of lactonase and catalase, focusing on the effects of oxygen and glucose. Important in their studies is the glucose oxidase negative mutantgoxC. None of the three enzyme activities was induced in this strain under conditionsthat normally would induce the entire system (pH 5.5, high oxygen, high glucose). This indicates that an active glucose oxidase is necessary for induction of lactonase and catalase. Subsequent experiments in wild type showed that all three activities can be induced by the addition of hydrogen peroxide, even though no glucose is present, which is normally obligatoryfor induction. A central rolefor hydrogen peroxide in the induction is in agreement with the observationthat optimal induction conditions are also optimal conditions for enzyme activity and thus for hydrogen peroxide production. Considering the low affinity of glucose oxidase for oxygen, it is thento be expected that at low oxygen concentrations no induction of glucose oxidase is found. Also, the requirement for the presence of a substrate for glucose oxidase (glucoseor 2-deoxyglucose) for induction fits in this model. The unusual situation that the product of a reaction is the inducer of the enzyme catalyzingthat reaction doesnot result in overinduction because the simultaneous induction of catalase takes care of the breakdown in the inducer hydrogen peroxide. A summary of the induction pattern of glucose oxidase, catalase,and lactonase activities is given in Table 3. It shows that in the goxB background the induction of all three enzymes is glucose-and oxygen-independent. The goxB mutants were the only onesto have an effect on the oxygen dependency ofthe induction.All other complementation groups, includinggoxE, have no effect on catalase induction. As hydrogen peroxide the gene product is also able to induce catalase, it is hypothesized that goxB is somehow involved in mediatingthe hydrogen peroxide induction.Effects ofthe goxE gene are limitedto the glucosedependentglucose oxidase

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Table 3 Dependency of the Various Strains on Either the Presence of Glucose or onHigh Oxygen Levels for theInduction of Glucose Oxidase,Lactonase, and Catalase Activities oxidase Glucose Lactonase Catalase Strain Glucose Oxygen Glucose Oxygen Glucose Oxygen Wild type + + + + + + goxB + + + + goxE

and lactonase induction. This shows that besides the hydrogen peroxide effect thereis still another parameter relatedto the carbon source, which has an effect on the induction of glucose oxidase and lactonase, especially. Northern analysis showedthat the regulation of glucose oxidase expression by oxygen takes placeat the transcriptional level (Fig. 10).

Flgure 10 Northern analysis of the effect of the oxygen levelon glucose oxidase mRNA levels in A . niger wild-type N400 (lanes 1 to 3) and the A . niger strain NW103 (lanes 4 to 6)which carries the goxB21 mutation. The strains were grown under conditions optimal for induction (10% glucose; pH 5.5; aeration above 30% air saturation). After 20 h, mycelial samples were taken and the dissolved oxygen concentration was then lowered to approximately 7% air saturation. mRNA levels in induced samples (lanes1 and 4) and samples of mycelia6 h (lanes 2 and 5) and 12 h (lanes 3 and 6) after lowering the oxygen level are shown. An AvuZ fragment of the gene was used as a probe. (From Ref. 294.)

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7.7 GlucoseOxidaseGene Four recent publications described the isolation of the glucose oxidase gene (203,294-296). The methods used involved the use of protein sequence-derived oligonucleotide probes(203,295,296) or the screening of a cDNA expression library using antibodies (294). Kriechbaum etal. (295) and Frederick et al. (203) used a cDNA library constructed fromthe A. niger ATCC 9029 strain. Whittington et al. (296) isolated the glucose oxidase gene directly froma genomic library of theA. niger B60 strain (297). Witteveen et al.(294) isolated the gene from the CBS 120.49 strain. The sequence of Whittington et al. (296) shows some minor differences in the promoter comparedto the sequence of the ATCC9029 strain (at positions -476, -464, and - 395). Comparison of the cDNA sequence and the genomic sequence revealed that no introns are present inthe glucose oxidase gene. S1 nuclease mapping ofthe 5’ end ofthe gox transcript indicated the initiation of transcription to be at position - 38,43 bp down(296). Protein sequence data showed that stream of the TATAA sequence the matureenzyme is preceded bya 22-amino acid prepro-sequence which (203; see also Section6). The mature enhas a monobasic processing site zyme, which consists of 583 amino acids, contains eight potential sites for N-linked glycosylation and three cysteine residues. Comparison of the glucose oxidase amino acid sequence with a protein sequencedatabase (Swisspir) resulted in five proteins with more extended homology:E. coli choline dehydrogenase, twoDrosophila glucose dehydrogenases, and two yeast methanol dehydrogenases(298-302). All five proteins are flavoproteins. Figure 11shows alignments of these sequences with glucose oxidase (of the glucose dehydrogenasesand methanol oxidases, only onewas used in the alignment). The stretches of the highest homology are involved in FAD binding (262). Near the N-terminus the p-cr-p motif is involved in ADP binding (303). A second(AA243-289) and a third stretch (near the C-terminus) contain conserved blocks of amino acids as well and are also involved in the FAD binding. Besides these stretchesa number of amino acids are conserved in all four sequences which are described by Hecht et al.(262) to be important for the tertiary structure. These amino acids form salt bridges buried in the protein or strong H-bridges. Theyare indicated inthe figure. Forthe activity of the protein it is essential that the FAD group be nonplanar. Asn 107 is involved in preventingthe planarityof the FAD group.A r g 226 is enforcing this effect by forming a strong bond to the Asn 107. Both residues are conserved in all four sequences. Five ofthe eight potential N-glycosylation sites are indeed glycosylated. All these sitesare in regions with lower homologyto the other proteins.

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This is in agreement with previously published results that enzyme activity and stability are not effected by deglycosylation (see Section7). The region that is involved in substrate binding is less homologous than theFAD binding parts. However, in these areas there are two stretches (around A A 325 and 434) that show a series of conservedamino acids. The function of these parts is not known. The quite extensive overall homology between the six proteins, even though they are from evolutionary distant organisms, is remarkable. They probably form a distinct classof structurally related flavoproteins. 7.8 Overexpression of GlucoseOxidaseand Expression of the Glucose Oxidase Gene in Other Organisms

In filamentous fungiit is often possible to achieve overproductionof an enzyme just by increasing the copy number of the encoding structural gene, which can be achieved by (co)transformation (see, e.g., Section 4.7). The transformationof strains with different gox genetic backgrounds was made possible by constructing double mutants of gox and pyrA (294). Table 4 showsthe activities of some of thesetransformants. The data show that in multicopytransformants of the strains witha wild-type or goxC genetic background onlyan increase in glucose oxidase activity of a factor of 3 or 4 was achieved, even though morethan 50 copies of the gene were present. Instrains with a goxB background, higher levels of glucose oxidase were observed,but this had avery negative effecton growth, probably dueto the formation of Hz02,since thesestrains were grown under gluconate-producing conditions. However, we cannot exclude reasons other than HzOztoxicity for the discrepancy between copy number and enzyme levels. These results make it questionable whetherit is possible to produce still higher levels of glucose oxidase A. in niger in the presence of glucose. Vectors containing the glucose oxidase gene have been usedto transform Saccharomyces cerevisiae and Aspergillus nidulans. S. cerevisiae (203,304) was transformed withthe plasmid pSG02, in which the glucose oxidase gene (includingthe preprosequence) is under the control of the regulated ADHZ-GAPDH hybrid promoter.This resulted in a production of glucose oxidase of 3 g/L. The glucose oxidase produced this way was strongly overglycosylatedand had aMW of 350to 400 kDa, as estimated by gel permeation chromatography. This enzyme had a normal activity but showedan increased heat resistanceand pH stability.

9.1

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Table 4 Glucose Oxidase and Catalase Activities and Dry Weights after 24 h of Growth under Inducing Conditions in A. niger N400 (W) and NW103 (goxB), and in wt, goxC, and goxB Strains Which Were Transformed witha Plasmid Containing the Structural Gene for Glucose Oxidase Glucose Mycelium Catalase oxidase dry weight Strain Strain activity activity nr description (8) (Wmg DW) (Wmg protein) Wild type 1.7 98 1.5 N400 Wild-type transformant, 111 1.2 4.0 G1 high copy number G15 5.5 98 0.5 Wild-type transformant, high copy number c35 4.5 95 1 .o goxC17 transformant, high copy number C36 0.8 93 1.5 goxC17 transformant, low copy number 381 goxB21 1.8 2.7 NW103 B24 113 0.12 goxB21 transformant, high copy number B7 3.9 233 0.44 goxB21 transformant, low copy number

7.9 Glucose Oxidase Gene Used in

a Reporter System

The detectability in intact mycelium, the stability of the enzyme, and the relative easyand sensitive detection ofthe enzyme activity allowthe glucose oxidase geneto be used as a reporter system for studying promoter activities of enzymes that are more difficult to assay. De Graaff et al. (159) made a construct in which a promoter element derived from the xlnA gene of A. tubigensis which encodes an endo-xylanase was ligated to a fragment containing the goxC structural gene and the core promoter (TATA box and transcription initiation site) but without regulatory sequences of the glucose oxidase gene. This construct was used to transform a strain with the goxC mutation. Inthis way they were able to show that a 158-bp element contains theinformation for xylandependent inAlso, the effect of other carbon duction and glucose repression (Fig. 12). sources and other potential inducers could be investigated very efficiently using this system.A gox reporter system that includes the gox core promoter is particularly useful to monitor transcription activation elements.

286

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8 CONCLUDING REMARKS Gene expression studies, particularly in the filamentous fungusA. nidulam, have reached a stage where precise and sophisticated knowledge about the mode of binding of activatorand repressor proteinsto the corresponding target genes is now being generated rapidly. It is to be expected that x-ray crystallographyand high-resolution NMR spectroscopy DNAwill further unravel the precise molecular interactions between binding domains in fungal regulatory proteins and nucleic acid target sites. Understanding the physiological consequences of gene expression regulation for fungal metabolic pathway control requires in addition a detailed analysisof the various levelsat which, for example, widedomain control systems interact with pathway-specific regulator genes and/or structural genes. The double-lock control system mediated by creA in the ethanol regulon ofA. niduluns is one of the most illustrative examto modples presentlyknown (57). Similarly, other factors which are able ulate transcription levels will be found and identified, for instance by conservation of sequence motifs. This is already indicated for the CCAAT

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motif inthe amdS gene (63),for the relatedHAP2/3/4like binding motif found in several polygalacturonase gene promoters (151), and other boxes, such as the “gpd” box (73). Regulation of gene expression in fungi of industrial interest is expected to follow basically the same general principles as those discovered A. in nidulans and N. crassa. We have recently identified, for example, mutants in A. niger which are equivalent mutations to the widedomain control genes creA,paeE, and areA of A.nidulans (unpublished results). However, regulation of specific genes hasto yet be described in full detail. For many extracellular polysaccharide degrading or modifying enzymes that are of industrial importance, the structural genes have been cloned and analyzed, but the genetic system, including the regulatory components, is still missing. Moreover, the identity and structure of the low-molecular-weight signals responsible for the induction of subsets of enzymesrequired for the breakdown of complex polymeric substrates such as pectinare usually unknown, although thereare a few exceptions. Induction of the cellulolytic complex in T. reesei by sophorose (glucose-~-l,2-glucose)is, of course, well known (305,306). In A. terreus heterodisaccharides composed of glucoseand xylose have been shownto induce both cellulolyticand xylanolytic enzymes, whereas the homodisaccharides induce selectively either cellulases (in the case of sophorose) or xylanases (inthe case of xylose-@-1,2-xylose) (307). Identification of inducers and regulatory genes will become an important topic, particularlywhen complex substrates are requiredfor fermentation as is the case for the production of plant cell wall-degrading enzymes. Organic acid formation in A. niger is studied primarily with respectto citric acid formation, butno regulatory components have been identified. Only the applied aspects of gluconic acid formation have been investigated. Our studies on glucose oxidase gene expression have identified two important regulatory genes ( g o d and goxE) which mediate a response to hydrogen peroxideand the carbon source, respectively (285,286,294). We have used this system further to illustrate the potentialof industrial strain improvement by mutagenesisand recombination, in combination with genetic manipulation (286,294). We expectto see an increasing trend in strain breeding strategiesto avoid empirical approaches and to exploit, rather, the recombinant DNA technology available in combination with traditional genetic techniques.

ACKNOWLEDGMENTS We gratefully acknowledge financial support by CIBA-GEIGY AG,Basel, by the Netherlands TechnologyFoundation (STW), Utrecht grant WBI

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47.0637, and by the European communities BIOT-CT 90-0169, enabling us to carry out the experimental work on glucose oxidase and pectinase

gene expression described in this chapter.

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sugar chains on glucose oxidase from Aspergillus niger.Biochem Cell Biol 1989; 67:460-464. 254. Takegawa K, Fujiwara K, Iwahara S, Yamamoto K, Tochikura T. Primary

structure of an 0-linkedsugar chain derived from glucose oxidaseAsperof gillus niger. Agric Biol Chem 1991; 55:883-884. 255. Takegawa K, Kondo A, Iwamoto H, Fujiwara K, Hosokawa Y, Kat0 I, Hiromi K, Iwahara S. Novel oligomannose-type sugar chains derivedfrom glucose oxidase of Aspergillus niger. Biochem Int 1991; 25:181-190. 256. Hayashi S, Nakamura S. Comparison of fungal glucose oxidases: Chemical, physicochemical and immunological studies. Biochim Biophys Acta 1976; 438:37-48. 257. Hayashi S, Nakamura S. Multiple forms of glucose oxidase withdifferent carbohydrate compositions. Biochim Biophys Acta 1981; 657:40-51. 258. Nakamura S, Hayashi S, Koga K. Effect of periodate oxidation on the structure and properties of glucose oxidase. Biochim Biophys Acta 1976; 445:294-308. 259. Kalisz HM, Hecht HJ, Schomburg D, SchmidRD. Crystallization and pre-

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1952; 50331-341. 265. Adams EC, Mast RL, Free AH. Specificity of glucose oxidase. Arch Biochem Biophys 1%2; 91:230-234. 266. Feather MS. A nuclear magnetic resonance study of the glucose oxidase reaction. Biochim Biophys Acta 1970; 220:127-128. 267. Kleppe K. The effect of hydrogen peroxide on glucose oxidasefrom Aspergillus niger. Biochemistry 1966; 9139-143.

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26~1455-1464. 291. Muller HM. Gluconate accumulation and enzyme activities with extremely nitrogen-limited surface cultures ofAspergillus niger.Arch Microbiol 1986; 1441151-157. 292. Heinrich M, Rehm HJ. Formation of gluconic acid at low pH-values by

free and immobilized Aspergillus niger cells during citric acid fermentation. Eur J Appl Microbiol Biotechnol 1982; 15:88-92. 293. Wirsel St, Kriechbaum M, KoopmannJO, Heilmann HJ, Gassen HG. Glucose oxidase of Aspergillus niger: Characterization of the structural gene and comparison of the expression in wild type and transformedAspergillus strains. In: DECHEMA Biotechnology Conferences 4. Weinheim: VCH Verlagsgesellschaft, 1990:333-336. 294. Witteveen CFB, van de VondervoortPJI, van den Broeck HC, van Engelenburg FAC, de Graaff LH, Hillebrand MHBC, Schaap PJ, Visser J. The induction of glucose oxidase, catalase and lactonase in Aspergillus niger Curr Genet 1993; 24:408-416. 295. Kriechbaum M, Heilmann HJ, Wientjes FJ, Hahn M, Jany KD, Gassen HG, Sharif F, Alaeddinoglu G . Cloning and DNA sequence analysis of the glucose oxidase gene from Aspergillus niger NRRL-3. FEBS Lett 1989; 255~63-66. 296. Whittington H, Kerry-Williams S, Bidgood K, Dodsworth N, Peberdy J,

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6 Gene Expression in Recombinant Streptomyces Richard H. Baltz

Lilly Research Laboratories, a Division of Eli Lilly and Company, Indianapolis, Indiana

1 INTRODUCTION Streptomycetes are gram-positive filamentous bacteriafound commonly in soil. They have chromosomes ranging in from size about 3.3 to 5.2 md (1-7). Although geneticand physical mapping experiments initially suggested that streptomycete chromosomes were circular (1,2,8,9), recent physical studies indicatethat streptomycetes chromosomesare generally linear (10-12). Streptomycetes genomescontain a guanine plus cytosine (G + C) content averagingabout 73% (13-15), approaching the genetic code upper limit(16). Because of the high G + C content, streptomycetes use a subset of the codon catalog containingG or C in the third position of codons much more frequently than codons containing A or T in the third position (17-19). Since their genomes are about twice as large as those of Escherichia coli, the streptomycetes have the coding potential for many functions beyond those required for normal growthand metabolism. Most streptomycetes encode complex secondary metabolite biosynthetic pathways, and the correspondinggenes are generally clustered (18). Streptomycetes harbor many different plasmids, and most have been 309

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shown to be self-transmissible. These include both circular and linear plasmids that can replicate autonomously or integrate into the streptomycete chromosome (see, e.g., Refs. 7 and 20-39). Streptomycetes are saprophytes and obtain nutrients from the breakdown of organic matter in soil by the secretion of hydrolytic enzymes. Streptomycetes grow as branching mycelia that can differentiate into aerial mycelia and spores (40,41). During differentiation, streptomycetes produce secondary metabolites, including antibacterial agents, antitumor agents, anthelminthic agents, immunomodulators, and herbicidal agents. Sincestreptomyceteshaveexceptionalabilities to producesecondary metabolites and to secrete hydrolytic enzymes, they have the potential to be exploited for the production of natural or hybrid secondary metabolites and for the production and secretion of homologous or heterologous proteins. The different biotechnological applications of streptomycetes present different challenges. For instance, the cloning and expression of a gene encoding an enzyme that catalyzes a rate-limiting step in the biosynthesis of a secondary metabolite has at least three requirements: (a) the gene must be expressed at the proper timeand at the appropriate level in the fermentation cycle;(b) the introduction of the gene per se must not cause deleterious effects on secondary metabolism; and (c) the gene must be stably maintained in the absence of antibiotic selection. These requirements may be best addressed by inserting the cloned geneinto a neutral site in the chromosome. Cloning streptomycete or other actinomycete genes in heterologous streptomycetes to produce hybrid secondary metabolites may have different requirements. For instance, the enzymatic conversions to produce the hybrid structures may be inefficient since they utilize unnatural substrates. Therefore, it may be important to express high levels ofthe enzymes requiredto carry out the conversions. Multiple copies of the gene(s) may be required,or the gene may needto be fused to a highly efficient promoter. High-level expression of the heterologous gene@) must be accomplished ainmanner that avoids deleterious effects on secondary metaboliteproduction, and the recombinant strain should be stable inthe absence of antibiotic selection. The application of streptomycetes for the production of homologous or heterologous proteins need not be concerned with maintaining efficient secondary metabolite production. However, the requirement for highly efficient secretion of the product is critical if the process is to compete with other expression systems. High-level expression may be facilitated by maintaining the gene on a multiple-copy plasmid,and fusing the gene to highly efficienttranscription, translation, and secretion sequences.

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In this chapter I address the problem of gene expression in streptomycetes with these applications in mind. Rather than attempt to be comprehensive, I refer to other reviews when possible and emphasize key studies that have a direct bearingon the applications outlined.I have not attempted to review the regulationof primary metabolism or how the expression of secondary metabolite biosynthetic pathways is influenced by carbon, nitrogen, or phosphate metabolism. The interested reader is referred to reviews (42-46) for detailed discussions on these and related topics. I have also not attempted to review the extensive literature on antibiotic resistance mechanisms in streptomycetes, although some may play a direct role in secondary metabolite production by facilitating transport or by rendering the producing streptomycete resistant to its toxic secondary metabolite.This subject has been reviewed elsewhere (18,47,48).

2 GENE CLONING SYSTEMS A major advantage of the streptomycetes is that many different species with different metabolic capabilities to produce secondary metabolites are available. This dictates that the molecular genetic techniques must readily be adapted to many species to derive the maximum benefit from the metabolic diversity.I discuss somebroad host range cloning systems in this section, then discuss several broadly applicable gene transfer systems in the following section.

2.1 StablePlasmidReplication Many streptomycete plasmid and bacteriophage cloning vectors have been developed, and the properties and uses of these vectors have been reviewed extensively (20-22,39,49,50). Of the vectors described, only a few have been used widely. The most extensively used vector is pIJ702 (20,21,39,51,52), a derivative of pIJ101, a plasmid that has been extensively characterized (22,33,53,54; Table 1). pIJ702 contains a thiostrepton resistance gene( W ) and a tyrosinase (mel)gene (encoding melanin production) for insertional inactivation; therefore, it has beena convenient cloning vector even though it doesnot contain bifunctionalityfor replication in E. coli. Many genes have been clonedand expressed in pIJ702, and it appearsto be a useful vectorfor the overproduction of single-gene products (see Section 4.4.4). pIJ702 and other pIJlOl derivatives can have severe negative effects on secondary metabolite production (55-58), and as such maynot generallybe useful for the constructionof secondary metabolite production strains.

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pIJ922 (Table 1) and related plasmids are derived from SCP2* (39, 59,60). pIJ922 has a lower copy number than pIJ702, does not contain E. coli replication functions, but is self-transmissible (pIJ702 is not). Like pIJ702, pIJ922 also caused decreased biosynthesis of nemadectins (56). Other derivatives of SCP2* bifunctional for Streptomyces and E. coli have been constructedfor ease of cloningand analysis (61-63). One of these is a cosmid, pKC505 (49,63; Tablel), which is self-transmissible. pKC505 has been usedto clone large clusters of secondary metabolite biosynthetic genes (63-67).

2.2 Chromosomal-IntegrationPlasmids For the addition of a second copy of a gene encoding a rate-limiting step in secondary metabolite productioninto a production strain, the stable insertion of the gene into the chromosome may be a most prudent approach. Some chromosome integration systems have beenreviewed recently (22,50). I discuss three examples of integration systems drivenby plasmid integration functions, phage integration functions, or homologous DNA in this section, and by transposons in the following section.

2.2.1 pSAM2 pSAM2 is an 11. l-kb self-transmissible plasmidfrom Streptomyces ambofaciens that can integrate into the chromosome or replicate autonomously (22,34,68-73). Derivatives of pSAM2 can integrate site-specifically into the chromosomes of other streptomycetes (69-71). The site of integration for pSAM2 overlapsa putative tRNAPro genethat is highly conserved in streptomycetesand other actinomycetes(71). The pSAM2 inte(70-73) and gration (int)and excision (xis)functions have been localized show strong similarities to int/xis functions of temperate bacteriophages of gram-negative bacteria (73). A fragment containing the xis, int, and attP functions has been incorporated into vectors bifunctionalfor E. coli and streptomycetes;the vectors replicate inE. coli and integrate instreporiT tomycetes (74,75).A versatile pSAM2 derivative, pPM927, contains from plasmidRK2 and can be transferredfrom E. coli to streptomycetes by conjugation (74; Table 1; see also Section3.3); it also contains a site for cloning genes downstream of the thiostrepton-inducible tipA promoter for regulated expression(see Section 4.3.2.b). pPM927 is usefulfor introducing single copies of genes into the chromosomes ofdifferent streptomycetes, but it is not known if such insertions cause deleterious effects on secondary metaboliteproduction in industrial strains.

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A number of other streptomycete plasmids integrate site-specifically in streptomycetes (50), most notably plasmid SLPl (38,76-80),which integrates into a functional tRNA'Y. As with pSAh42, it is not known if site-specific insertion ofSLPl is deleteriousto secondary metaboliteproduction inindustrial strains.

2.2.2 4C3 l Integration 6 3 1 is a temperate bacteriophage that has broad host specificityfor Strepinto many cloning vectors tomyces species (81). 4C31 has been developed (39,81). Kuhstoss et al. (75) identified a 4-kb segment of 4C31 DNA that was sufficient to cause nonreplicating plasmids containing the segment to integrate into the chromosome ofS. ambofaciens at very high frequencies following protoplast transformation. The frequenciesof transformation were 60- to 600-fold higherthan those mediated by pSAM2 xidint/ attP functions (75). Plasmid pKC796 (Table 1)and cosmid pKC731, both containing 4C31 int and attP functions, were also shown to transform Streptomyces griseofuscus, Streptomycesfradiae, Streptomyces Iividans, Streptomyces Iipmanii, and Streptomyces thermotolerans. The 4C3 1 int gene was localizedto a 2. l-kb segment of DNA,and the integration process and int function were analyzed in some detail (82). Other vectors combining the 4C31 integration functions with the RK2 oriTfunction have been constructed (83; see Section 3.3). These cosmids and plasmidsare useful for constructing stable recombinant strains.It is not known if insertion of cosmids or plasmids into the 4C31 attB site per se causes deleterious effectson secondary metabolite biosynthesis in different streptomycetes. This isan important issue that needs to be addressed for both the plasmid and bacteriophage integration systems.

2.2.3 HomologousRecombinationfrom Nonreplicating Plasmids MacNeil etal. (84) describedan integration vector that contains no replil), cation functionsfor Streptomyces species. The vector, pVE616 (Table replicates in E. coli, but transforms Streptomyces avermitilis only after S. avermitilus DNA has been inserted; recombinants form by homologous recombination. The recombinant frequency increased proportionately with the second power of DNA length between 1.8 and 18 kb, and apparently with a higher power below 1.8 kb. Thusthe transformation frequencies increasedabout 2000-fold as the length of the insert was increased from1.1 kb to 18 kb. If this relationshipis generallytrue (which cannot yet be determined for lack of published studies inother strepto-

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mycetes), it must be taken into account in developing strategiesto use homologous recombinationto insert homologous genes into the chromosome. For instance, it may be very difficult to inser.t a large block of hoto generate nonmologous genesinto a different smaller homologous site tandem duplications. Hillemann et al.(85) demonstrated that the frequency of homologous integration into the chromosome of Streptomycesviridochromogenes was increased 10-to 100-fold by using single-strandedrather than doublestranded plasmid DNA. They used an E. coli plasmid (pDH6) containing the bacteriophage f 1 replication origin to prepare single-stranded DNA. It will be interesting to see if single-stranded circular plasmid DNA gives higher transformation frequencies in other streptomycetes. If so, this method might be used to facilitate the insertion of cloned genesinto specific sites in the chromosome.

2.3 Transposition Transposons are versatile molecular genetic tools that have several applications in streptomycetes (86,87), including the stable insertionof cloned of transposons have genes into the chromosome(88). Recently, two types been developed for streptomycetes which may be usefulfor this application.

2.3.1 Tn4556andDerivatives Tn4556 is a 6.8-kb transposon isolated from the neomycin-producing S. fradiae (89,90). DNA sequence analysis indicated that Tn4556 was related to Tn3 (91,92). A viomycin resistance gene was insertedinto Tn4556 in different locations to generate Tn4560 and Tn4.563 (W, Table 2). Tn4560 or Tn4563 can transposeinto different sitesin the chromosome or plasmids in S. avermitilis (93,94), Streptomyces coelicolor (60,95,96), the neomycin-producing S. fradiae (89), Streptomyces lincolnensis (88), S. Iividans (89), and Streptomyces tendae (97). to insert heterChung and Crose (88) showed that Tn4560 can be used ologous or homologous DNA into the chromosome by first inserting Tn4560, then exchanging a Tn4560 derivative for the resident Tn4560by homologous double crossover, or transposon exchange,from a plasmid delivery vector. They inserted 13 kb of S. lincolnensis DNA into the S. lividans chromosome, and 6.6 kb of S. lincolnensis DNA into the S. lincolnensis genome using this method. Transposon exchange has advantages for constructing high-yielding secondary metabolite production strains.

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Table 2 StreptomyceteTransposons

Size Transposon Refs. Feature9 (kb) Tn5096 Tn5099 Tn5099-IO Tn5100-4 Tn4560 Tn4563 Tn5351 Tn5353

Origin

3.0

AmR

4.4

IS493 IS493 Tn5099 3.3 IS493 1.8 Tn4556 8.8 8.8 Tn4556 Tn4563 11.1 8.8 Tn4556

HmR,VIE, RRS HmR, MCS, RRS, HT HmR, MCS, RRS, AORF A B , HT VmR VmR VmR, I W B NmR, I W B

7, 86, 87 86, 87, 100 87, 103 103

90 90 98, 99 98, 99 * A m R , apramycin resistance gene; HmR, hygromycin resistance gene; VIE, catechol dioxygenase gene; RRS, contains rare restriction sites; MCS, multiple cloning site; HT, hypertransposition; AORF AB, lacks ORF A (unknown function) and ORFB (transposase gene); VmR, viomycin resistance gene; luxAB, luciferase genes.

First, many different insertion mutants can be screenedfor stability and for the lack of deleterious effects on secondary metabolite production. The most sutiablemutants can then beused as recipientsfor transposon exchange to insert genesinto the neutral site. Transposon exchange also circumvents the problem of inserting homologous genes directly by transposition. With Tn4560 and TO096 (see the next section), homologous recombination occursat higher frequenciesthan transposition when homologous DNA is contained in the transposon. Schauer and co-workers constructed derivatives of Tn4.556 and Tn4563 that contain promoterless luxAB genes from Vibrio harvii close to the right-end inverted repeatand reading inward(98,W;Table 2). These promoter probe transposons allowfor the detectionof light emission over a 103-fold range (see Section 4.3.3.bfor a discussion ofluxAB) and should be useful to identify and clone promotersthat display differentpatterns of regulation of gene expression.

2.3.2 Tn5096 and RelatedTransposons M 0 9 6 (7) and Tn5099 (100) were derived from the 1.6-kb S. Iividans insertion sequence IS493(101). Tn5096 is a 3.0-kb transposon that contains an apramycin resistance gene ( A m R ) inserted between the two ORFs of IS493(Table 2); Tn5096 can be delivered to Streptomyces on temperature-sensitive plasmidsby transformation, transduction, or conjugation from E. coli (86,87; see Section 3). Tn5096 inserts relatively randomly

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317

and has a broad host range, including S. ambofaciens, Streptomyces cinnamonensis, S. coelicolor, S. fradiae, S. griseofuscus, Streptomyces mseosporus, and S. thermotolerans (7,87,102). Analysis of Tn5096 transposition in a tylosin (Tyl) production strain of S. fradiae indicated that about one-halfof the transposition events had no effectTyl on yields(86). This suggests that Tn5096 neutral insertion mutants might be useful recipients for the insertion of heterologous or homologous genes by the transposon exchange method described above. Two Tyl genes were inserted into Tn5096 and the resulting transposon (Tn5098) was delivered into S. fradiae by conjugation from E. coli: the plasmid integratedinto the chromosome by homologous recombination in the tyl gene cluster (86; P. J. Solenberg and R. H. Baltz, unpublished). However, the same transposon inserted into the S. griseofuscus genome by transposition (7). Therefore, Tn5096 can be used directly to insert heterologousgenes by transposition, and like Tn4556 derivatives, can probably be useda as target for transposon exchange for the neutral insertion of homologous or heterologous DNA. Tn5099 isa 4.4-kb transposon constructedby inserting a promoterless xylE gene and a hygromycin resistance gene (HmR) closeto the left end of IS493 (100; Table 2); Tn5099 thus functions as a portable promoter probe. An advantage of Tn5099, well as as of the IwcAB promoter probe transposon described above, is that promoter activity canbe measured in situ in the chromosome under different physiological conditions. For example, Tn5099was used to identify promoters inS. griseofuscus that are sensitiveto or insensitiveto high phosphate concentration(100). Tn5099 may be useful to clone promoters with particular attributes since it is E. coli. Tn5099 also relatively smalland since the HmR gene expresses in contains several restriction sites found infrequently in streptomycetes (AseI, DraI, and SspI); thus Tn5099 insertions can facilitate physical (100). mapping of genes by pulsed-field gel electrophoresis analysis A hypertransposing derivative of Tn5099 lacking the xylE gene has been identified (87,103). This derivative, Tn5099-10 (Table2), is useful in preparing libraries of transposition mutants in different streptomycetes because of its elevated transposition rates. Another hypertransposing derivative, Tn5100-4 (Table2), contains only the inverted repeats from Tn5099-10, HmR, a multiple cloning site and restriction cleavage sites found infrequently in streptomycetes(AseI, DraI, PacI, SpeI, and SspI). The transposable cassette transposes at elevated frequencies from a ts plasmid that contains ORFAand ORFB from IS493 outside the cassette. Tn5100-4should be useful to make very stable insertion mutations, for

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stable insertion of the cloned genes into the chromosome, to map sites of insertion, and to clone DNA flanking sitesof insertion (103).

3 GENETRANSFERSYSTEMS 3.1

Protoplast Transformation

Protoplast transformation induced by polyethylene glycol was the first method to introduce plasmid DNA into streptomycetes (104,105), and has been adapted for many different species (106-1 14). Protoplast transformation can be used to introduce relatively small plasmids containing cloned genes into many species. However, many streptomycetes express restriction systems(1 15)that can lowerthe efficiency of protoplast transformation, and in some cases blockit completely (106,109,112,113). Restriction barriers can be circumvented by usingmutants defective in rea host that modifies striction (1 13,116)or by passaging the plasmid through the plasmid DNA to protect against restriction (106,113). However, restriction can be a significant problem if the objective is to introduce a complete chromosomal library of streptomycete DNA into a relatively wild-type strain.

3.2 ConjugationBetweenStreptomycetes

As mentioned in Section 2.1, some streptomycete cloning vectors are self-transmissible. An example is pIJ922, which can be used to transfer cloned genes betweendifferent S. coeficoforstrains for complementation analyses (95). A disadvantage of heterospecific conjugation is the need for counterselection against thedonor and the need to use combinations of strains that do not inhibit each other’s growthby their respective secondary metabolites. This method hasnot yet been widely usedfor interspecies transfers.

3.3 Conjugationfrom E. coli toStreptomycetes A powerful new method to introduce plasmid DNA into streptomycetes is by conjugation with E. coli (1 17,118). The method capitalizes on the broad host specificity of the IncP-type gram-negative plasmid RP4 or RK2. Transfer is dependenton inserting the originof transfer ( o r i n from RK2 into an E. cofi-Streptomyces shuttle vector, and maintaining the tra functions of RP4 or RK2 in E. coli. The systemwas based on earlier observations that demonstrated facile conjugation into gram-negative

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bacteria (1 19)and into single-cell, gram-positive bacteria (120). Mazodier et al. (117) showedthat plasmids containing the pIJlOl and pBR322 origins of replication could be transferred from E. coli S17-1 into S. lividans, Streptomycespristinaespiralis, and Streptomyces viridochromogenes. Smokvina et al. (74) constructed plasmidsthat contain the RK2 oriT and the pSAM2 attP, int, and xis functions; these plasmids conjugate from E. coli S17-1 into S. lividans and insert into the pSAM2 attB site (see Section 2.2.1). Bierman et al. (83) have extended these observations by constructing a series of plasmid vectors that have different combinations of versatile features: bifunctionality for E. coli and streptomycetes, markers that express in both E. coli and streptomycetes, monofunctionalityfor E. coli replication, oriT from RK2 to drive conjugal transfer from E. coli to streptomycetes, bifunctionality for E. coli replication and 4C3 1 att/intmediated integrationinto streptomycete genomes, and temperature-sensitive plasmid replication in streptomycetes. These plasmids can be used for gene disruption, gene replacement, or stable insertion of genes into the chromosome. Two examples for gene disruption (pOJ260) and cosmid cloning (pKC436)are shown in Table 1. The frequency of conjugal transfer of plasmids containingoriTfrom E. coli S17-1 to S. fradiae C373.17 is very high (83).S. fradiae C373.17 is highly restrictingfor plasmid DNA from E. coli or from other streptomycetes (106,116,121) and is difficult to transform by PEG-mediated protoplast transformation. Thus it appears that conjugation fromE. coli, which presumably is mediated by a single-stranded plasmid concatemer, effectively circumvents restriction in this strain. A recent study (122) has shown that cosmid pKC436 (83) containing large insertsof Saccharopolyspora DNA was transferred by conjugation fromE. coli S17-1 to Saccharpolysporaspinosa and formed stable recombinantsat high efficiency by recombinationinto the chromosome. SinceS. spinosa may be even more restricting than S. fradiae (123), the results suggestthat intergeneric conjugation from E. coli to streptomycetes and other actinomycetes may bean effective means of circumventing restriction barriers. Baltz et al. (86) have constructed plasmid pCZA186that contains the RK2 oriT and Tn.5096 (see Section 2.3.2)inserted into the temperaturesensitive plasmid pGM160 (124; Table 1). This plasmid conjugates from E. coli S17-1 into S. fradiae and can deliver TnZ096 effectively by a temperature-shift method. Similar plasmid constructions have been made with Tn5099-IO and Tn5Z00-4, and both have been conjugated from E. coli S17-1 into S. fradiae and S. griseofuscus (103) and transposed at ele-

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vated frequencies. It is likelythat these plasmidsw l ibe suitable to facilitate Tn5096, Tn5099-ZOY and Tn5ZOO-4transposition in other streptomycetes. Gormley and Davies (125) have shown that RSFlOlO can be transferred by conjugation from E. coli S17.1 to S. lividans and Mycobacterium smegmatis. This gram-negative plasmid replicated and expressed streptomycin resistanceand sulfonamide resistance the in recipients. Thisprovides further evidence that conjugation from E. coli can be an effective means to introduce DNA into streptomycetes and other actinomycetes. 3.4 Transduction of Plasmid DNA

Since plasmid pIJ702 hasa broad host specificity in streptomycetes, its utility as a cloning vector is limited primarily by the ease by which it can be introduced into different streptomycetes. As discussed in Sections 4.4.4 and 5.3, pIJ702 has been used in many studies on protein secretion in S. lividans, a convenient host for protoplast transformation (39). To broaden the utility of pIJ702, McHenney and Baltz cloneda segment of bacteriophage FP43 DNA into pIJ702 that caused the resulting plasmid, pRHB101, to be transducibleby bacteriophage FP43into many streptomycete species (126,127). Hahn et al. (128) showed that the segment of DNA that mediates transduction contains an origin for headful packaging of DNA (pac).This system is versatile in that the physiological state of the recipient cells can be manipulated before transduction to minimize restrictionbarriers(126,127,129).McHenney and Baltz(130)showed that DNA can be clonedinto pRHBlOl without disruptingtransduction or plasmid stability. A similar transducible plasmid, pRHB106 (102; Table l), was derived from pMT660, a temperature-sensitive derivative of pIJ702 (131). Transposon Tn5096 was inserted into pRHB106, and the resulting plasmid, pRHB126,was transducible at high frequencies (102). Tn5096 transposed into the chromosome of several Streptomyces species from this plasmid, including S. ambofaciensy Streptomyces cinnamonensis, S. coelicolor, S. fradiae, and S. thermotolerans (102). These results suggest that pRHBlOl and related plasmids might alsobe useful to introduce other genes into a wide range of streptomycetes.

4 GENEREGULATION 4.1 GlobalMechanisms

4.7.7 StringentResponse The stringent responseto amino acid starvation in E. coli results in the cessation of stable RNA synthesis and is mediated by ppGpp (132). The

Expression Gene

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stringent responseor ppGpp formation has been demonstrated in many Streptomyces species (133-140). An important question is whether the stringent response is involved the in transition from primary metabolism to secondary metabolism.If so, the stringent response might be exploited to overproduce secondary metabolites. Air and Vining (133) showed that many species of Streptomyces produce ppGpp and pppGpp but that it was unlikely that either controlled the initiation of streptomycin production in S. griseus. Ochi isolatedand characterized relC mutants of a number of Streptomyces species (140142). The generalfeatures of the "relaxed" mutants were reduced ppGpp accumulation duringamino acid deprivation, continued RNA synthesis during amino acid deprivation, reduced antibiotic production, delayed onset of aerial mycelium formation, temperature sensitivity, increased sensitivity to erythromycin, increased thiopeptin resistance, reduced growth rate, and loss ofthe ribosomal proteinST-L1 1. Since thesemutants were defective in ppGpp production and stringent response, Ochi suggested that the stringent responsemay be involved in signaling the onset of secondary metabolism. An alternative interpretation is that the negative effects on antibiotic biosynthesis and sporulation may be pleiotropic responses due to altered ribosome structure, not directly relatedto ppGpp formation. BascarAn et al.(137) showed inS. clavuligerus that RNA synthesis was under stringent control after nutritional shiftdown from complex medium, but that cephalosporin biosynthesis initiated whileppGpp levels remained at basal levels. They observed elevated ppGpp levels only ina medium that gave poor growth and poor cephalosporin biosynthesis. Their results didnot rule out a role for ppGpp inantibiotic biosynthesis; however, they demonstrated no obligatory relationship between the stringent response and cephalosporin biosynthesis. Strauch et al.(138) showed that amino acid depletion,or the addition of serine hydroxamate, ledto increased levels of ppGpp and decreased transcription of the rrnD rRNA set of genes S. in coelicolor A3(2). However, transcription of the actZZgene and productionof the blue pigmented antibiotic actinorhodin(Ar)did not necessarily occur immediately following elevation of ppGpp. Also a relC mutant did not behave differently from its parent strain in production of Ar and the red pigmented antibiotic undecylprodigiosin (Red), with or without a nutritional shift-down (see Sections 4.2.1 and 4.2.8 for more detailson the Ar and Red antibiotics). They concluded that elevatedlevels of ppGpp are not sufficient to initiate antibiotic biosynthesis inS. coelicolor A3(2), but that the transient increase in ppGppat the end of exponential growth,and its occurrence instation-

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ary phase cells, might indicate a requirementfor ppGpp for the expres-

sion of genes involved in secondary metabolism. proTakano et al. (143) have exploredthe relationship between ppGpp duction and activation of the Red biosynthetic pathway. They followed the transcription of the redD gene, a pathway-specific activator (see Section 4.2.8), the transcription of the redX gene, and the production of Red after nutritional shiftdown, or after the addition of serine hydroxamate. Under these conditions, they could not show a direct causal relationship between ppGppproduction and activation of the Red pathway. They also expressed the redD gene from a multicopy plasmid;and under these conditions, redD transcription, redX transcription, and Red production occurred in exponential phase, prior to the normal elevatedproduction of ppGpp. Theirdata showed that Red production was controlled by the expression ofredD, and that ppGpp was not a sufficient physiological signalfor the activation of transcription of Red biosynthetic genes. In E. coli, relA mutants are defective in the production of the stringent factor that is required for biosynthesis of ppGpp and pppGpp. It seems likely that the analysis of antibiotic production in relA mutants of streptomycetes might yield more conclusive results on the role of stringent responseand ppGpp or pppGpp production in secondary metabolite production. If there is a direct role, a cloned relA gene might be manipulated to give earlier and higher-level expression to enhance secondary metabolite production.The Streptomyces relAgene hasnot yet been described inthe literature but might bea useful future target to help clarify this issue. 4.1.2 Sigma Factors Since the provocative demonstration of the existence of two distinct forms Streptomyces coelicolor(144), it has of RNA-polymerase holoenzyme in become apparent that streptomycetes encode multiple forms of the sigma subunit of RNA-polymerase (145-153; for reviews, see Refs. 154-156). One sigma factor of particular interest, uwhiG,is involved in a very early stage of sporulation within aerial hyphae(41,147,153) and thus appears to operate a major developmental switch. Consistent with this notion was the observationthat overproduction of uWhacaused ectopic productionof spores insubstrate mycelium and inhibited Ar production (147). Tan and Chater (153) cloned two S. coelicolor promoters that require uwhiGfor transcription. The ,promoters were not expressed during early vegetative growth, and their maximum activity was observed when aerial mycelium was abundant. Both promoters resembled the Bacillus B promoter involved in motilityand the coli-form 6 promoter involved in chemotaxis.

Expression Gene

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They also resembledsix other streptomycete promoters involved antiin biotic resistance orantibiotic production. The functional significance of the latter observation is not known. Since these promoters are responsive to aWhiG, they might conceivably be applied to overproduce specific proteinsunderconditionswheretheispresentinmulticopyoroverexpressed. (147) A singlewhiG gene appearsto be present in most streptomycetes and in other sporulating actinomycetes (157),consistent with its crucial role in the sporulation process. In S. coelicolor, four genes, hrdA, hrdB, hrdC, and hrdD (145,148),potentially encoding sigmafactors similar to Ou' of E. coli (158),have been identified. BothhrdC and hrdD are nonessential for normal growthand metabolism, whereashrdB appears to be required for viability (150).Streptomyces aureofaciemalso has four genes (hrdA, hrdB, hrdD, and hrdE) that encode similar u70-like functions (152). The hrdA homolog was disrupted and the recombinant was indistinguishable from the parent strain in growth, morphology, differentiation, or antibiotic production (159). Both hrdB and hrdD were expressed in cultures growing in minimal medium. From the two studies it is not clear what the functions are for the hrdA, hrdC, hrdD,or hrdE genes, whereas hrdB may encode a principal u70-likeU factor. Also, it is not yet knownif any of the secondary metabolite biosynthetic pathways or secretion pathways in any streptomycetes utilize specific sigma factors during transcription, although thisseems likely. Thisarea is ripe for exploration and potential industrial application. 4.1.3 A-Factor A-factor (2-isocapryloyl-3R-hydroxymethyly-butyrolactone) is a small molecule regulatory factor involved in switching on secondary metabolism [both sporulation and streptomycin (Sm) biosynthesis] inS. griseus (160-166). Other autoregulators have been reviewed (162,163)and are not discussed here. A-factormutants are deficient inSm production and sporulation, but both deficiencies are readily relieved by the addition of a very low amount of A-factor to the culture medium.It appears that A-factor exerts its influence on secondary metabolism by binding to a specific A-factor-binding proteinwhich in the absence of A-factor acts as a repressorof certain genes. At least three mWAs encoded fromaphD, strB, and strR (a positive regulatory gene) were detectable inS. griseus only in the presence of A-factor, consistent withthe notion that the Afactor-binding proteinacts as a repressor. The promoters for aphD, strB, and strR were clonedinto a promoter probe vector using mdh the reporter gene fromThermusflavus. Only the strR promoter fusionshowed enhanced

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expression of the mdh message (four- to fivefold) and protein (about twofold) in response to added A-factor (161,164). Thus it appears that the major direct actionof A-factor may be mediated by the derepression or activationof the strR gene, whoseproduct is a positive regulatory protein(seeSection 4.2.5). Further experimentssuggested that A-factor may mediate its influence on strR expression through the action of yet another activator protein, distinct from A-factor-binding protein. Gel retardation studies identifiedthe putative activator protein (X) that binds upstream from the transcription start site of the strR gene. Horinouchi and Beppu (164) proposed that A-factor-binding protein blocksthe expression of protein X, the activator of the strR gene. In the presence of A-factor, protein X biosynthesis is derepressed; protein X activates the biosynthesis of StrR by bindingto a specific sequence300 bp upstream of the transcription start site; and StrR activates the transcription of Sm biosynthetic genes. A subtlety of this model, supported by additional experimental data, is that the aphD gene, a major Sm resistance gene, is turned on immediately after the strR gene is activated by read-through from the strR promoter. The general significance of A-factor or other related small molecules (164,165) in globalcontrol of secondary metabolism remains unclear. Afactor appears to be produced by greater than 50% of actinomycetes screened (166), but its role in activating secondary metabolism has been documented extensively only in S. griseus. In S. coleicolor and S. lividam, A-factor does not appear to be required to trigger antibiotic biosynthesis; rather, it appears to be a parallel pathway controlledby afsR (see below). 4.7.4 afsR The afsR gene appearsto be a global regulatory gene S. in coelicolorand S. lividans (164,167-169). Introduction of the clonedafsR gene into these strains caused enhanced antibiotic biosynthesis and increased A-factor production. The cloned afsR gene can also suppressthe effects of absA mutants (see below). The AfsR protein contains two consensus ATP(168). The binding sequencesand two consensus DNA-binding sequences purified AfsR protein was phosphorylated by membrane fractions of S. coelicolor and S. lividans by transfer of the y-phosphate ofATP (169). An adjacent gene, afsK, encodes a serinehhreonine kinase that phosphorylates the AfsK protein and phosphorylates serine or threonine residues in the AfsR protein (170). The AfsR protein and the AfsR-kinase (AfsK) appearto comprise a two-component systemfor signal transduction (Table 3), AfsK as a membrane-associated sensor, and AfsR as the

Evidence for function S. lividans S. Iividans S. coelicolor S. cQelicolor

s. CQeliCokM

coel~color coe~icolor coelicolor coelicolor coelicolor coelicolor hygroscopi~us S. p e u c e t i ~ S. ~ ~ u c e ~ i u s S. ambofaciens S. griseus S. glaucescens S. coelicolor S. S. S. S. S. S. S.

afR ufsK afsQ1 afsQ2 absA

absB abs-276 abs-364 abaA

actII-OW4 actII-OWl

brpA dnr1 dnr~ srmR strR tcmR redD

Two-component regulator Two-component sensor Two-component regulator Two-com~onentsensor Transcriptional activator TranscriptiQnalactivator ~nknown Unknown Unknown Two-component regulator Repressor Two-component regulator Two-com~Qnentregulator T~o-compQnent regulator Transcriptional activator scriptional activator Repressor Two-com~Qnentregulator

Refs.

164, 167-169 Genetic 170 Genetic, biochemical 164, 171 Genetic, biochemical, sequence 171 Sequence 172-174 Genetic 172- 174 Genetic 174 Genetic 174 Genetic 175 Genetic 181, 184, 186 Genetic, sequence 181 Genetic, sequence 189, 190 Genetic, biochemical, sequence 66, 193, 194 Genetic, sequence 193, 194 Genetic 195 Genetic, biochemical Genetic, b i ~ ~ ~ ~ c a l 197 204, 205 Genetic, biochemical Genetic, b i o c h ~ sequence ~ c ~ ~ 143, 212, 213

8

22. 0

326

Baltz

regulator that activates transcription. The deduced aa sequence of the AfsR protein showed similarityto the streptomycete-positive regulatory proteins actII-ORF4, dnrRI-ORF1, and redD-ORF1 (see Section 4.2), but not to OmpR or PhoB from E. coli. 4.1.5 afsQ Like afsR, the cloned afsQl gene of S. coelicolor caused enhanced A r , Red and A-factor production in S. lividans (164,171).The deduced aa sequence of afsQl showed high similarityto regulatory proteinsof twocomponent regulatory systems (Table 3). The afsQ2 gene, which showed sequence similarityto the sensor proteins, was located just downstream of the afsQl gene. The deducedafsQ2 protein contained the highly conis the siteof phosphorylation in sensor kinases. served His residue, which The TGA termination codon of afsQZ overlapped the GTG initiation codon of afsQ2, suggesting that the genes were cotranscribed (and possibly translationally coupled).The afsQZ gene contained sequences similar to the - 35 and - 10 consensus prokaryotic promoter sequences upstream of the start codon (see Section 4.3.1). The afsQI message was detected only after 3 days of growth, and increased at 4 days, just preceding the onset of Ar production. The afsQI gene contained the conserved Aspfor phosphorylation at residue 52 (171). Site-specific mutagenesis of the gene, causing the insertion of Glufor Asp, abolished its positive regulatory effect Ar onbiosynthesis. This result provided direct evidence that afsQI mediates its effects on secondary metabolism by sensory transduction. Gene disruption of afsQZ or afsQ2 in S. coelicolor did not blockAr, Red, or A-factor production, however. afsQI was able to suppress an absA mutation, just as afsR (see below). DNA sequences that hybridized to afsQl or afsQ2 were observed in many different streptomycetes. These findings suggest that S. coelicolor and S.lividans, and presumably other streptomycetes, use one or more twocomponent signaltransduction systems to sense the nutritional environment and to participate in the switch from primary to secondary metabolism. Much additional work is needed to identify the effector(s) that interact with the AfsQ2 putative sensor proteinand the sites of binding of the putative regulatory proteinAfsQl

.

4.1.6 abs Champness and co-workers (172-174)have identified two loci Sin . coelicolor that are involved in the regulation offour all S. coelicolor antibiotics

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327

(Table 3). Mutations inabsA resulted inthe complete loss of production of Ar, Red, methyleneomycin (Mm), and calcium-dependent antibiotic (CDA). TheabsB mutants hada similar phenotype but were slightly leaky on some media. The absAand absB mutations mapto different lociand appear to block the regulationof antibiotic biosynthesis ratherthan block a step common to the biosynthesis of these diverse structures.Introduction of the Ar-positive regulatory gene, actII-ORF4, on the high-copynumber vector pIJ702, overcame the block inAr biosynthesis in the absA and absB mutants, consistent with the notion that absB is a regulatory gene (174). Also, preliminary studies indicatedthat abs mutants transcribed the act1 and redX promoter-driven xylE gene (see Section 4.3.3.a) much less efficientlythan did thea h + parents. The abs mutations did not affect sporulation. Two classes of second-site absA suppressor mutations have been isolated and both produce all four antibiotics. Revertants containing class I1 suppressors (sap), which mapped close to or within absA, overproduced Ar and Red. Champnesset al. (174) have identified a segment ofS. coelicolor DNA (mia)that inhibits the production of Ar, Red, and CDA when present on pIJ702 butnot when present on a lowcopynumber plasmid. The mia gene appears to map at a different location than absA and may define another developmental locus. Three DNA segments have been shown to suppress the Ar and Red afsR (see above), nonproduction inabsA and absB mutants. These include abs-276, and abs-364 (Table 3). The nature of the latter two DNA segments is not known.The authors propose that absA and absB may exert their influenceon the biosynthesis of several different antibioticstranas scriptional activatorsfor pathway-specific regulatory genes, suchas act11ORF4 and redD (see Section 4.2). 4.1.7 abaA Another regulatory locus in S. coelicolor, a b d (Table 3), was identified by its abilityto stimulate overexpressionof Ar (175). Disruption of ORFB of the‘locusby insertional mutagenesisof the DNA cloned in4C3 a 1 vecAr production, nearly complete tor resulted in the complete blockage of blockage of Red production, significant reduction in CDA production, but no inhibition ofMm production. TheabaA locus mappedto a siteat about 2 o’clock on the S. coelicolor map, distinct fromabsA (10 o’clock), absB (5 o’clock), afsB (5 o’clock), or afsR (7 o’clock).The DNA sequence of abaA did not suggest any specific function. The results of these studiesare interesting but do not yet identify specific genesand mechanisms in all cases. It would be usefulto obtain DNA

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sequences of allthe genes and to search for homologs. Transposonmutagenesis might provide the means to connect the phenotype rapidly with specific DNA sequences. A better understanding of the precise mechanisms and interplay between the various genes and the identification of specific effectors will be important in defining the signal transduction pathways in streptomycetes, and to.determine how they interact with secondary metabolite production. 4.2

Pathway Specific Regulation

4.2.1 Actinorhodin Actinorhodin (Ar)is a blue-pigmented polyketide antibiotic produced by S. coelicolor A3(2). The entire set of 22 genesfor Ar biosynthesis, regulation, and export has been cloned, sequenced, and analyzed in some detail (176-185). The central segment of the act cluster, the actZZ region, contains a transcription activator gene, actII-ORF4, and a gene (actIZORF1) that encodes a repressor for two adjacent genes (actII-ORF2/3) involved in the export of Ar (181,183; Table 3). The introduction of extra copiesofactZZ-ORF4 into S. coelicolor caused overproduction of Ar (186). The actZZ-ORF4 geneappears to encode a transcriptional activator that shows strongaa sequence similarityto the redD gene productand to the N-terminal domain of the afsR gene product (183). Transcription studies indicatedthat the actZI-ORF4 mRNA levels increased dramatically during the transition from exponential to stationary phase. Thiswas followed bytranscription of the actIIZ and act VI--0RFl genes and production of actinorhodin (184). Genedisruption of actIZ-ORF4 causeda blockact messages and blockage age or strong reduction in transcription of several in Ar biosynthesis (183). Parro et al. (179) have shownthat the actZ-actZZZpromoter region has a region of dyad symmetry that might sequesterpart of the actzpromoter in a cruciform structure. They postulated that this structure might make the promoter inaccessible to RNA polymeraseand impede progressionof the actZZZ transcript. A positive regulatory protein might interact with this region to allow transcrption from both promoters. 4.2.2 Bialaphos Bialaphos is a linear tripeptide herbicide producedby Streptomyces hygroscopim. The geneticsand biosynthesis of bialophos have been studied extensively (for reviews, see Refs. 18 and 187). The bialophos biosynthetic pathway is subject to activation by the product of the brpA (188-190; Table 3). The brpA gene is transcribed from two promoters within an

Expression Gene

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329

intergenic regionand at least one more promoterfarther upstream within the bialophos gene cluster (189). Transcriptionfrom all three promoters increased just before the stationary phase. The BrpA protein contains a helix-turn-helix DNA binding motif found in the regulator component of twocomponent regulatory systems. Analysis of proteins expressed in S. hygroscopicus using two-dimensional gel electrophoresis of a brpAdeficient mutant and a brpA-proficient strain indicated that 27 proteins were dependent on brpAfor optimal expression, including 10 that mapped to a 10-kb fragment from the 35-kb bialophos gene (bap) cluster (190). Expression of the bap gene cluster coincided with the elevated synthesis of the stringent response mediator ppGpp (but see Section 4.1.1).

4.2.3 Daunorubicin Daunorubicin (DNR) is an anthracycline antitumor antibiotic produced by Streptomyces peucetius. Genes encoding DNR biosynthesis and resistance have been cloned in cosmidpKC505 (65,66,191,192). Two segments of cloned genes, the dnRI locus containing dnrl and dnrJ genes and the dnR, locus containing two open reading frames, caused about a 20-foldoverproductionofc-rhodomycinone (RHO), an intermediate in DNR biosynthesis, when expressed from a low-copy-number vector; the dnRz segment also caused a twofold increase inDNR production (66). The effects onRHO and DNR biosynthesis were more pronounced when dnRI and dnR, were expressed from high-copy-number plasmids. The two open reading frames located in dnRI (dnrl and dnrJ) were sequenced: the dnrl gene product showed significant aa sequence similarityto the S. coelicolor actll-Orf4, afsR, and redD-Orfl gene products. The functional relatedness of the dnrR, locusto that of actll-Orf4 was established by showing that the cloned dnrIJ gene fragment caused overproduction of blue pigment (assumed to be Ar) in S. lividans and complemented an actll mutation inS. coelicolor. It appeared from these and other studies that the dnrl gene product acted in trans. Insertional inactivation of dnrl in the wild-typeS. peucetius strain blocked RHO and DNR production and reduced the level of resistance to DNR; however, DNR production was restored by introducing a functional dnRI fragment on a lowcopy-number plasmid.The dnrl gene, which functions as a positive regulator, also appears to be under positive regulatorycontrol ,by yet another response regulatory gene dnrN (193,194; Table 3). 4.2.4 Spiramycin Spiramycin (Srm) is a 16-member macrolide antibiotic produced by S. ambofaciem. SeveralSrm biosynthetic genes have been identified by cosmid

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cloning of Srm resistance genes, and analyzing cloned genesfor heterospecific complementationof a tylB mutation in S. fradiae, or by gene disruption and gene transplacement in S. ambofaciens (64). Two mutants were defective inboth prelactone and postlactone biosynthesis; these defined a regulatory locus srmR. A 9-kb DNA fragment containing the srmR locus was introduced into S. fradiae PM73, atylB mutant that produces tylactone,an intermediate inTyl biosynthesis (121); the recombinant strain produced four times the level of tylactone as PM73 (64). Geistlich et al. (195) explored the mechanism srmR of regulation using a fusion of a promoterlessxylE gene (see Section 4.3.3.a)to the srmG gene. In a srmR mutant backgrown, the srmG-xylE fusion gave no expression of the xylE gene. However, whenthe srmR gene was inserted into the chromosome at the 4C31 attB site using cosmid pKCl163, the colonies expressed the xylE function. The results indicated that srmR encoded a trans-acting function required for srmG transcription. The srmR gene has an open readingframe that should encode a 64.8kDa polypeptide that does not share significant aa similarity with any reported protein sequences. The srmR gene contains one TTA codon (see Section 4.4.2). The SmR protein is required for the transcription of srmG and srmX, but not for srmR or srmB, a spiramycin resistance genethat is inducible by Srm. The srmG and srmX promoter regions show similarity to each other in the - 39, -27, and - 10 regions, but notto other streptomycete promoters (195). Introduction of srmR into a stable derivative of the wild-type S. ambofaciens caused an increase in Srm production from about 100 pg/mLto 500 pg/mL. This observation, coupled with the fourfold enhancement of tylactone productionS. fradiae in PM73, that can suggests that srmR encodes a transcriptional activator (Table 3) function in related 16-member macrolide biosynthetic pathways. It is not known if the srmR gene product can activate more distantly related macrocyclic lactone pathways. 4.2.5 Streptomycin The molecular genetics of streptomycin (Sm) biosynthesis has studbeen ied in Streptomyces griseus and Streptomyces glaucescens. Of the 25 to 30 genes directly involved in Sm biosynthesis, or 1819 have been analyzed in one or both streptomycete species (196,197). The organization of the Sm gene clusters is similar. The overall DNA sequence homology between genes of identical function ranged from 60 to 85% identity, indicating that the pathways diverged in the distant past. Both gene clusters contain a positive regulatory gene, strR (197; Table 3), which encodes proteinsthat are 62.5% identical in aa sequence. The

Expression Gene

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331

StrR gene products are DNA-binding proteins that activate transcription of at least the strBI genes. The StrR proteins bind to at least two specific DNA sites within thestr gene clusters in both species. One site was located upstream of strBZ promoters and the other within the strR reading frames.The DNA binding sites contained conserved inverted repeats. StrR was shownto be sufficient and required for the activation of strBl transcription. The strR gene expressed in trans in S. lividans resulted in a reduction in xylE expression from strD or strS promoters, suggesting that StrR may exert negative regulatory effects on other genes. In these cases,no StrR-binding siteswere observed upstream ofthe strD or strS genes. Both strR genes, as well as two other genes that are the first in their respective transcription units(strA and strN), contained single TTA codons (see Section 4.4.2). Like S. coelicolor (198), the Sm-producing S. griseus contains a single bldA gene encoding tRNA leumA,and a bldA mutant gave a nonsporulating (bald) phenotype (199). Distler et al. (196) suggested that independently expressed key genes in Sm biosynthesis could be regulated coordinatelyat the translationallevel bybldA gene expression (see Section 4.4.2). Further work is needed to confirm this. In summary, Sm biosynthesis appears to be controlled by pathway-specific positive regulation (strR), by A-factor (see Section 4.1.3), and possibly by bldA (but see Section 4.4.2).

4.2.6 Tetracenomycin Tetracenomycin (Tcm) isan antitumor agent that is assembled by a type I1 polyketide synthase. The structural organizationtheoftcm gene cluster is wellestablished, and many of the gene functions have been determined (200-203). Resistanceto TcmC is inducible by TcmC. Guilfoile and Hutchinson (204,205) showed that the resistance gene tcmA and a repressor gene tcmR (Table 3) are regulated by the TcmRprotein, which binds to an operator regionthat encompasses the divergently transcribed promoters for tcmR and tcmA. The deduced TcmR protein showed sequence similarity to the actZ1-ORF1 repressor and to the tetR regulatory genes from E. coli. 4.2.7 Tylosin nlosin (Tyl) is a 16-member macrolide antibiotic produced byS. fradiae. Much is known about the genetics and biochemistry of Tyl production, and many ofthe Tyl biosynthetic genes have been cloned and characterized (18,121,206-209). The case for positive regulation of Tyl biosynthetic genes remains indirect but compelling: certain tylG mutants that retain

332

Baltz

many intact Tyl biosynthetic genes do not express those genes (18,208, 210); the cloned tylF gene that contained about 500 bp of upstream sequences, including a promoter, did not express in an S. fradiae mutant (JS87) deletedfor most or all Tyl biosynthetic genes (206); and the cloned @IF gene expressed in S. lividans at a low level when cloned on a highcopy-number vector (206). The enhancement of tylactone production in strain PM73by the cloned srmR gene from S. ambofaciens (64) suggests a srmR homolog. that one positive regulatorfor Tyl biosynthesis may be

4.2.8 Undecylprodigiosin Undecylprodigiosin (Red) is a pigmented antibiotic produced by S. coelicolor A3(2). The genetics and biosynthesis of Red have been studiedextensively (18,211) and a complete set of genes for its biosynthesis have been cloned (212). Expression of the red pathway is positively regulated by the product of the redD gene (143,212,213; Table 3).Introduction of extra copies ofthe cloned redD gene into S. coelicolor on lowcopy-number (pIJ941) or highcopy-number (pIJ702) vectors caused about a 25-fold increase inRed production in liquidculture relative to S. coelicolor containing pIJ941 (213). The clonedS. coelicolor redDgene also caused overproduction of Redin S. lividans when expressed on the low-copy-number plasmid pIJ940 (212). The redD gene is expressedlate in the growth cycle, and precedes the transcription of redX and Red production in stationary phase (143). Increased copiesof redD resulted in higher levels ofredD and redX transcripts and Red production in exponential phase. Takano et al. (143) suggested a possible role for a minor RNA polymerase holoenzyme containingan alternative U factor in redD transcription. They notedthat the redD gene shows significant sequence similarity to other secondary metabolitepathway activations, actll-ORF4, dnrI, and the 5' end of afsR, but that alignment of the predictedaa sequences failedto reveal a convincing common DNA-binding motif. They suggested that these genes may encode a family of regulatory proteins with a novel means of recognizing specific nucleotide sequences in DNA. Signal transduction schemes employing two-component systems, comprised of sensor and response regulator proteins, are used extensively in bacteria to sense environmental conditions and to respond accordingly (214-221). From previous sections it appears that two-component regulatory systems may function in streptomycetes to regulate secondary metabolism. In some cases, the evidence for regulator functionis compelling, but no evidence exists for the corresponding sensor function (Table3).

Expression Gene

in RecombinantStreptomyces

333

It is possible that the pathway-specific regulator functionsare sufficient alone, or that they are coupled with other sensor proteins that are not pathway specificand therefore not clustered withthe antibiotic pathway genes. For instance, they may couple with sensors for carbon, nitrogen, or phosphate limitation. This is an area that deserves much more work to reap the full benefits in practical applicationsto yield enhancement. 4.3 Transcription

4.3.7 Promoters Strohl (222) compiled 139 DNA sequences apparently associated with transcriptional start sites in streptomycetes. Some of the promoter sequences have been characterized more stringently than others, and a few were studied genetically or biochemically in enough detail to establish the inthe - 35 and - 10 regions for RNA polymerase binding. Some of ferences are from sequence comparisonsand not from functional analyses. I summarize the main points of the review and refer the interested reader to the original article for more detailed discussion. Of the 87 genesanalyzed, 27 had multiple promoters, and 13 contained overlapping divergent promoters,a structure postulated to be involved in complex regulation. In some cases the overlapping divergent promoters were located within or partially within open reading frames. Two promoters initiatedtranscription from the middle of multigene operons that also had promoters upstream of the first gene of the operon, and at least six promoters were located within ORFs. Strohl(222) noted that 29 of the 139 promoters contain - 35 and - 10 regions very similarto the consensus prokaryotic promoterthat is recognized by RNA polymerase containing u70-like subunits. The - 35 and - 10 regions were separated by 16to 18 nucleotides. The consensus streptomycete promoter contained 'ITGAC(Pu)at the - 35 regionand TAg(Pu)(Pu)T at the - 10 region, and was similar to the consensus TTGACA (- 35) and TATAAT (- 10) for E. coli, which also has a 16- to 18-nucleotide spacing. Some of the streptomycete promoters in thisgroup were functional inE. coli. The other 110 promoters did not conform to consensus prokaryotic promoters. Some showed sequence similarities to other promoters in the group in the- 35 or - 10 regions, but the functional significance is largely unknown. Also, some had sequences similar to the - 10 consensus sequence but not to the -35 consensus sequence for u70-like responsive promoters.

334

Balk

Some streptomycete promoters expressed only late the in growth cycles of the respective organisms,but three such promoters showed no significant sequence similarities from-40 to + 1. Therefore, itis not clear what constitutes temporally regulated promoters, much less what U factors might (see Section 4.1.2). interact with them, withthe exception of uwhiG Strohl(222) also reviewed some mutational analyses of promoter function. The important observations in a limited number of examples are summarized as follows: the highly conserved (invariant in the 29 promoters analyzed by Strohl) 3' T of the - 10 consensusprokaryotic sequence may be very important for promoter activity; a spacing of17 nucleotides between the - 10 and - 35 regions may give higher transcription than a spacing of 16 nucleotides in consensus prokaryotic promoters, and interaction of RNA polymerase with the- 35 region maynot be veryimportant with somepromoters. However,no broad generalizations canbe made to predict the timing or level of transcription for particular promoter sequences. On the other hand, most heterologous prokaryotic promoters tested in streptomycetesare functional, and some relatively strong (e.g., ermEPIa; 223) or regulated promoters (see below) have been identified. Furthermore, several promoter probes (see Section 4.3.3)are available to so the opportunity study transcription in situ or after cloning in plasmids, to make rapid progress onthe understanding of regulation of transcription exists.

4.3.2 RegulatedPromoters a. Chitinase. Deli6 et al. (224) described the mutational analysis of two highly regulated chitinase promoters from Streptomyces plicatus. Both promoters have- 35 and - 10 regions that fall within the grouping of consensus prokaryotic promoters. Both chitinase promoter regions also contain 12-bp direct-repeat sequencesthat partially overlap the putative RNA polymerase binding site. Regulation both of genes is repressed by glucose and derepressed by chitin. A single base substitution in one of the 12-bp direct repeats of one of the promoters, which was studied by fusion of the xylE gene (see Section 4.3.3.a), rendered the expression of xylE no longer subject to glucose repression or chitin derepression. This indicated that glucose repression was exerted at the level of transcription initiation, and that the 12-bp direct repeat was involved inbinding a transacting repressor molecule. This system may be usefulfor the regulated expression of single-gene products. b. tipA. tipA is a gene in S. lividans that is inducible at least 100fold by very low levels ofthe peptide antibiotic thiostrepton (225). Thio-

Expression Gene

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335

strepton is produced by Streptomyces azureus but not by S. lividans. The tipA gene appearsto encode two proteins,an 18-kDa protein (Tip&) and a 31-kDa protein(TipA,) which contains a 109-aa N-terminal extension ofthe 18-kDa protein(226).The N-terminal extension in TipA, contains an aa sequence that shows significant similaritiesto the transcriptional activatorsSoxR ofE. coli, MerR ofPseudomonas aeruginosa,and NolA of Bradyrhizobium japonicum. TipA, shows enhanced bindingto the tipA promoter, suggesting that the C-terminal region contains a site for thiostrepton binding. The tipA promoter can be used for regulated expression of genes in S. lividans. Since thetipA promoter is activated by TipA,, its utility may be limited to streptomycetes that produce TipA,-like proteins. Alternatively, the S. lividans tipA gene might be added to other streptomycetes by methods described in earlier sections to ensure the expressionof the tipA promoter in the presence of thiostrepton. c. galPI. The galactose utilization operon of S. lividans, which encodes three genes,galT, galE, and galK, is transcribed from two promoters, galPl and galP2 (227 and references therein). galPl is induced by galactose and galP2 is expressed constitutively. Galactose induction can be partially repressed by glucose, perhaps by inhibiting glucose uptake. galPI directs the transcription of a polycistronic RNA which contains the galT, galE, and galK coding sequences. Two direct-repeat sequences were observed withingalPl(227). Mattern et al. (228)have studied the regulation of galPl by oligonucleotidedirected mutagenesis in the potential operator region using primer extension and transcriptional fusions using thexylE reporter system (see Section4.3.3.a) to measure expression. The putative operator region contains two overlapping motifs that may be involved in regulation. These include the two pairsof imperfect direct repeats mentioned above and a series of six hexamers that conform to the sequence TNTNAN elements that may interact with the helix-turn-helix motif of DNA-binding proteins. They made triple base substitutions at all threeN positions of hexamer I1 and hexamer IVYeither by transitions or transversions. Transition mutations in hexamerI1 caused about a fivefold increase inxylE expression over wild-type in galactose medium, with virtually no expression on glucose or glycerol media. Transversions in hexamer I1 caused about a 10-fold increase in xylE expression on galactose, with little expression (20-fold lower) on glucose and very little on glycerol. Multiple transversion mutations in hexamers I1 and IV resulted in relatively high levels of xylE expression on galactose, glucose,or glycerol.

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In another study, Mattern et al. (229) evaluated the effects of base substitution mutations in the - 10 and - 35 regions of the galPZ promoter. Certain mutations in positions - 11, - 34, and - 36 caused severe reductions in the expressionfrom galPZ, thus establishing that the - 10 and - 35 regions of this promoter function inRNA polymerase interacthat it contains a string of six G residues tions. The - 35 region is unusual in from - 37 to - 32. Most ofthe mutations in this region caused increased expression of galPZ-xylE fusions on galactose medium, some 10-fold higher than control. The results of these studies suggest that the galPZ promoter and operator, and specific mutant derivatives, may be useful for regulated gene expression in streptomyces, using galactose an as inducer. 4.3.3 Promoter Probes and Reporter Genes A number of promoter probe plasmids and reporter genes have been used to analyze transcription in streptomycetes. These include antibiotic resistance reporter genes (230,231), ampC (232), galK (233), E. coli lacZ (234), S. lividans 0-galactosidase (lac; 235), Vibrio harviae luciferase ( I d B ; 236), firefly luciferase (luc; 237), xylE (238,239), and reporter genes that specify secreted enzymes(240)or pigments (241). I will focus on xylE and luxAB, which appear to be well suitedfor different typesof transcriptional analyses. a. VIE. The VIE gene from Pseudomonasputida encodes catechol(2,3)dioxygenase, which converts colorless catecholto a yellow 2-hydroxymuconic semialdehyde (238). The utilityof the xylE gene in streptomycetes was first demonstrated by Ingram et al. (239), who showed that galPZVIE fusions could be used to measure the expression ofthe galPZ promoter under different nutritional conditions. They showed that this system can be used as a visual screen for different levels of expression and can also be used to quantitate enzyme levels in crude extractsof cells. Mattern et al. (228,229) used galPI-xylE fusions to study the effects of different mutations in the galPZ promoter region on gene expression (see Section 4.3.2.c). Bruton et al. (242) have inserted a promoterlessxyIE gene containing a strong ribosome binding site into bacteriophage 4C31 vectors that facilitate cloning of segments of DNA upstream of the xylE gene. The attP deleted derivations can be used to insert the xylE gene so that it is controlled in situ by the regulatory signals that normally act on the DNA inserted in the vector. They demonstrated the utility of the system by cloning a 0.9-kb segmentinternal to an Ar transcription unit in the same orientation as xylE. The phage was used to lysogenize a bldA mutant and its

Expression Gene

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337

bldA+ parent by homologous insertion into the act cluster. The bldA strain expressed catechol dioxygenase (yellow), whereasthe bldA strain did not. This demonstrated that the transcription of an antibiotic biosynthetic gene couldbe monitored by xylE expression, and that the tranbldA gene (see scription of the act gene in question required a functional Section 4.4.2). An advantage of xylE in this study was that it contains no 'ITA codons (see Section 4.4.2). Bruton et al.(242) havealso constructeda V I E promoter probe phage vector that can be inserted site-specifically in single copyinto the 4C31 attB site. Since +C31can lysogenize most streptomycetes, this vector may permit a rapid analysis of expression from specific promoters in many different streptomycete species or in derivatives of a particular species. Other xylE promoter probe plasmids have been constructed (243,244), Srm gene clusand one(244) has been usedto monitor transcription in the ter in situ (195). Also, a promoterless V I E gene located in transposon Tn5099 (100) can be usedto generate transcriptional fusions in situ(see Section 2.3.2). b. luxAB. The marine bacterium V. harveyi produces a light-emitting luciferase enzyme that is encoded bythe luxAB operon (see Ref. 236 and references therein). The luciferase enzyme catalyzes the oxidation of long-chainaldehydes and reducedflavinmononucleotide to generate photons. The long-chain aldehyde can be added exogenously the form in of n-decanal to stimulate light production in mutants unable to synthesize this substrate. Schauer et al. (236) cloned a promoterless luxAB operon probe plasmid pIJ486. The reinto a multicopy streptomycete promoter sulting plasmid, pRSllO5, contains a multiple cloning site upstream of the luxAB operon for the insertion of DNA-containing potential promoters. This plasmid can be usedto analyze the temporaland spatial expression of promoter sequences duringthe process of colony growthand cellular differentiation inS. coelicolor. TheluxAB operon has been incorporated into promoter-probe transposons (98,99; see Section2.3.1) that can be used to assess transcriptional activity in situ. +

4.3.4 LysR Homologs The LysR family of positive regulatory elements is employed fairly extensively by prokaryotes (220,245,246). LysR genes are often adjacent to the genes they activate, are transcribed divergently, and modulate their own expression by repression. Recent studies on 0-lactamase and protease genes has provided strong evidence for lysR-like regulation in streptomycetes (247-251).

ile 4 Potential LysR Boxes Within the Intergenic Region Between lysR Nornolog Genes and Metalloprotease or P-Lactamase Genes Strains

Gene

a unction

S. cacaoi

bla

P-Lactamase

S. coelimlor

mprA

Protease

Umtream seauenc@

Refs.

G C CG T T T C C G CA T A T AT C GC A G G T T C G C A T -

247 250

-.__I

S. lividans ~ t ~ ~ p t o m yC5 ces

slpA

Protease

S. caca~i

SnP blaA

Activator

S. coelicolor

~ p r R Activator

S. lividans

slpR

Protease

C 66 C C C T AT _A_ -T- - A G C C A T A G G G C GGC CCT AT G T C G C G G C C T G G A C -----

248,249

250

-

-

-

-

_

I

-

251

-

S ~ r ~ p ~ ~ mC5 y c e s SnpR

ActivatQr

C T A G C C T C G G T C C GT T C AC C TGG C G C T T AC C C T C C C AG G

1ysR

Consensus

/T N N N N N N N N N N N A "/A245 -

Activator

ahsitions displaying dyad symmetry are underlined.

A T A A A C -

247

248 251

Expression Gene

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339

Streptomycescacaoi expresses a P-lactamasethat is regulated by a LysRlike protein. ThelysR homolog, blaA, is located adjacent to the P-lactamase gene (bla) and transcribed divergently (247). Urabe and Agawara (247) have shownthat the BlaA protein bindsto the intergenic region between blaA and bla, consistent with a potential activator function for the BlaA protein. I have scanned the intergenic region and have noted two potential LysR boxes (T-NI,-A; Table 4), one upstream of the bla transcription start site and one overlapping the - 10 region and transcription start site for blaA. It will beinteresting to see if theseare indeed sites for BlaA binding. Metalloprotease genes from S. lividans (248,249), S. coelicolor (250), and Streptomyces sp. strain C5 (251) have been cloned and sequenced. Each of these genes isadjacent to a lysR homolog that is transcribed divergently. In S. lividans, the cloningof a fragment containingthe metalloprotease gene (slpA ) and the lysR homolog (slpR)on a multicopy plasmid caused the largest increase in protease secretion. Cloning of either of the genes alone also resulted in higher protease production, supporting al. noted the notion that slpR is a positive regulatory gene. Butler et(248) the presence of similar inverted repeat sequences the in intergenic region between slpA and slpR in S. lividans and between snpA and snpR in Streptomyces sp. strain CS, and suggested that there may be sitesfor the binding of regulatory proteins. Upon inspection, I have noted that both of these sequences contain the conserved sequence for LysR binding, the LysR box T-NII-Awhich usually contains some limited dyad symmetry (245). Table 4 shows these sequences. Upon further inspectionof the DNA sequences, it appearsthat the intergenic region inS. coelicolor contains a similar sequence. Furthermore, potential LysR boxes have been found very close to the potential Shine-Dalgarno sequences upstream of the respective lysR homologs (Table 4). These observations are consistent with the notion that the LysR homologs may activate the transcription of the adjacent metalloprotease genes, and possibly repress their own synthesis. Much more work is needed to establish the role of these lysR homologs in protease secretion. This could have a direct on bearing the use ofstreptomycetes for heterologous secretion.An important question is how ubiquitous this type of regulation is in streptomycetes, particularly as it may relate to secondary metabolismand secretion.

4.4 Translation and Protein Secretion

4.4. l Initiation Strohl(222) analyzed 44 streptomycetes genes for their complementarity to the 3’ end of the 16s rRNA of S. lividans and for spacing relative to

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the translational start codon. The genes were chosen to reflect a wide range of functions. The Shine-Dalgarno sequences ranged from 5 to 12 nucleotides (averaging 8.5) upstream ofthe start codon. The calculated binding strength ranged from -2.2 to - 22.2 kcal/mol (averaging - 11.3). The conserved sequence was 5'-(a/g)GGAGG-3'. Strohl noted that streptomycetes express a variety ofE. coli genes, includingampC, which has a relatively poor Shine-Dalgano sequence (5'-TATGGAAJ').In this regard, streptomycetes are similar to E. coli in that they cantolerate a wide range of degrees of complementarity between the Shine-Dalgarno sequence and the 3' end of 16s rRNA. Eleven streptomycete genes do not contain apparent Shine-Dalgarno sequences; transcription and translation are proposed to initiate at the same site. Eight of the genesare involved in antibiotic resistance or cellular differentiation, and at least two ofthe gene products are highly expressed inS. lividans (aph and rph). Jones et al.(252) have studiedtranscription and translation of the aph gene by adding short leader sequences to the message, with or without new ATG start sites. Theaddition of two or four nt untranslated leaders to the message caused reduced translational efficiency. Theaddition of a leader containing an AUG resulted in the use of the new upstream AUG,to the apparent exclusion ofthe normal AUG. The results suggest that the start codon position relative to the 5' terminus is a determinant of translational efficiency in messages that contain no Shine-Dalgarno sequences. In summary, it appears that many different mRNA species, with or without Shine-Dalgarno sequences, can betranslated in S. lividans. It is not knownif a particular sequence gives optimal translation of manydifferent genes in different streptomycete species. This problem is ripefor further analysis and could have important applications for high-level gene expression. 4.4.2 tRNA and lnfrequentCodons As noted previously, streptomycetes displaya very strong bias in codon usage, using predominantly those codons containing G or C the in third position (17-19). Some codons are used veryinfrequently: the least abundant are TTA (Leu), CTA (Leu), and TTT (Phe) (19). The infrequent 'ITA codons have been found predominantly in antibiotic resistance genes, antibiotic regulatory genes, andgenes involved in cellular differentiation, whereas CTA and IITT codons are more widely dispursed among genes of different function (253). The TTA codonis apparently recognizedby a single tRNA encoded by the bldA gene (198,253-255). S. coelicolor mutants containing bldA mutations are defective in aerial myceliumfor-

Expression Gene

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mation and in the production of A r , Red, Mm, and CDA. Additional studies have suggestedthat the bfdA gene may play a regulatory rolein antibiotic biosynthesis bycmtrolling the expression ofantibiotic regulatory genes or resistance genes (254-257). Inthe Ar pathway it was suggested that bfdA exerts developmental control at actIbORF2, encoding a putative transmembrane exportprotein, and actU-ORF4, a transcriptional activator(258). The block inAr production ina bldA mutant could be overcome by convertingthe single TTA codon actll-ORF4 in to TTG; however, Ar was not secreted into the medium, presumably due to the unchanged TTA codon in actII-ORF2. Despite the impressivedata demonstrating that the bfdA gene is needed only for secondary metabolism and for cellular differentiation in S. coelicolor, it is not clear that it has a normal regulatory function. Whereas one recent study showed that mature bfdA-encoded tRNA accumulates only relativelylate in growth (255),another study showedthat functional bfdAencoded tRNA is presentthroughout the S. coelicoforgrowth cycle in liquid cultureand that the lack of production ofAr and Rd in rapidly growing cultures is due solely to the absence of pathway-specific activators (184,259). Also, the expression of the firefly luciferase gene (luc), which contains 11 TTA codons, is quite high in S. lividans, suggesting that the Leu tRNAmAis not normally limiting for translation (237). It may be necessaryto explore the effects of bfdA ina number ofother streptomycetes before its role in the regulation ofantibiotic biosynthesis can be firmly established.

4.4.3 Protein Assembly and Chaperonin Function Molecular chaperones are defined as “a family of unrelated classes of protein that mediate the correct assembly of other polypeptides but are not themselves components of the final functional structures” (260). Molecular chaperones function by binding specificstructural features of proteins that are exposed in early stages of assembly and inhibit incorrect assembly pathways (260,261). Molecular chaperones may play important roles in proper folding during protein synthesis, protein transport across membranes, assembly of multisubunit enzymes, biosynthesis of organelles, and correctionof damage induced by stress (260,261).Chaperonins, one class of molecular chaperones,are found in all prokaryotes or prokaryoticlike organelles examined. Chaperonin 60 (GroEL), a 60-kDa protein, assembles in 14 subunits to form a “double donut.” Chaperonin 10 (GroES), a 10-kDa protein, assembles in a seven-subunit single ring. These two chaperonins often interact functionallyto mediate polypeptide assembly (260,261).

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The chaperonin GroEL is one of the three major heat-shock proteins expressed in E. coli, the others being Lon and DnaK (262). GroEL is believed to function in correct folding, oligomerization, stabilization, and export of proteins in E. coli. The GroEL and GroES functions are essential at all temperatures inE. coli, and proper expression is assured by the presence of two promoters: a constitutive promoter that is recogandapromoterrecognized by nizedby the major E. coli U factor E d 2 , a U factor induced by heat shock (263). GroEL in prokaryotes also appears to be associated with other specialized functions, including stationary-phase metabolism, the stringent response, and cellular differentiation (Ref. 264 and references therein). Guglielini etal. (264) observed four major heat-shock proteins in each of four different Streptomyces speciesthat corresponded in sizeto Lon, DnaK, and two homologs of GroEL (56and 58 kDa). In addition, they observed 16- to 18-kDa heat-shock proteins. The N-terminal sequences of HSP56 and HSP58 of Streptomyces albus were similar to those of other GroEL-like proteins; and the N-terminus of HSP18 was identical to that of HSP58. HSP94 and HSP70 of S. albus were transiently expressed after heat shock, similar to that observed with heat-shock proteins inE. coli that are transcribed by RNA polymerase holoenzyme containing the heat shock induced by thermolabile$2. In contrast, HSP56, HSP58, and HSP18 were highly expressed for more than 2 h after the temperature shift up, and comprised greater than 20% of total protein. The authors noted that continuous expression is not typical for prokaryotic heat-shock proteins but has been observed in the differentiating Caulobacter crecentus.It was suggestedthat an alternative heat-stableU factor might be involved intranscription of the groEL genes inS. albus. S. Mazodier et al. (265) identified two groEL-like DNA sequences in albus. groEL1 encodes HSPlS and HSP58; groEL2 encodes HSP56. The nucleotide sequencesof groELl and groEL2 predicted proteins of 56,680 Da that contained 70% aa sequence identity. ThegroEL1 gene, but not groEL2, was adjacent to a groES-like gene. ThegroEL1 and groES-like genes were separated by a long inverted repeat with a motif also found in the groE gene regions of three speciesof Mycobacteria. Two groELlike genes were observedin all 12 Streptomyces species tested, and in no case werethe two genes closely linked. Servant et al. (266) have carried out gene disruption analyses using nonreplicating vectors transferred by conjugation from E. coli S17-1. aa of GroELl They showedthat GroEL2 (HSP56) and the 249 N-terminal (HSP58), correspondingto HSP18, are essential for growth. A mutant

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lacking the291 C-terminal aa of HSP58 showed normal growthand differentiation. by the groESGuglielmi et al.(267) identified three transcripts encoded groELZ and groEL2 genes that are present at low temperature and elevated at high temperature. The transcripts corresponded to groES, groESgroELI, and groEL2. Two promoters were identified, P1 upstream of groELl and P2 upstream of groEL2. Both appearedto be typical streptomycete vegative promoters. Farther upstream of the promoters were conserved sequences (GCACTC9N GAGTGCTAA) that are also found upstream of other heat-shock genes in gram-positive bacteria, including Mycobacterium tuberculosis. They suggested that this highly conserved sequence may be a recognition motif for a protein of fundamental importance to survival. TheP1 and P2 promoters have been usedfor hetero(267), and the P1 promoter has logous gene expression in streptomycetes been used for expression ofthe nefgene of the HIV-1 virus inMycobacterium bovis BCG (268). Mazodier et al. (265) suggested that two genesand three functions for groEL-like proteins maybe required for substrate recognition, subcellular localization of proteins, and physiological changes associated with cellular differentiation.It is interestingthat streptomycetes and other actinoas macromycetes frequently produce complex secondary metabolites such cyclic lactones orcyclic peptides, which are likely to be assembled by very large, complex, multifunctional, and multisubunit enzymes (269-271). Also, streptomycetes are very effective in secreting hydrolytic enzymes to obtain nutrients from the environment. It is possiblethat multiple GroEL proteins providea mechanism to assure proper protein assembly and secretion duringthe various phases of cell growth. If so, streptomycetes may be particularly well suited for heterologous protein secretion aswell as secondary metabolite biosynthesis. 4.4.4 ProteinSecretion Streptomycetes secrete many different hydrolytic enzymesand other proteins. Many ofthe genes encoding extracellular proteins have been cloned, expressed, and characterized to varying extents, primarily inS. lividans. These include agarase(146,272-274), several a-amylases(275-282), DDcarboxypeptidase (283,284), chitinases (285-287), esterase (288-290), &galactosidase (291), 6-lactamases (247,292-294), lysozyme (295), proteases (248-25 1,296,297), lipase (298), xylanases (299-302), a-amylase inhibitor (303), and protease inhibitor(304,305). In several cases the genes have been sequenced, and the regulatory and signal peptide sequences

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have been studied in some detail. Some of these might be further developed as heterologous secretion systemsand are considered below. a. Agarase. The agarase gene fromS. coelicolor (dagA) was the first gene encodingan extracellular hydrolase cloned, expressed, and secreted in S. lividans (272). The dagA gene was cloned inthe multicopy plasmid pIJ702, and recombinants produced up to 860 times as much secreted agarase as normally secretedby S. coelicolor (272,273). The yield of secreted agarasewas about 60 mg/L, or 50% oftotal secreted protein.The expression ofthe dagA gene issubject to catabolite repression (273).The dagA gene encodes a 309-aa preprotein containing a 30-aa signal sequence that is cleaved to yield the 32-kDa secreted form (274). The dagA gene contains four promoters andis transcribed byat least three different RNA polymerase holoenzymes (146).It contains a potentially strong ribosome binding site (5‘-AAGGAG-3’) that shows perfect complementarity to the 3’ end of the16s ribosomal RNA ofS. lividans (274). The signal sequence has features characteristicof other signal peptides: a positively charged N-terminal region, a central hydrophobiccore followed by residues capable of inducing a beta turn, and a carboxy terminus of Ala-xxx-Ala. b. a-Amylase. The a-amylase genes from several Streptomyces species have been cloned and characterized. The a-amylase gene from S. hygroscopicus, an industrial producerof a-amylase for starch hydrolysis, has been studied in detail (275). It is transcribed from two promoters; and translation is presumably facilitated by a potentially strong ribosome binding site, 5’-GAAGGAG-3‘. The translation product contains a 30-aa leader sequencethat is cleavedto form the mature a-amylase of M, 47,980. The leader sequence containsa positively charged amino terminus, ahydrophobic core,and two helix-breaking proline residues near the carboxy terminus. The cloned gene also has a palindromic structure ( - 32.4 kcal/ mol) distalto the 3’ end that may act as atranscriptionalterminator. The a-amylase gene cloned in pIJ702 was expressed in S. lividans, but the yield of secreted a-amylase(1940 units/mL) was about fivefold lessthan that secreted by the original S. hygroscopicus production strain. Introduction of the cloned gene back into the production strain caused afourfold increase in a-amylase production (37,700 units/mL). Therefore, the ability to secrete high levels of a-amylasewas not determined solely by the regulatory sequences associated withthe gene cloned fromthe highproducing strain. It is not known if any yield-enhancing mutations are located within the regulatory or signal sequences, sincethe wild-type aamylase gene sequence hasnot been analyzed.

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The a-amylase gene from Streptomyces limosus, a high producer of a-amylase, was also cloned inS. lividans in pIJ702(276,277). It encodes a protein larger than that of S. hygroscopicus (M, 57,065) and is transcribed as a monocistronic message from a single promoter that shares significant sequence similarityto the consensus eubacterial promoter in the - 35 and - 10 regions.It has a presumptive strong ribosome binding site identical to those of the S. hygroscopicus a-amylase and the S. coelicolor agarase (5’-AGGAGG-39, and contains a signal sequence 28 ofaa containing two positively charged residues at the amino terminus, a hydrophobic core, and two proline residues preceding the amino terminusof the a-amylase sequence(276). A possible transcriptionterminator comprised of two overlapping inverted repeats of24 and 26 bp was present on the transcript. Expression of the gene was inducible by maltose in S. limosus and in S. lividans, and was repressed by mannitol inS. limosus or by glucose inS. lividans and S. coelicolor. The glucose repressionwas at the level of transcription and was not observed in a glucose kinasedeficient mutant of S. coelicolor (277). The levels of secreted a-amylase observed in S. lividans containing multiple copiesof the a-amylase gene on pIJ702 were similar to the levels observed in the original S. limosus strain. The absolute quantities of a-amylase inthe supernatant were not reported (277). S. venezuelae also produces high levels of an a-amylase that is subject S. .venezuelae a-amylase to glucose repression and maltose induction. The gene was cloned in S. lividans in pIJ702(278); it is transcribed as a monocistron using a promoter identical to the S. limosus a-amylase promoter. The putative ribosome binding site is identical in sequence to those of the S. hygroscopicus and S. limosus a-amylase genes(see above). The S. venezuelae a-amylase geneproduct has a signal peptide sequence of 28 aa of 61.5 kDa. The deduced that is cleavedto give a mature secreted protein aa sequences of the S. venezuelae and S. limosus a-amylase were 75% identical. Both a-amylases also showed greater than 50% aa sequence identity to the S. hygroscopiucus a-amylase (278). These three a-amylases share onlyvery limited sequence similarity with other prokaryotic, fungal, or plant a-amylases, but show a high degreeof similarity to a-amylases from mammalsand invertebrates (276,279). S. grkeus IMRV 3570 overproduces several extracellular enzymes (280, 281), including an a-amylase. The a-amylase gene of S. griseus differs by ORF from that of S. limosus, encoding aproonly two nucleotides in its tein that differs by two aa from the corresponding S. limosus a-amylase. The S. griseus signal sequence and putative ribosome binding site are

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identical to those from S. limosus. The high degree of DNA sequence identity of the a-amylase genes ofS. griseus and S. limosus suggests that these strains may belong to the same species. The S. griseus a-amylase gene cloned in pIJ699 inS. lividans secreted about fivefold higher levels of a-amylase (83 pg/mL) thanthat in S. griseus. Vigal et al. (282) showed that secretion of S. griseus a-amylase by S. lividans could be increased two- to threefold by substituting an arg for ala at position 6of the signal sequence. The modified signal sequence was coupled with several different promoter substitutions,and two of the constructions gave additional increases in secretion. Substitution of the Tn5 neo gene promoter and S. lividans the S. grismsaf gene promoter (see below) caused the resulting recombinants to produce about 600 pg/mL and 800 pg/mL a-amylase, respectively. The S. lividam recombinant expressing the a-amylase gene from the saf promoter had 10-fold higher a-amylase transcript than the strain expressing the a-amylase gene from its native promoter. The data suggested that neither the translation nor the secretion machinery were limiting in S. lividans. c. Esterase. Streptomyces scabies, a plant pathogen, secretes an esterase that helps the organism penetratethe outer layers of underground plants, such aspotatoes and radishes. The esterase gene has been cloned and sequenced (288)and expressed and secreted inS. lividam from plasmids pIJ486 and pIJ702 (289). Transcription of the esterase gene is regulated by zinc in S. scabies and S. lividans (289,290), and esterase is produced when the culture reaches late exponential and early stationary phases of growth. It appears that expression ofthe esterase gene may require the binding of a transcriptional activator protein that requires zincfor binding. The binding site for activation has been localized by DNase I footprinting to a 23-bp sequence located between bp -82 and -59 relative to the transcription start site. Deletion of the 23-bp sequence abolished esterase production in S. lividans. However, the putative zinc-binding activator protein has not yet been identified. The esterase gene encodesa protein containing a 39-aa signal peptide that is cleaved during secretion. The signal peptide contains four positively charged aa at the N-terminus, a hydrophobic core of 15 aa, and small unchargedaa at the - 1 and - 3 positions relativeto the signal sequence cleavage site. Deletion of 23the of 39 aa residues abolished secretion. Also, deletion of the first twoor second two chargedaa caused approximately 100-fold reductions in esterase secretion, whereas deletion of all four charged aa caused a sixfold reduction in esterase secretion. Deletion of various sets of the four hydrophobic aa, or eight aa in the ,

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hydrophobic core, resulted about in a 500-fold decrease in esterase secretion. Removal ofthe signal peptide cleavage site resulted ainsimilar decrease in secretion. The results demonstrated that the cleavage site, the positively charged N-terminus, and the hydrophobiccore are critical for normal secretion of esterase. Optimization of secretion will requirea more extensive analysis of aa substitutions in the signal sequence. Regulation of the esterase promoter by zinc might also be exploited for purposes other than secretion. d. Other Signal Sequences. Several other signal sequences have been identified that may be useful for heterologous secretion of proteins in streptomycetes. LEPlO is a protease inhibitor secreted in largequantity from S. lividans, and LT1 is a related protein secreted in largequantity from Streptomyces longisporn (306). The LT1 promoter was relatively strong comparedto several other promoters usinggalK (see Section 4.3.2.c) as a reporter gene. The LEPlO promoter was much less active in this assay as the LT1 (306). In this analysis,the galPZ promoter was nearly as active promoter. The LEPlO preprotein contained a 35-aa signal peptidethat a hydrophobic core, and a putahad a positively charged amino terminus, tive cleavage site of ala-pro-ala. The LT1 gene is synthesized as a preproprotein; the propeptideis removed by an extracellular protease. The LT1 signal sequence was very similar to the LEPlO sequence in the Nterminal and hydrophobic core regions,and it contained an ala-leu-ala putative signal peptidase recognition site. Brawner and colleagues (306, 307) have also described the properties of a P-galactosidase signal sequence that has been used in secretion studies. The DD-carboxypeptidase gene (dac) has also been used to develop a secretion vector derivative of pIJ702 (308). The system employs the dac signal sequenceand dac or other promoters. The dac promoter was able to drive the secretion of 30 to 40 mg/L of secreted DDcarboxypeptidase inS. lividans. The authors have attemptedto substituteother strong promoters, including the ermE promoter from S. erythraea, but have as those obtained with the dac pronot yet shown secretion yields as high moter. Tendamistat is a potent inhibitor of human a-amylasethat is secreted by Streptomyces tendae (303). The gene encoding tendamistat has been cloned and its use for secreting proinsulin has been described (309,3 11) and will be discussed in Section 5.3.2. e. sa$ Daza et al. (312,313) have cloned a segment of DNA (saf) from S. griseus ATCC 10137 into pIJ702 that caused S. lividans to over-

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produce several extracellular enzymes, including amylase, protease, pgalactosidase, alkaline phosphatase, and lipase, but not DNase, cellulase, or xylanase. The cloned gene caused enhanced secretion of the same enzyme activities in S. coelicolor but did not enhancethe secretion of agarase. Introduction of the saf gene on pIJ702 into S. coelicolor was also associated witha delay inAr production, but the effectof pIJ702 alone was not presented. Intracellular alkaline phosphatase and amylase activities increased in the S. lividans recombinant containing the cloned saf gene, suggestingthat increased secretion inthis strain is due to increased gene expression rather than to more efficient secretion. The saf gene, which apparently encodes a protein of M, 10,500, is expressed from a strong phosphate-regulated promoterthat has a nearly perfect 46-bp inverted repeat overlapping the - 35 region, as well as several short direct and inverted repeats. The saf promoter exhibited significant similarity to several other streptomycete promoters,and the saf gene contains sequences that could functionas a ribosome binding site and a transcription terminator. The deduced aa sequence of the saf gene product is a basic protein that shows significant sequence similarityto DNA-binding proteins. The results suggest that the saf gene productis a positive regulator for the expression of certain extracellular proteins. It is not known if the inclusion of extra copies of the saf gene will further enhance secretion in other recombinant strains, for example, those containing the a-amylase constructions containing modifications in the promoter and signal peptide sequences (see above).

5 INDUSTRIALAPPLICATIONS 5.1 HybridSecondaryMetabolites One application of recombinant DNA technology in streptomycetes is the production of novel or hybrid antibiotics (314-319). Since the first demonstration of production of a hybrid antibiotic by this methodology (320), a modest number of other examples have been publishedand the results of some studies have been reviewed (193,269,321,322). Rather than review eachof these, I will discussa few examplesthat demonstrate specific principles or important inferences.

5.1. l

4"-lsovalerylSpiramycin

4"-isovaleryl spiramycin is a hybrid antibiotic produced by cloning the carE gene (isovaleryl transferase) fromS. therrnotolerans,a carbomycin

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producer, into S. ambofaciens, a Srm producer(323). The recombinant, which contained the carE gene in the bifunctional plasmid pKC796 inserted into the S. ambofaciens chromosome at the +C31 attB site, was stable in the absence of selectionand produced high levels of 4”-isovalerylSrm. This application demonstrated the feasibility of constructing stable, high-producing recombinantsfor potential industrial scale-upby inserting the cloned DNA into the chromosome.

5.7.2 lsomacrocin Cox et al. (324) demonstrated the production of a novel molecule bythe overexpression of a homologous Tyl biosynthetic gene in a mutant of S. fradiae blocked in Tyl biosynthesis. The last two steps in Tyl biosynthesis are methylations of the2‘”-OH and 3”’-OH of the 6-deoxy-D-allose moity of demethyl macrocin. Normally, mutants defective in 2”’-O-methyltransferase activitybut proficient in 3”’-O-methyltransferase activity fail to methylate either position (121,210,325). Therefore, prior O-methylation of the 2”‘-OH is normally required to generate a suitable substrate for the 3”-O-methyltransferase. However, when multiple copies of the tyfF gene, which encodes the 3”’-0-methyltransferase,were introduced on the highcopy-number vector pIJ702into a tyfEmutant, which lacked 2”‘-O-methyltransferase activity, the recombinant strain produced a novel compound 2“-O-demethyl Tyl (isomacrocin). This demonstrated that a poor substrate for 3“’-O-methylation can be effectively methylated by overexpression of the 3‘”-O-methyltransferase.This concept may have general utility inother systems to drive enzyme reactions using substrates structurally relatedto the normal substrate.

5. 1.3 Modified Erythromycins Erythromycin (Erm) is an attractive model for genetic manipulation since the parent molecule is clinically relevant and since much is knownabout the biosynthesis (326) and the structural organization and sequence of the DNA encoding many ofthe biosynthetic enzymes (327-332). Weber et al. (333) used a targeted mutagenesisapproach to block the conversion of 6-deoxyerythronolide B (DEB) to erythronolide B (EB) by gene disrupting the cytochrome P450 hydroxylase gene (eryF) that encodes an enzyme that converts DEB to EB. The recombinant strain produced 6A deoxyerythromycinA @EA), an acid-stable derivative of erythromycin (EA). The results are significant for several reasons.First, the data indicate that the hydroxylation of the carbon 6 positionof the lactone can be bypassed to produce the acid-stable shunt metabolite. This derivative

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might be further modified to produce a clinically superior derivative of Erm, since acid liability has been an undesirable feature. The more recent derivatives of Erm have attempted to address acid liability by introducing chemical modificationsto increase acid stability (334).The results are also important inthat they demonstratethat targeted mutagenesis using cloned genes can be. usedto make specific desired mutations that have not been observed by screening randomly mutagenized cultures. Since antibiotic biosynthetic genes are generally clustered, the approach of targeted mutagenesis may generally be applicableto many other actinomycete secondary metabolites. a complex of multifuncThe lactone portion of Erm is synthesized by tional enzymes, the polyketide synthase (PKS) encoded by about a 33-kb segment of DNAthat contains three large ORFs. EachORF encodes two repeat unitsor modules, and each module containsthe coding sequences for all of the enzymatic steps associated with theaddition of 'one shortchain carbon unit to the growing fatty acid chain (330,331). A module can include all or some of the following enzymatic activities: actyltransferase (AT), acyl carrier protein (ACP), &ketoacylACP synthase (KS), &ketoreductase (KR), dehydratase (DH), enoyl reductase (ER), and thioesterase (TE).Donadio etal. (330) introduced an 813-bp in-frame deletion into the KR domain of module 5, and the recombinant strain produced the predicted 5,6-dideoxy-3c~-mycarosyl-5-oxoerythronolide B, a derivative of Ermthat contains a keto functionat carbon 5 rather than the hydroxyl function, the normal substrate for the glycosylation with the amino sugar desosamine. The experimentimportant is in that it showedthat the reductiion of the C-5 keto function can be bypassed and a functional lactone can be synthesized by the mutant strain to yield a novel polyketide. Donadio et al. (330,332,335) have speculatedthat the modular organization of the Erm biosynthetic genes, and the likely modular organization of other macrolide biosynthetic genes, including Tyl, Srm, and avermectin genes, should facilitatethe construction of specific recombinants withpredictedmacrocycliclactonestructures.Thisgeneral approach may be applicable to other secondary metabolite biosynthetic pathways that employ large multifunctional enzymes containing modules that dictate the incorporation of specific precursorsat particular steps in secondary metabolite biosynthesis. 5.1.4 AromaticPolyketides The aromatic polyketides incompass a number of diverse structures and biological activities. Prominent members of this group include benzoisochromanequinones (Ar,medermycin,granaticin),tetracycline, and

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anthracyclines (DNR, Tcm). The first reported production of a hybrid antibiotic involvedthe introduction of Ar biosynthetic genesinto a medermycin producerto generate a recombinantthat produced mederrhodins A and B, and into a granaticin producerto produce dihydrogranatirhodin (320). More recently, detailed studies on the genetics and biochemistry of aromatic polyketide biosynthesis indicate that thetype I1 polyketide synthases contain multiple subunits, manyof which are interchangeable between pathways (319,203,336-341). Indeed, a recombinant S. galilaeus strain containing the S. coelicolor act1 and actV11 loci produced a shunt product of the Ar pathway aloesaponarin 11, apparently by a hybrid polyketide synthase (336). In the same studies, the authors also generated two other novel compounds, by introducing desoxyerythrolaccin and 1-O-methyldesoxyerythrolaccin, a different segment of the Ar DNA into a strain of S. galilaeus. The authors speculated that interspecies cloning may be generally applicable to generate new chemical structures. Hopwood et al. (342-344) have described a general approach to produce hybrid aromatic polyketides using genes clonedfrom six different biosynthetic gene clusters, including those for Ar, granaticin, frenolicin, griseusin B, oxytetracycline, and Tcm. All of these polyketidesare produced by multienzyme complexes, and the corresponding genes have fairly similar arrangements in the producing streptomycetes. Several examples of hybrid polyketides have been generated using this methodology(343,344). The successes of producing hybrid type I1 polyketide synthases are particularly encouraging since they document heterologous complementation in complex multisubunit enzymes. This implies that at least Some features of the individual proteins requiredfor the bindingand assembly of complex multisubunit polyketide synthases have been conserved during evolution.

5.2 ImprovedSecondaryMetabolite Yield A second applicationof recombinant DNA technology in streptomycetes is the improved production of secondary metabolites(193,315,317,318, 345). This might be accomplished by increasing the levels of key intermediates or cofactors from primary metabolism, by blocking competing pathways, by improving the overall metabolismor robustness of the fermentation, by increasing the levels of secondary metabolic enzymes associated with rate-limiting steps, orby improving the coordinated expression of sets of secondary metabolic enzymes by manipulating the regulatory systems (e.g., activators, U factors).

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Although there appearsto be real potentialfor applying recombinant technology to yield improvement, significant published examples of success are almost nonexistent. Part of this may be due to the proprietary nature of many industrial fermentation processes, and part may be due to the technical difficulties associated with further improving product yields in highly developed strains by implementing (or retrofitting) recombinant technology late in the strain development cycle. Also, many of the best cloning methods, including those that facilitate the construction of recombinant strains in ways that avoid inadvertent negative effects onthefermentationyields,have just becomeavailable.Nonetheless, some encouraging resultsare summarized below.

Gene Dosage to Increase the Level of a Rate-Limiting Enzyme An industrial-scale application of this approach has been pursued for Tyl production in S. fradiae (121,315). Highly productive strains of S. fradiae accumulate relatively high levels of macrocin, the immediate precursor of Tyl (325,346). Macrocin lacksthe O-methyl group that is normally present at the 3” position of mycinose moiety of Tyl,so it appears that the macrocin O-methyltransferaselevels may besuboptimal for efficient conversion of macrocin to Tyl in highly productive strains (346). The macrocin O-methyltransferase gene was cloned (206,207)and introduced into a production strain on the freely replicating plasmid pIJ702 and on the integrating plasmidpKC796 (57).In both casesthe recombinant strains expressed higher levels of macrocin O-methyltransferase and of either vector per produced higher levelsof Tyl. However, the presence se appeared to cause a decrease intotal macrolide yield (i.e., the sum of Tyl, macrocin and demethylmacrocin), thus reducing the overall potential yield of the process. It remains to be seenif this problem can be overcome by methods (desdribed in Sections 2.2 and 2.3) for the neutral insertion of cloned genes into the chromosome. Hutchinson (193) has noted examples of improving polyketide and @-lactam yieldsby gene dosage in laboratory strains of S. glaucescens and S. clavuligerus. In S. glaucescens, introduction of extra copies of the acyl carrier protein (ACP) gene for polyketide biosynthesis on a multicopy plasmid caused a large increase in intermediates to TcmC (194). In S. clavuligerus, insertion of a second copy ofthe lat gene into the chromosome caused a two- to fivefold increase in production of P-lactam antibiotics(347).The lat geneencodedlysine€-aminotransferase, an enzyme involvedin the biosynthesis of a-aminoadipic a acid, a precursor 5.2.l

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in penicillin and cephalosporin antibiotic biosynthesis. These examples illustrate that both precursor flux and antibiotic biosynthetic enzyme levels can contribute to rate limitations in specific systems, and that gene cloning can address either limitation.

5.2.2 ImprovedMetabolicRobustness The transfer of oxygen in highly viscous fermentation broths may be (348) limiting in some large-scale batch fermentations. Magnola et al. introduced a bacterial hemoglobin gene from Vifreoscillainto S. coelicolor and showed that the recombinant produced about 10-fold higher yields of Ar than the control strain under conditions of oxygen limitation. They suggestedthat the actively expressed hemoglobin protein may allow the internal cells in the mycelial clumps to acquire oxygen more efficiently. This,in turn, may leadto improved protein synthesis because of better ATP production, as has been shown in E. coli cells containing the cloned hemoglobin gene (349). It will beimportant to see ifthe hemoglobin gene can be used to improve product yields in highly developed production strains under large-scale production conditions with full aeration and agitation. 5.2.3 Gene Dosage to Increase the Expression of Positive Regulatory Factors As discussed in Section4.2, the introduction of additional copies of positive regulatory genes has been shown to increase product yield in a number of different cases, generally using laboratory strains grown in laboratory media.It is not yet known if this approachwill be successful in highly developed production strains grown in complex production media. Since improved production yields may be associated with a number of different factors, the early applicationof positive regulatory genes in the process of strain development might facilitate the subsequent detection of mutations that influence the flow of precursors into the pathway and speed the process of yield improvement. This approach is appealing since positive regulators are generally clustered withantibiotic biosynthetic genes and can be readily cloned. 5.3 Secretion of Homologous and HeterologousProteins Several potentiallyimportant homologous or heterologous proteins have been cloned, expressed, and secreted in streptomycetes,and some examples have already been discussed (e.g., a-amylase). I summarize several others below.

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5.3.1 Hemicellulases Hemicellulases suchas endoxylanases have potential applications as bleaching agents in the manufacture of paper (350,351). Hardwoods contain up to 20% xylan, the main hemicellulose. Microorganisms that degrade hemicellulose produce different combinations of lignocellulose degrading enzymes, including endoglucanases, exoglucanases, ligninases, and cellulases. Endoglucanase activity is sufficient for biobleaching, whereas cellulase activity is undesirable. Xylanase, an endoglucanase, is produced in good quantities by S. lividans (350,351). Whereas S. lividans normally produces three xylanases, a mutant deficient inthe production of all threewas isolated and used to clone the individual xylanase genes, using pIJ702 (299,301,302,350,351), and each produced 40- to 50-fold more xylanase than the wild-type S. lividans strain containing pIJ702. Fermentation optimization studies using inexpensive medium ingredients have achieved xyalanse yields of2 to 3 g/L after 120 h in the fermentation (351). The authors noted that the excellent stability of the cloned xylanase genes in pIJ702 and the efficient secretion may facilitate the development of a continuous fermentation process. All three xyalanses are translated as preproteins containing typical signal sequences. Each signal sequence contains a positively charged N-terminus, a hydrophobic core, a helix breaking prolineat - 4 or - 5, and an Ala-=-Ala for signal peptidase cleavage. These signal peptidases are also being studied for the secretionof heterologous proteins. Becauseof the high levels of xylanase produced, these signal peptides present additional options to those presented in Section 4.4.4 for heterologous secretion. 5.3.2 Proinsulin Koller and colleagues (303,309,311) have applied the cloned tendamistat (a-amylase inhibitor) gene from S. tendae to construct fusions with a monkey proinsulin gene in pIJ702 for secretion in S. lividans. Initially, the tendamistat gene was fused with the proinsulin gene usingan 1l-aa spacer peptide to allow for independent folding of the two proteins. A methionine codon was introduced to facilitate CNBr cleavage to release proinsulin. Transcriptionwas driven by tandem tyrosinaseand tendamistat promoters; and secreted product yields rangedfrom 20 to 100 mg/L. The product contained active tendamistat, but the proinsulin portion contained incorrect disulfide bridges. Koller et al. (31 1) obtained correct disulfide-bridge formationby replacing the connecting peptide with a single residue. Thisform of proinsulin can be enzymatically processed to a fully’

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active insulin derivative. They note that the yields with this derivative (30 to 40 mg/L of insulin) are superior to the yields obtained in a yeast secretion system. Also, the presence of the active tendamistat inthe fusion product offers a means to identify improved strains or improved fermentation conditionsby monitoring a-amylase inhibitor levels. 5.3.3 OtherProteins The previous examplesillustrate the feasibility of using S. lividans as a cloning host for the secretion of high levels of homologous and heterologous proteins. S. lividans has also been used for the secretion of the human solubleCD4 receptor (306),the Flavobacterium parathion hydrolase (352-354), human interferons a 1 (355) and a 2 (356), human interleukin-lfl (357), human interleukin-2 (358), Streptomyces subtilisin inhibitor (359), the Fv domain aofmonoclonal antibody (360), the aculeacin A acylase from Actinoplanes utahensis (361), and the plant sweet-tasting protein thaumatin (362). These studies have generally used pIJ702 as a cloning vector and have employed a number of different combinations of promoters, ribosome binding sites, and signal sequences.It is not yet clear what combinations of host, promoter sequence, ribosome binding site, signal sequence, and cloning vector will give optimal expression and secretion of desired proteins. It appears that systematic studiesof these parameters, coupled with in-depth nutritional and fermentation studies, could lead to many fruitful applications of streptomycetes as protein producers in large-scale fermentation.

6 SUMMARYAND PROSPECTS Molecular methods used to manipulate streptomycetes have improved dramatically over the past decade, and it is now relatively easyto clone and express streptomycetegenes in different Streptomyces species. Furthermore, thegenes can, in principle, be expressed without causing negative effects on secondary metabolism. Important advances have been made in understanding the various layers of regulation that control secondary metabolite production and secretion, and major pieces ofthe intricate puzzle have been cautiously positioned,but much remains to be discovered. It appears, however, that the field is poisedfor major new insights in the fundamental understanding of the coupling of nutritional signaling and regulation of gene expression, and the molecular tools to make the needed advancementsare largely available.Also, the successes to date in hybrid secondary metabolite production, improved product yield, and in

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homologous and heterologous secretion of proteins, achieved largely using somewhat rudimentaryand nonoptimized methods, suggeststhat the future applications will be more rewarding and will come more quickly, particularly as the newest molecular genetic technologiesare coupled with a more complete understandingof gene regulation. The challenge isfor academic and industrial scientists to build on the impressive accomplishments of the past decadeand to focus on the key questions that will provide the basis for further exploitation of streptomycetes as premier industrial microorganisms.

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7 Recombinant Microorganisms Safety and Regulatory Aspects Anthony J. Taylor*

Health and Safety Executive, London, England

1 FIRST CONTROLS

As many will recall, the early days of the development of techniques involving recombinant DNA (rDNA) were times of uncertainty. Concerns sprang from the feeling of many within the scientific community that their work might exposeboth them and the wider human communityto unforeseeablehazards.Woulddisease-producingmicrobesbecreated accidentally or maliciously that could cause devastationto plants or animals? Would modified bacteria be capable of transmitting cancers to humans? Could scientists really predictthe outcome of their “engineering” of nucleic acids? It has to be said that with the benefit of hindsight, after close to two decades’ experience of rDNA work, we can now view suchfears for human health and safety ina more rational way. However, at the time they were absolutely correctand proper mattersfor concern. It was only by addressThe views expressed in this chapter are those of the author and do not necessarily reflect the policy of HSE or of any other U.K. government department. *hesent affiliation: Department of Health, London, England.

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ing these questions that rDNA technology was able to progress to the level of commercial exploitationthat we have today. The period from 1973 to 1975 saw what became and has remained an unusual sight inthe development of occupational safety;an industry, in this case the scientific community, calling for a halt to their own work so that safety issues could be addressed. This contrasts with so many health and safety issues worldwide, where significant progress has occurred only on the back of an existing and tangible problem. Concerns regarding the developments by which restriction endonucleases could be usedto produce hybridDNA witha biological activity of an unpredictable nature were first raised at the Gordon Research Conference on Nucleic Acids held in 1973. Singer and Sol1 (1) called for the U.S. National Academyof Sciences to consider the potential hazardsof rDNA to workers and the public. The following year saw members of the Committee on Recombinant DNA Molecules within the academy respond by recommending, ina letter published inScience, a voluntary haltto certain types of rDNA experiments. The “Berg letter”(2) highlighted two areas of work: (a) the creationof new autonomously replicating plasmids that might transfer determinants of drug resistance or of toxin production, and (b) the linkageof oncogenic and animal viral DNA to autonomously replicating elements. The authors called for the National Institutes of Health (NIH)to establish an advisory committeeto oversee rDNA work and to devise guidance for safe systems of work. The Berg letter promoted a rapid and mixed set of responses. There was a strong commitmentfrom NIH to the proposals,but outright opposition camefrom some withinthe scientific community who saw them as an inhibition of free investigation. Journal editorials debated whether the moratorium should be strengthened by restrictions on publication of “suspect” work. Nevertheless, scientific authorities did respond to the call for a moratorium and action was taken internationally. Within a month, the United Kingdom Research Councils had established a review to assess the potential benefits and hazards of “genetic manipulation” and the U.K. Medical Research Council instructed all its staff to comply with the moratorium. A working party under Lord Ashby reported in 1975 with recommendations for the United Kingdom that were seen by the scientific media as “an amber lightfor genetic manipulation”(3). It recognized the enormous potential benefit in the technology which needed to be developed properly, with due regard for safety. This was reinforced by the International Conference on rDNA Moleat Pacific Grovein cules held in1975 at the Asilomar Conference Centre

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California. As a consequence, Asilomar has becomepart of the history and occasionof science. Aftera meeting in which discussion was intense ally heated, it was concluded that certain defined genetic manipulation work could go ahead provided that strict safety precautions were adopted. For other work, the moratorium was to remain until the question of safety guidelines could be fully and properly addressed,and some classes of experiment were judged to be potentiallyso hazardous that they shouldnot be allowed to proceed under any circumstances(4). The Asilomar debate broughtto a wider audience the concept of biological containment,that is, the use of viralor bacterial hostsfor genetic research that are incapable of surviving without the provision of special culture conditions as a result of mutations encoding such properties as temperature sensitivity, cell wall deficiencies, and nutrient dependency. Asilomar also recognizeda need for classes of physicalcontainment, three graded levels ofsafety precautionsthat should be applied to genetic manipulation experiments, depending on the level of hazard. Thus the concept of defined control replaced that of the moratorium. The temporary halt had allowed time for consideration of the risks and for the first stepsto be taken toward the establishment of national advisory bodiesto provide the oversight essential for safe development of the technology-in all, an acceptable balancethat allowed progress while safeguarding workers and the public. The National Institutes of Health established the Recombinant DNA Advisory Committee (RAC), which held its first meeting immediately following the Asilomar conference.Draft guidelines were producedthat were issued inJune 1976 (5). The original guidelines described four levels of biological containmentfor E. coli K12 host-vector systemsand specified appropriate levels of physical containment basedon hazard assessment of the type of work being done. These guidelines were revised in 1978 to take account of classes of experiment that were deemed to pose no hazard as a result of the genetic manipulation. In general, therewas also a lowering ofthe physical containment required for most other experiments still coveredby the guidelines, which also dealt withthe roles and responsibilities of employers, researchers, and institutional biosafety committees. It will be of interest to European readers in particular to note that from their inception, the NIH guidelines have included consideration of protection of the environment as well as human safety. The process of relaxation of the guidelines was continued in1980when the requirementfor registration with NIH of certain typesof experiments was removed. This trend toward a more pragmatic approach to control

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has continued. Today, it is recognized that the vast majority of laboratory experiments involving rDNA organisms are intrinsically safeby virtue of the host strains employed. As a result, such work is permitted in microbiology laboratories without special containment requirements (6). The NIH guidelines, it should be noted, are not federal regulations and do of laboratory facilities. not require licenses,permits, or statutory inspection 2 DEVELOPMENT OF GUIDELINES FOR LARGE-SCALE WORK The developmentof large-scale applicationsof rDNA technology posed new questions to be answered by regulatoryauthorities worldwide. Both the RAC in the United States and the Genetic Modification Advisory Group established in the United Kingdom recommended a 10-L cutoff point in their definitionsof what constituted large-scale work, and both bodies established procedures by which large-scale experiments would be reviewed on a case-by-case basis. Both committees also had quickly to develop a systemto deal withdata that might contain commercially sensitive information. Case-by-case review continued in the United Kingdom under voluntarycontrol until 1989, but following the issueof large-scale standards in 1980, RAC votedthat it did not wish to continue to receive and review in detail information on large-scale containment facilities.It was content to pass this roleon to local biosafety committees, reviewing data on the characterization of recombinant organisms only. The initiative in developing guidance on safety shifted significantly in the early 1980s away from national authorities into a truly international arena when the Organisation for Economic Cooperation and Development (OECD) began a work program to examine various aspectsof biotechnology. OECD is a body comprising 24 national governments (19 from Europe, plus Australia, Canada, Japan, New Zealand, and the United States). It was an immediate priorityof OECD’s Committee for Scientific and Technological Policy to examine safety aspects of biotechnology, and in 1983, an ad hoc Group of National Experts on Safety and Regulations in Biotechnology was created to establish scientific criteria by which to evaluate safety in the use of rDNA organisms in industry, agriculture, and the environment. The group’s remit was primarily to examine the application of organisms rather than the act of constructing them. The latter it judged to be well covered by existing provisions and controls. The report of the OECD Group was issued after 3 years of work and after agreement by the council of the OECD, where nations are represented

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at cabinet level. The significance of the report “Recombinant rDNA Safety Considerations” (7) cannot be understated, for it provided a structured framework on which harmonizedstandards could betaken forward, and as will be described, has formed the basis for much of the European Commission’s initiative on safety in biotechnology. A major recommendation of the report was that the vast majority of industrial rDNA applications should use organisms of intrinsically low risk, and as such would warrant only minimal containment. Good Industrial Large-scale Practice (GILSP) may prove to be the lasting monument of the OECD report. For those organisms that could not be handled with GILSP, the OECD Group produced additional criteria for physical containment, compatible withthat used to handle pathogens. When considering organismsto be usedin environmental and/or agricultural applications, the OECDGroup recognized that this area of the technology had reached only an early stage of development. This promoted the use of data from work with “traditionally derived” organisms and a step-by-step development from research and development trials of rDNA organisms for field use. The report listed the factors that should be taken into account in the risk assessment of such new applications.

2.1 GoodIndustrialLarge-scalePractice The OECDreport, like theNIH/RAC guidelines beforeit, promoted the essential use of biological containment as the primary tool of the technologist in ensuring safety. Only when this is inadequate will physical containment be required,and in the GILSP concept, biological containment was stressed as the key. The 1986 report describes criteria for the host organism,for the vector/insert employed,and for the rDNA organism itself. 1. The host should be “non-pathogenic” with an “extended history of

safe industrial use” or with “built-in environmental limitations.” 2. The vector/insert mustbe “well characterised and free from known harmful sequences,” ‘limited in size,” and “poorly mobilisable.” 3. The final modified organism should be as “safe as the host organism.” The wording of these criteria is sufficiently flexible to allow considerable national variation in interpretation, and as a result,the principles of GILSP within the OECD report became incorporated in a number of pieces of national guidancehegulation: for example, those in Denmark,

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Japan, and the United Kingdom. Host organisms that have been assigned GILSP status in more than one country include Escherichia coli K12 derivatives, Saccharomyces cerevisiae, Aspergillus oryzae, and Bacillus species,including B. licheniformis, B. subtilis, and B. amyloliquefaciens. In most cases these organismsare widely recognized as nonpathogens. In the caseof E. coli K12,the most commonly used hostfor rDNA studies, strains of a known human pathogen are utilized that exhibit a number of stable factors that markedly reduce the organism’s ability to colonize humans or to survive beyond the fermentation vessel. Such factors include loss of part of a lipopolysaccharide side chain, reduced adherence abilities, inabilityto produce enterotoxin,and enhanced susceptibilityto lysis in human serum. Enhanced biological containment been has achieved by the utilization of auxotrophic strains and those with cell wall mutations, all of which reduce the ability to survive in the environment. Further guidance was issuedfrom OECD in1992elaborating and illustrating the GILSP criteria beyond those set out in the 1986 report (8). This, and the inclusionof the current OECD criteria within the European Community Directive on the Contained Use of Genetically Modified Micro-organisms (g), will undoubtedly leadto a refined specificationfor GILSPcompatible organisms that will help develop a greater harmonization in the oversight of large-scale work in rDNA technology. Beyond GILSP, the OECD’s establishment of three sets of physical containment and organizational factors for large-scale work with more hazardous organisms has also become an unofficial international standard. They have beenincorporated in EC legislationand have been widely introduced into large-scale practice inthe United States (10) and Japan. The major features of large-scale containment can be divided underthree general headings: 1. Primary containment, by which closed systems

are used to minimize or prevent the release of process organisms (e.g., sealed fermentors, treatment of exhaust gases, etc.) 2. Secondarycontainment, in which the fermentor plant is housed within a controlledarea that may require air pressure differentials to be established, HEPA filtrationto be used, and effluent inactivation regimes put into force 3. System of work, which deals with personnel activities and monitoring arrangements withinthe facility (11)

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2.2 Release of rDNA Organisms to the Environment Just as technologists and regulators had cometo terms with the need to deal withthe safety aspects ofthe transfer of rDNA technologyfrom the laboratory scaleto the industrial setting,so a new and more sensitive issue was raised with regard to the introduction of modified organisms into the environment. In1985, Day (12) described his feelings over thedebate a case of ddjd that had developedon the issues raised by release work as W, a rerun of the debate at Asilomar 10 years earlier. Kornberg(13) reinforced this by describing the topic of release of rDNA organisms as generating several simultaneous debates: an essentially scientific debate in which no consensus had yet developed on whether the introduction of rDNA organism would cause harm,a regulatory debate among national and international agencies trying to develop the right regulatory framework in which this technology would be controlled, and a third debate in terms of public perception. Several years later, this is still the case, although some progress towardthe “post-Asilomar” stageof regulatory development has occurred. National and international concern over release issues had its impact on early release proposals inthe United States.An application by Monsanto to the Environmental Protection Agency (EPA) in January 1985 to field test a strain of Pseudomonasfluorescensmodified to express the Bacillus thuringiensis toxin (Bt) was followed by a year of discussion of supporting data and a further 5 months before, in April1986, an experimental use permit under the Federal Insecticide, Fungicide and Rodenticide Act(FIFRA) was denied. During this period, Advanced Genetic Sciences’ (AGS) non-ice-nucleating bacterium,a modified Pseudomonas sp., despite having received an experimental use consent a year after ing, was further delayedby legal actions. Eighteen months elapsed before AGS could carry out its field tests and then only in an atmosphere of public alarm. At about the same time in the United Kingdom, the Advisory Committee on Genetic Manipulation (ACGM) had produced guidelines on planned releasesto the environment(14) and had considereda first application for the release of a modified organism-in this case a modified virus to be used as an insecticide. The review of this proposaltook only three months,and after consideration of additional data, the trial release occurred in the autumnof 1986. Until 1989, release proposals in the United Kingdom were reviewed bya subcommitteeof ACGM under a purely vol-

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untary scheme for notification. This changed withthe passing into law of the Genetic Manipulation Regulations1989, which required notification to the Health and Safety Executive (HSE) at least 90 days in advance of work commencing of the intention to “introduce into the environment” (release) a “genetically manipulated organism.” Worldwide the number of releases of such “novel” organisms has been increasing rapidly. Since the first trials in 1986, over 600 releases have been reviewed viathe U.S. Department of Agriculture (USDA) alone.In the United States and elsewhere the majority of these have involved the introduction of group plants modified to express traits such as pest or herbicide resistance, and these do not seem to have caused such public and media fever as that which surrounded the Pseudomonas releases in 1986 and 1987. Nevertheless, concerns still exist and it is to promote public confidence as well as meeting genuine safety issues that regulatory authorities have issued comprehensive guidanceto experimenters. There are concerns that need addressing with regard to the introduction of novel organisms whatever their type. These are certainly good examples where introduced alien species of plant, animal, insect, and so on, have become establishedand insome cases become seriouspests. Could rDNA species similarly fill vacant ecological niches or displace existing natural species? Certainly,the trait of weediness in plantsis a matter for serious consideration: inparticular, the exchange of genetic material between modified cropsand their wild weedy relatives. Characteristics such as herbicide resistance could, it is feared, become a serious problem if so transferred. The outcome of such exchange might beto promote the stability of undesirable characteristics inthe environment and raisesthe fear of environmental damage. Calling upon experience since1986, there has beena continual updatingof guidanceto promote safe release experiments. The fundamental questions that must be addressed on all releases can be summarized as follows: Will the organism survive? Will it multiply? Will it spread to other sites? Will it cause harm? Will it transfer genetic material to nontarget organisms? (15) In the United Kingdom, ACGM produced comprehensive new guidelines in 1990 (16) which contained a risk assessment procedureto address the following key areas: thenature of the organism and its novel genetic material, the release site and its habitat, the survival and dissemination characteristics of the released organism, and safety precautions and contingency plans. The scheme sets out a number of questionsthat are answered witha series of “points

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to consider.” The ACGM scheme was designed not to be restricted to crop plant releases but also to be applicable to microbial or animal species. The USDA issued ”proposed guidelines for research involving the planned introduction into the environment of organisms with deliberately modified hereditarytraits” in 1991 (17). These developed the concept of levels of safety concern for particular rDNA organisms and appropriate confinement levels for safe field research. The release of rDNA organisms has been the trigger for an entire new regulatory initiative worldwide in which environmental considerations are at least as important as those of effects on workers or on the general public. In many respects the early 1990s sees the rDNA debate back in 1976. Questions are being asked, but neither scientists nor regulators have sufficient experimentaldata to provide reasonable answers in all circumstances. A careful step-by-step development ofthe technology will have to take place during this “learning phase,” and for this the developing regulatory framework will be of critical importance.

3 REGULATIONWORLDWIDE The international picture with regard to the regulation of rDNA technology is that of a spectrum of approaches. The most specific legislation exists in the member states of the European Communities, where five countries hadstatute law in place bythe end of 1992 that specifically addressed activities involving rDNA organisms and implemented the two EC Directives (9,18).

3.1 EuropeanCommunityDirectivesonBiotechnology

In 1988, three European Community Directives relatingto the safety of biotechnology were published. The significance of the European Directives should not be underestimated.The economic community ofthe 12 member states comprisesa total population of 320 million, a figure not far below that of the U.S. and Japanese populations combined. It has been the intention of the community since its inceptionto remove controls that restrict free movement of people, money, and goods across national frontiers. As of the end of 1992, each member state was obliged to incorporate mechanisms into its own laws to achieve this. In the area of biotechnology, one directive deals with protection of workers from the risks related to exposure to biofogicalagents at work. The Biological Agents Directive, adopted at the end of 1990 for imple-

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mentation by the endof 1993 (19), seeks to deal with risksto workers from all biological agents except higher plants and animals. Its main provisions are for risk assessment, classification of agents into risk categories, and a variety of practical measuresfor action when exposure cannotbe avoided. The Biological Agents Directive is accompanied by two directives specifically dealing with gene technology. These reached formal adoption in 1990 and should have been implemented into the national law of each member state by October 1991. The directiveon the contained useof genetically modified microorganisms (GMMOs) (9) establishes a regime by which both human healthand environmental protection can be achieved for work with GMMOs in the laboratory and in industry. “Contained use” covers those situations where, by the use of physicalor physical plus biological barriers, GMMOs do not escape to the environment. Much of the approach within this directive is based on the OECD’s 1986 paper (7). Operators of contained use facilities will be required to conduct a safety assessment, apply appropriate containment and safety measures, and notify the national competent authority of the facilityand of certain details of work carried out. The directive requirements divideon both the scale of work and the level of risk posed by the microorganisms used, and both factors are taken into account in determining whether record keeping, notification, or consent is required from the national competent authority. All “first use”of facilities will requirenotification, andif other than “low-risk” agents are used, consent will be needed. All individual commercial operationswill require prior notification at least and consent will again be required for each use of non-‘low-risk” agents. The directive calls upon competent authorities to organize inspection of facilities and to make a minimumsummary of information availablefor public records, although thereis provisionto protect commercially sensitive information beyond that summary. The second biotechnology directive deals with the deliberate releaseof genetically modified organisms (GMOs) to the environment (18). The directive isin.two parts, controlling both releases for the purposes of research and development and controls on products that are, or contain, GMOs. Those wishing to undertake releases for research purposes will be required to notify the national competent authority in advance. An exchange of information on such proposedtrial releases will operate between member states,but the decisionto give consent will remain national. For the foreseeable future each release will need specific individual approval, but there is provision for a simplified procedure as experience develops.

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In the case of the marketing of products that are, or contain, GMOs, a Part C notification will be made to the individual national authority, which after reviewing the proposal, will circulate it to all other member states authorities who will haveto agree to the product being authorized for release. If one or more objectionsare received, discussionswill take place between authorities to a fixed timetable. Failing agreementat this stage, the proposal is then to be submittedto the European Commission, which will put itto an international committee acting asthe final arbiter. This scheme is intended to produce a crosscommunity system of approval so that a product developed in one memberstate can be marketed in all 12 after a single entry to the regulatory system. The Deliberate Release Directive does provide exemption for those products that are subject to community-based product law, which contains “similar” provisions. for human and environmental risk assessment. To date (end of 1993) no such product legislation was yet in force, and for the foreseeable future the clearance system described abovewill operate. Product-based “vertical legislation” does, however, exist indraft form for the following sectors: pesticides, novel foods, medicinals, transgenic plants and animals, and seeds. At the end of 1993, two years after full implementation of these two directives was intended, it is still difficult to predict how well they will function. Only five applications had been made under Part theC procedure for the marketing of products, and two clearances issued. The first of these, for a veterinary vaccine, was approvedafter a delay of 3 months caused by the opposition of a single member state. Certainly, industry lobby groups in Europe have expressed major reservations over the deliberate release directive, fearingthat it will prove to 1989 be little morethan a disguised moratorium on releases (as sought in and 1990 by factions withinthe European Parliament),as a result ofPart C not working efficiently and not allowing for product clearances. The directive is seen by someas establishing an overlap between existing product approval schemes (national and community) and creating a clumsy system of multiple hurdles based on the technology used (20). These may be political points, but thereis still widespread unease over the way that the process of rDNA technology in Europe is being regulated by this directive rather by product considerations. This impliesthat the risks associated with GMOs are different from those of the same organism modified bytraditional strain selection -a quite opposite view to that expressed within the United States in 1991-1992 (see below).

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The aim of industry is to introduce its products in the marketplace. The new biotechnology based on rDNA techniques is still only a small part of the bioindustry as a whole. It has not yet developedto a stage of self-survival-and there is a danger that an overly restrictive system of control driven by public and political pressures may suffocate the baby at birth. At least partiallyas a result of these concerns and in recognition ofthe lack of existing coordination for biotechnology policy,a new committee, chaired by the European Commission’s Secretary General, the Biotechnology Coordination Committee (BCC), has been established in Brussels to cover all ofthe Commission’s services with responsibilitiesfor sectors of biotechnology (21). During 1993 as at least someof the European nations gained experience in operating under the directives, discussion had begun between the competent authorities with a view to reducing the bureaucratic burden on users, especially with regard to simplified operating procedures, especially for research trials.

3.2

European Regulations

3.2.1 UnitedKingdom The United Kingdom was the first nation in the world to implement speas long agoas 1978, with the passing of the cific law dealing with rDNA, Health and Safety (Genetic Manipulation) Regulations made under the Health and Safety at Work etc. Act,1974 (HSWA). The regulations made it obligatory to notify the Health and Safety Executive (HSE) ofan intention to carry out genetic manipulation work,so that HSE inspectors could use their existing powersto inspect facilities and ensure that standards laid down in guidance from the Genetic Manipulation Advisory Group (GMAG) were met. GMAG was the sourceof U.K. guidance in this field from 1978 to 1984. In 1984 the Advisory Committee on Genetic Modification (ACGM) replaced GMAG and has producedto date 11 separate guidance documents to cover all aspects of rDNA work. This guidance provides a benchmark by which compliance with the general duties of care underthe HSWA can be judged. The 1978 regulations were revised, and the Genetic Manipulation Regulations 1989 extended the obligation for notification to three areas: construction/modification (as in the 1978 law), use,and intentional introduction release to the environment. Provisions were made for simplified retrospective notification of low-risk work following risk assessmentand review by a local safety committee.

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Implementation of the two EC directives on biotechnology has been achieved through new legislation, the Genetically Modified Organisms (Contained Use) and the Genetically Modified Organisms (Deliberate Release) Regulations1992. These are made under the powers of HSWA and the Environmental Protection Act 1990, along with the European Communities Act 1972. The U.K. regulations follow the EC directives very closely and go beyond them in two areas only: (a) the inclusion of all modified organisms rather than the restriction to microorganisms found in the contained use directive, and (b) the provision of statutory public registers for information about releases and products. In 1990, a second U.K. advisory committee was established. The Advisory Committee on Releases to the Environment (ACRE) is charged with reviewing all applications made under the Deliberate Release Regulations. Meanwhile, ACGM continuesto oversee all contained applications.The United Kingdom also has several product-oriented pieces of legislation that can be used to clear proposals involving rDNA technology. In particular, the Ministry of Agriculture, Fisheriesand Food operates a notification schemefor pesticides underthe Food and Environment Protection Act, 1986, and a license scheme for work with plant pests under plant also exists that the Medicines Control Agency health legislation. Legislation uses to deal with new medicines under the Medicines Act.

3.2.2 Denmark The United Kingdom’s initiative in setting up a regulatory framework was followed in1986 by Denmark, whichbrought into force its Environment and Gene Technology Act (EGTA). The Danish legislation differed from that then in use inthe United Kingdom by being concerned with environmental protection and being dealt with by an agency within the Danish Department of the Environment. The act also deals with human health aspects of such work. The EGTA covered research scale and production scale work and specifically prohibited the deliberate release of modified organisms without specificapproval of the Minister for the Environment. In 1989, the Danish legislation was updatedto bring the law into line with the then developing EC directives. Although the present law is somewhat more burdensomeon GILSP users than the contained use directive demands, Denmark could reasonably claim to have beenthe first EC state to have fully implementedthe new requirements. 3.2.3 Germany During 1990, Germany becamethe third country in Europe to implement statute law, with the successful passage through parliament of its Genetic

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Engineering Act. Thisact regulates contained work inboth research and large-scale operation, the deliberate release into the environment of GMOs, the placingon the market of products containing or consisting of GMOs, and the transportation and storage of GMOs. The act deals with both human and environmental health and provides protection by defining safe working practices, implementing systemsof state control, monitoring, and regulating liability under civil law. The German legislation was reviewed before it passed into law by Schubert (22). The German legislation has been described as containing many provisions that are “either virtually incomprehensible or mutually incompatible” (23). The act provides for permit requirementsfor construction, operation, and modification of commercial-scale facilities. Under certain circumstances, permission for commercial operations is subject to public hearings. The act is controlled by the Lander (the 16 states). The 1990 law has suffered from inconsistent application in different Lander, and during 1992, German industrialists and academics became increasingly vocal in their frustration over what‘was seen as a complex and overbureaucratic set of procedures for even the lowest-risk work. Up to 15 local agencies (such as fire, sewerage, water, etc.) may be involved and over 60 pages of data were required evenfor small-scale work. As a result, lengthy delays in gaining approval were being encountered. These concerns have apparently been heeded, and a federal decisionwas reported (24)that will bring the “gene law” into line with the directives. The Bundestag (lower house)of the German parliament approved a revised Genetic Engineering Law in October 1993. The amendmentsto the 1990 legislation aimto ease the licensing requirementsfor both research experiments and industrial production plants. Once the revision passes the Bundesrat (upper house), the position of Germany will be closer to that of the rest of the EC. While this was occurring within Germany, the German competent authorities presented a case to the European Commission for modifications to Directive 90/219/EEC that would allow further deregulation of genetic engineering.

3.2.4 TheNetherlands The year 1990 also saw the implementationof statute law in the Netherlands to control rDNA technology. The Chemical Substances Act was amended with effect from March 1990 to cover both contained work and releases of modified organisms.The Netherlands Directoratefor Chemicals and Risk Management, part of the Directorate for Environmental Protection, operates the oversight function for all rDNA work. Further

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revision of the Dutch law occurred in 1993 with a new Environmental Protection Act coming into force which simplified proceduresfor notifications. Similar updating of the law covering contained work came into force in June 1993.

3.2.5 France France has operated a systemof oversight of rDNA work through two committees based in two agencies, the Ministries of Agriculture and of Research and Technology. In 1992these were put onto a statutory footing by passagethroughout the French legislature of a law intended to implement the two directives.The law will be supplemented by the application of standards issued by AFNOR, the French standards body. Apart from the countries mentioned above, the majority of the EC nations have not yet implemented specific law to cover rDNA work. What exists in some countries are systems of oversight on based guidelines rather than law, or utilizing as a statute base, when necessary, existing legislation drafted to deal with "traditional" products. By July 1993 the European Commission was taking action for nonimplementation of the Biotechnology Directives against Spain, Greece, and Luxembourg. However, Italy, Ireland, and Portugal had only framework law in place, with no implementing regulations yeton their statute books. The remainingmember state, Belgium, had law in placefor the Flanders regions but had not completed the task in other regions.

3.3 Regulations in the United States

As described above,the United States has had comprehensive guidelines to cover the construction of rDNA organisms at a laboratory scale for some time. Beyond the research stage, the system of dealing with commercial applications has generated a series of overlapping areas of responsibility that willbe familiar worldwide-a number of regualtory agencies each operating product-based review schemes. Three principal agencies are involved in controlling rDNA products the in United States. 1. The Food and Drug Administration (FDA) has responsibility for human and veterinary medicines,food, and food additives. By 1989, the FDA had approved rDNA products in the field of therapeutics: for example, proteins for human use, such as interferon, insulin, and human growth hormone, and in the field of in vitro diagnostic reagents, such as monoclonal antibodies and DNA probes. Over 650 genetically en-

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gineered products were undergoing clinical trials at that time (25), although these wereproducts of rDNA organismsrather than those classified as in the EC as containing genetically modified organisms. 2. The U.S. Department of Agriculture (USDA) uses two principal pieces of law to regulate the products of the “new biotechnology,” although, as with the FDA, these were not designed with rDNA technology in mind. a. The Virus-Serum-ToxinActdealswithveterinaryvaccines and other biologicals.The USDA has reviewed a proposal to test a vaccinia virus modified to act as a vaccine against rabies under this legislation (26). -b. The Federal Plant Pest Act and the Plant Quarantine Act enable the agency to demand review of a risk assessment prior to field testing plants and microorganisms that may be potential plant pests. These acts are overseen by the USDA’s Animal and Plant Health Inspection Service (APHIS). The USDA issues permits for the release into the environment of a “regulated article.” The commonest reason for plant releases to be so considered has been the use of Agrobacterium tumefaciens as a vector or the incorporation of cauliflower mosaic virus regulatory sequences. An “environmental assessment” is evaluated by APHIS, which confirms that the release will not pose a risk of introduction or dissemination of plant a pest or present a significant impacton the human environment-afinding of no significant impact(FONSI). APHIS has 120 days to process the application, and a permit will be denied if the requirements noted above are not met. In such a case, APHIS must produce its reasonfor denial and the decision may be appealed. Permits granted by APHIS may be qualified by conditions beyond those submitted the in initial application. The USDA’s responsibilities in the area of field testing of rDNA organisms was set out in 1990 in a guide to those undertaking such experiments (27). A set of detailed guidelines designed for the promotion of safety and to allow researchersto design safe experiments was published in 1991 (17). The year1992 saw the issue of proposed new rulesthat would greatly simplify the regulatory system controlling the field testing of plants. Six major crops-tomatoes, corn, cotton, soybeans, potatoes, and tobacco-modified by insertion with certain genes would be subject to a simplified notification procedure(28).

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3. The Environmental Protection Agency (EPA) administers two relevant pieces of legislation. The Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) requires submission both of health and environmental data prior to the registrationof any pesticide product. The Office of Pesticides Programs (OPP) operates FIFRA. Prior to 1984, notification was not required for trial tests of biological pesticideson plots of lessthan 10 acres. Sincethat time, notification to field-test rDNA microbial pesticides has been required regardless of scale. Experimental use permits (EUPs) may be required, and these are reviewed on a case-by-case basis byOPP. The Toxic Substances Control Act (TSCA) is also used to deal with substances, including microorganisms that are not regulated by other federal statutes. TSCA is essentiallya notification scheme requiringthe submission of health and safety data before commercial manufacture begins. Originally, TSCA exempted small-scaleproduction for research and development purposes, but EPA has established a review of smallscale environmental releases of modified organisms within the TSCA procedures. In July 1991, the Science Advisory Board, an independent body that which advises the EPA, met to review a proposed regulation under TSCA was intended to regulate the microbial products of biotechnology. A draft of the proposed regulation and accompanying discussion papers ran to over 300 pages and was not expected to be ready for public comment until 1992. A report was published (29) in 1992 which dealt with the delays faced by EPA in theirefforts from1984 onward to issue a TSCA biotechnology regulation. The failure of the draft to progress was tied closely to the federal government’sapproach to regulation.

3.3.7 FederalGovernmentPolicy With three main agencies all workingthe in field of rDNA,there is adanger ofconflicting review, and this was recognized in1985 when a cabinetlevel group was established to review the regulatory oversight provided for rDNA technology. At this time no new law was judged necessary, but to encourage consistency, the Biotechnology Science Coordinating Committee (BSCC) was established to include members from the three agencies together with NIH and the National Science Foundation (30). Unfortunately, BSCC was dogged by controversy in a number of areas, such as its ties with industry, which led to the resignationof its chairman in 1988, the closed nature of its discussions,and its dominationby medical interests at the expense of environmental expertise. Such problems

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did not assist progress(25). Many saw its purpose asthat of minimizing unnecessary overlap between the regulatory bodies,yet 1990 saw parallel reviews by the NIH and FDA to regulate gene therapy. The year 1990 also saw the publication, after much delay, of the BSCC’s scheme on how the key agencies willevaluate proposalsto release rDNA organisms. The paper described what werereferred to as ‘’principlesfor the scope of oversight for planned introduction into the environmentof organisms with modified hereditary traits.” The BSCC supported six categories of such organismsthat might safely be tested without regulatory assessment (31). Such a system harks back to the gradual deregulation of intrinsically safe work with rDNA organisms in laboratory-scale operations that occurred in the 1970s. The “scope” documentwas effectively rewrittenas a result ofthe work of the President’s Councilon Competitiveness. This body issuedreport a on national biotechnology policy (32) that called for work with rDNA organisms not to be subject to federal oversightthrough legislation. The “scope” document took the form of a policy white paper issued from the Office of Science and Technology Policy in February 1992. Now titled “Exerciseof Federal Oversight within Scope of Statutory Authority: Planned Introductionsof Biotechnology Products into the Environment,” the paper develops the principle promoted by OECD, that biotechnology does not per se pose a risk to health or to the environment (33). The paper calls for risk-based review, where risk is dependent on the characteristics anduse of individual productsrather than on the process of production. Deleted from the final version of the paper are the former “categories for exclusion,” whichare not considered to be consistent with the concept of risk-based approach. “Scope” is considering biotechnology under a broad definition, and under this, “traditional” applications are treated in the same way as the products of rDNA technology. The issue ofthe “scope” report was seen as the first phaseof a threepart initiative, phase2 being a review of existingand projected rulesfor the oversight of biotechnology products, and phase 3, the development of detailed “road maps” basedon the principle of “one-stop shopping,” which will set out the process of obtaining federalapproval for marketing. It remains to be seen how great an impact the “scope” report will have. In that existing FDA controlsdo not appear to penalize rDNAproducts, it might be expected that the EPA and USDA policies would have been more affected. However, such predictionswere thrown into the melting pot with the change of administration after 1992 the elections. At the time of writing, no clear indication had emerged to as direction of the Clinton

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presidency in the area of biotechnology. Certainly, the “scope” report was not received by many ina positive light. Bothit and subsequent developments have been the subject of criticismfrom lobby groups representing both industrial and environmental groups(34). As well as the major federal agencies’ roles in overseeing rDNA products, a number of individual state initiatives have been proposedor enacted, particularly with regard to the environmental release of rDNA organisms. Legislation has reached the statute books in at least three states: West Virginia, Minnesota, and North Carolina.

3.4

RegulationinOtherCountries

3.4.1 Australia Gene technology wasthe subject of review inAustralia in1987. The Genetic Manipulation Advisory Committee (GMAC) was established by the Australian government asa nonstatutory body to oversee all GM activities. It has produced guidelines updating thoseof an earlier committee, the Recombinant DNA Monitoring Committee. Australia has a range of state-enacted product-based legislation which covers some biotechnology products. A review dealing with regulatory oversight of biotechnology in both Oceania and Asia was published in 1990 (35). In 1992 the Australian House of Representatives recommended the establishment of a new authority to oversee the release of rDNA organisms to the environment. This proposal has a great similarityto the committee structure of the United Kingdom. In September 1992 the existing GMAC issued itsdraft guidelines covering planned release experiments. 3.4.2 Canada Canada has had national guidance issuedby its Medical Research Council since 1977 mirroring that of the NIH for rDNA research. A range of product-based legislation provides coverage that can be used to control rDNA products in the areas of pharmaceuticals, plant health, and animal health through the Food and Drug Act, PestControl Products Act, and Animal Diseaseand Protection Act. Recombinant DNA work is implicitly covered byfurther legislation, suchas the Seeds Actand Fertiliser Act. In 1988, the Canadian Environmental Protection Act (CEPA) became lawand the products of gene technology have been included as one area in which assessment of a “new substance” canbe reviewed. CEPA will control releases of modified organisms into the environment. The Canadian regulatory scheme is detailed ina single review (36).

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3.4.3 Japan Japan’s oversight of rDNA work exists within a framework of guidance rather than regulation (37). As in most countries, the first guidance for rDNA experiments was issuedfor laboratory work, in this case in1979. Like the guidelines issued by the NIH/RAC, these were significantly relaxed in a series of revisions, notably in 1983. Today, research and development aspects of this technology are dealt withby the Japanese Ministry of Education, Science and Culture as regards work in organizations such as universities and by the Science and Technology Agency (STA). STA has a rDNA committeethat advises on work below a 20-L level. Oversight of large-scale production involving rDNA technology in Japan falls to three agencies: the Ministry of International Trade and Industry (MITI), which since 1986 has issued guidelines for industrial applications primarily in theareas of chemicalsand fertilizers; the Ministry of Health and Welfare (MHW), which regulates the manufacture of medicines and food products; and the Ministry of Agriculture, Forestry and Fisheries (MAFF). The Japanese guidelines deal specifically with the preparation, propagation, and use of viable genetically modified organisms that have been generated invitro using vector DNA.This strict definition is narrower than that used in many European countries and excludes the generation of transgenic organisms using techniques of cell fusion or microinjection of nucleic acid. The schemeof review is not underpinned by statute, yet by 1989, 133 industrial proposalsand 51 medical proposals had been reviewed by MITI and MHW, respectively.This voluntary scheme is supported by the Committee on Genetic Manipulation, whose role is to promote observation ofthe review process. The most recent agency to enter the stage inJapan is MAFF. Following productionof draft guidance in1986, it issued guidelines in 1989 concentrating mainlyon the use of transgenic plants. These are available inthe English languageand are entitled “Guidelinesfor the Application of rDNA Organisms in Agriculture, Forestry, Fisheries, Food Industry and Other Related Industries.’’ The state of regulatory developmentsin the field of rDNA technology was reviewed with respect to the member countriesof the OECD in 1990 (38). This survey includes details from 18 nations and is accurate as of July 1990. The United Statesand the United Kingdom,to name just two a wide range of technologiesto recnations, have moved forward across ognize an increasing rolefor self-regulation. When genetic manipulation was first transferred to the industrial sector, it was felt appropriate to

Safety Aspects and Regulatory

403

require the prior notification of individual proposals. This was accompanied by the issue of guidanceon health surveillance, which calledfor particular care in large-scale installations. Once industry supportedthe it was possiconcept of GILSP, with its use of intrinsically safe organisms, ble to relax the more stringent requirements. The EC directive on contained use, however, returns all large-scale useto a system ofprior notification, and any large-scale work with what are described as group I1 organisms (higher-risk organisms) will bethe subject of a positive consent system. Uncertainty is in itself as damaging as any overrestrictive regulation on the statute books. The development of modified organisms for the marketplace is a long processand industry is rightly very sensitiveto the potential for change in public perception the of technology and the regulatory systemto be used to control it. The first half of the 1990s may be seen as the watershed in the development of rDNA technology.The potential benefits from the products of genetic modificationare enormous, but realization of such benefits can come only an in atmosphere of confidence that the safety issues have been addressed properly. Through the introduction of effective safety controls via a pragmatic regulatory approach, such benefits can be obtained with minimum risk.The challenge will be to determine the right balance between safety needs and regulatory excess.

APPENDIX

Suggested Criteria for r-DNA GILSP (Good Industrial Large-scale Practice) Micro-organisms andCell Cultures* organismHost Non-pathogenic characterised Well Non-pathogenic and

Vectorhsert

organism r-DNA

free from known harmful sequences No adventitiousagentsLimitedinsize as much Assafe inindustrialsetas possible to the ting as host organism, DNA required to per-orwithlimitedsurform the intended vival, and without adfunction; should not verse consequences in increase the stability the environment of the construct in the environment (unless that is a requirement of the intended function)

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Appendix (Continued)

A tor/insert organism Host Extended history of safe Should be poorly mobiluse or Built-inenvironmental Should not transfer any limitations permitting resistance markers to optimal growth in inmicroorganisms not dustrial setting but known to acquire limited survival withthem naturally out adverse consequences in the environment Source: Safety Considerationsfor Biotechnology, OECD, Paris, 1992. *Revised Appendix F to r-DNA Sofety Considerations, 1986.

REFERENCES 1. Singer M, Sol1 D. Guidelines for DNA hybrid molecules. Science 1973; 198: 11 14. a h e n S N , Davis RW, HognessDS, Natbans 2. Berg P, Baltimore D, BoyerW , D, Roblin R, Watson JD, Weissman S, Zinder ND. Potential biohazards of recombinant DNA molecules. Science 1974; 189303. 3. Amber light for genetic manipulation (editorial). Nature 1975; 253:295. 4. Berg conference favours useof weak strains (editorial). Nature 1975; 2546-7. 5. National Institutes for Health. Guidelines for Research Involving Recombinant DNA Molecules. Washington, DC: US Department of Health, Education and Welfare, 1976. 6. National Institutes for Health. Guidelines for Research Involving Recombinant DNA Molecules. Federal Register, 51 FR 16958, 1986. 7. Organisation for Economic Co-operation and Development. Recombinant DNA Safety Considerations. Paris: OECD, 1986. 8. Organisation for Economic Co-operation and Development. Safety Considerations for Biotechnology 1992. Paris: OECD, 1992. 9. Council Directiveon the contained use of genetically modified micro-organisms (90/219/EEC). Off J Eur Communities 1990; L117:l-14. 10. US Department of Health and Human Services, National Institutes of Health. Recombinant DNA Research: Action Underthe Guidelines. Federal Register, 33174-33183,1991. 11. Advisory Committeeon Genetic Manipulation. Guidelineson the Large Scale Use of Genetically Manipulated Organisms. ACGM/HSE/Note 6. London: Health and Safety Executive, 1987.

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12. Day PR. Engineeredorganismsin the environment: a perspective on the problem. In: Halvorsen HO, Pramer D, Rogul M, eds. Engineered Organisms in the Environment: The Scientific Issues. Washington, DC: American Society for Microbiology, 1985:4-10. 13. Kornberg H. Opening remark. In: Sussmon M, Collins CH, Skinner FA, Stewart-Tu11 DE, eds. The Release of Genetically Engineered Micro-organisms. London: Academic Press, 1988:l-5. 14. Advisory Committee on Genetic Manipulation. The Planned Releaseof Genetically Manipulated Organisms for Agricultural and Environmental Purposes. ACGM/HSE/Note 6. London: Health and Safety Executive, 1986. 15. Alexander M. Ecological consequences: Reducing the uncertainties. Issues Sci Techno1 1985; 157-68. 16. Advisory Committee on Genetic Manipulation. The Introductionof GeneticallyManipulatedOrganisms into the Environment:Guidelines for Risk Assessment and for the Notification of Proposals for Such Work. ACGM/ HSE/Note 3, revised. London: Health and Safety Executive, 1990. 17. US Department of Agriculture. Proposed guidelines for Research Involving the Planned Introductioninto the Environment of Organisms with Deliberately Modified HereditaryTraits; Notice. Federal Register 1991; 56:41344151. 18. Council Directive on the deliberate release into the environment of genetically modified organisms (90/220/EEC). Off J Eur Communities 1990; L117: 15-27. 19. Council Directive on the protection of workers from risks related to exposure to biological agents at work (90/679/EEC). Off J Eur Communities 1990; L374:l-12. 20. Ager BP, Poole NJ. Impact of EC legislation on the development of biotechnology in agriculture. In: Pests and Diseases, Vol. 3. Farnham, Surrey, England: British Crop Protection Council, 1990. 21. New Biotechnology Co-ordination Committee. Eur Biotechnol News1 1991; 108~1-2. 22. Schubert G. Much more discussion needed: The current state of debate on the West German genetic engineering bill. Proceedings of the European Workshop on Law and Genetic Engineering. Bonn: BBU Verlag, 1990:28-31. 23. Fritsch K, Haverkamp K. The German Genetic EngineeringAct. Bio/Technology 1991; 9:435-437. 24. Abbott A. Germany will ease requirements of gene technology laws in bow to researchers. Nature 1992; 360:286. 25. US Biotechnology. A Legislative and Regulatory Roadmap. Washington, DC: Bureau of National Affairs, 1989. 26. Engineered rabies vaccine to be tested in wild. Washington Post, August 15, 1990:A8. 27. AgriculturalBiotechnology:Introduction to FieldTesting.Washington,DC: US Department of Agriculture, Office of Agricultural Biotechnology, 1990.

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28. US propose relaxing rules on trials of biotech crops. Nature 1992; 36094. 29. US General Accounting Office; Resources, Community and Economic De30.

31. 32. 33.

34.

velopment Division. Biotechnology: Delays in and status of EPA’s efforts to issue a TSCA regulation. 1992. OfficeofScience and TechnologyPolicy. Co-ordinated Framework for Regulation of Biotechnology; Establishment of the Biotechnology Science Co-ordination Committee; Notice. Federal Register 1985; 50:47174-47195. Revamped deliberate release scope principles are aired. Am SOC Microbiol News 1990; 56:457-458. President’s Council on Competitiveness. Report on NationalBiotechnology Policy. Washington, DC: Office of the Vice President, February 1991. OfficeofScience and TechnologyPolicy.Exercise of FederalOversight Within Scope of Statutory Authority: Planned Introductions of Biotechnology Products into the Environment. Washington, DC: US Office of Science and Technology Policy, February 1992. Charles D. White House changes rulefor genetic engineering. New Sci 1992;

No 1815:14. 35. Millis NF. Biotechnologyin veterinary science: regulations in Asia and Oceania. Rev Sci Tech OIE 1990; 9:715-732. 36. Biotech Regulations:A Users Guide. Ministry of State for Science and Technology, Government of Canada, 1988. 37. Uchida H.Current situation and trendsof legislation inJapan. In: Defaye J, de Roissart H, Vignais PM, eds. Risk Management in Biotechnology. Paris: Technique et Documentation, 1990:175-180. 38. Organisation for Economic Co-operation and Development. International Survey on Biotechnology Use and Regulations: OECD Environmental Monographs No. 39. Paris: OECD, November 1990.

Index

Conjugation from E. coli to Streptomyces, 318 between streptomycetes, 318 Cyclic AMP, 14

Fermentation to high cell density, 227 media for pectinase, 250 of methylotrophic yeasts, 220

Gene@) activation of, 328 copies of, 210, 227, 246, 352 disruption, 159,216 fusions, 26

[GenWl for metallothionein, 145 of methanol metabolism, 202 regulation, 6 4 , 263, 320, 324, 337 A-factor and, 323 replacement, 123 reporter, 3, 285, 336 toxic, 7, 21 Gluconic acid, production of, 271 Glucose oxidase applications of, 270 expression of, 268 gene for, 281 induction of, 278 localization of, 274 overproduction of, 277, 284 properties of, 272 407

408

Index

Homology intramolecular, 7 in polygalacturonase gene, 258

[Pectinases] multiple forms of, 251 overproduction of, 255 Plasmid copy number of, 3,7,9, 6 6 , 126,

Inclusion bodies, 31 Integration bacteriophage system use of, 314 multiple insertions, 123 single copy, 123,313

temperature selection of, 127 replication, origin of, 3,126,143 stability of, 7, 8 , 141, 152 Posttranslational modifications,

Large-scale containment, 388 good industrial practice, 387, 403 guidelines for, 386 10-liter cutoff and, 386

Messenger RNA, stability of, 20 Methanol metabolism dihydroxyacetone synthetase and, 198

formaldehyde dehydrogenase and, 198

formate dehydrogenase and, 198 methanol oxidases and, 196 peroxisomes and, 196, 201

OECD, safety considerations of, 386

Pathogenicity, factors affecting, 388 Pectinases applications of, 249 cloning of, 252 expression of, 247 214

139

27,135,174

Promoters constitutive, 130, 151 CUP 1system autoregulation of, 148 in expression of r-hirudin, 148, 151

in Escherichia coli, 14-19 hybrid, 16, 131 probe@), 317, 336 regulated, 14,17,130,207, 222, 334

in Saccharomyces cerevisiae, 131132,145

strength, 3, 8 in streptomycetes, 333 Protease@) deficient strains, 29, 62 degradation of r-hirudin and, 159,163

Kex2, 175,217 suppression of, 63 Proteins folding of, 136,173,219 fusion cleavage of, 2, 268 glycosylation of, 135,172, 218 heat shock, 342 heterologous, production of, 14, 19, 21, 58, 64, 144,159,207, 213 hybrid, 26, 32-33

intracellular expression of,

409

Index

[Proteins] proteolytic degradation of, 17, 28,65,137,155

carboxypeptidases and, 138, 155

repressor, 7, 14 stability of, 137

[Secretion] of hemicellulases, 354 of heterologous proteins, 30, 55, 133,153,174, 355

216, 225, 353,

presequences and, 134, 217 of proinsulin, 354 prosequences and, 134, 217 of proteins, 19, 34,57, 172, 339, 343, 353

Recombination, homologous, 123, 212, 314

Regulation of biosynthetic pathways in streptomycetes, 328-332 of fungal metabolism, 243 of methanol metabolism, 1% Regulations in Australia, 401 in Canada, 401 EC directives and. 391 in Europe, 394 in Japan, 402 in the United States, 397 Release guidelines for, 390 of rDNA organisms, 389, 392 Ribosomes binding site of, 22 stalling of, 11, 24 RNA polymerase, 11, 17, 322

signals, 3,32,57,133,173,

216,

347

for acid phosphatase, 134, 148, 154, 217

for alpha factor, 134, 174, 217 cleavage of, 135, 217,265 recognition particle and, 57 Selection, markers for, 3, 7, 127, 172, 207, 214, 245

Sigma factors, 322 Strain@) field testing of, 389 fungal breeding of, 261 host, 3, 17 GILSP and, 388 stability of, 346

Transcription, 7, 8, 128, 333 promoter elements and, 128 signals, 11, 64 terminator elements, 3,13, 20, 128,148, 207

Secondary metabolites hybrid molecules, 348 stringent response and, 320 yield improvement of, 351 Secretion of acid phosphatase, 164 of agarase, 344 of alpha amylase, 344 chaperones and, 56, 341 of esterase, 346

upstream activating sequence, 148

Transduction of plasmid DNA, 320 signal system, 326 Transformation of Aspergillus niger, 245 of Hansenulla and Pichia, 209

of streptomycetes, 3 18

410

Translation, 132, 339 codon biashage, 133, 340 efficiency of, 22, 24 of heterologous mRNA, 132 initiation of, 2, 21 Transposition, frequency of, 7, 315

Index Vectors based on alpha amylases, 39 based on centromeres, 127 based on proteases, 63 bifunctional, 3 13 integrative, 10, 123, 210, 246, 313 plasmid based, 3 two-micron, 125, 139, 154, 210 yeast,122,139