Gerd Cellissen (Editor) Hansenula polymorpha Biology and Applications
Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Yerlag GmbH, Weinheim ISBN: 3-527-30341-3
Gerd Gellissen (Ed.)
Hansenula polymorpha Biology and Applications
^WILEY-VCH Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Veriag GmbH, Weinheim ISBN: 3-527-30341-3
Editor Cerd Gellissen
Rhein Biotech GmbH Eichsfelder Str. n 0-40595 Diisseldorf Germany
This book was carefully produced. Nevertheless, authors and publisher do not warrant the information contained therein to be free of errors. Readers are advised to keep in mind that statements, data, illustrations, procedural details or other items may inadvertently be inaccurate. Library of Congress Card No.: applied for British Library Cataloguing-in-Publication Data:
A catalogue record for this book is available from the British Library. Die Deutsche Bibliothek - ClP-Cataloguingin-Publication Data
A catalogue record is available from Die Deutsche Bibliothek © Wiley-VCH Verlag GmbH 0-69469 Weinheim, 2002 All rights reserved (including those of translation into other languages). No part of this book may be reproduced in any form - by photoprinting, microfilm, or any other means - nor transmitted or translated into a machine language without written permission from the publishers. Registered names, trademarks, etc. used in this book, even when not specifically marked as such, are not to be considered unprotected by law. printed in the Federal Republic of Germany printed on acid-free paper
Cover illustration, micrograph of H. polymorpha: courtesy of E. de Bruin and F. de Wolf, Wageningen, The Netherlands.
Composition Alden Bookset, Oxford, England Printing betz-druck GmbH, Darmstadt Bookbinding J. Schaffer GmbH & Co. Kb, Griinstadt ISBN
3-527-30341-3
Dedicated to Gerhard Ernst (1943-2001)
Preface
The methylotrophic yeast Hansenula polymorpha has attracted increasing interest as a useful system for fundamental research and applied purposes. Members of a growing scientific community working with this organism have organized themselves in a world-wide Hansenula polymorpha network (HPWN). A first international conference held in August 2000 in Dusseldorf, Germany, provided a comprehensive look at the scientific achievements and research underway with this yeast. It was the participants of this conference who expressed their desire to initiate and contribute to this first Hansenula polymorpha handbook. Hansenula polymorpha has become a preferred organism for the production of recombinant proteins on an industrial scale. Product examples range from therapeutics such as hepatitis B vaccines to industrial enzymes like the feed additive phytase. The significance of this yeast in basic research stems largely from studies focused on peroxisome homeostasis. This book is intended to provide an indepth, up-to-date overview of the status of Hansenula polymorpha research, applications and methods. Aspects of the organism ranging from systematics, genetics, methanol metabolism and peroxisomal function to its use as a technology platform for the production of recombinant proteins are covered. A detailed chapter on laboratory protocols is also included. The handbook is addressed to all researchers and scientists working with this organism as well as to interested advanced students. I would like to express my gratitude to the authors for their fine efforts. I would also like to acknowledge the assistance of Adam Papendieck and Laura Guengerich in the preparation of this book, as well as the continuous support of Karin Dembowsky and her staff at WILEY-VCH. Diisseldorf, November 2001
Gerd Gellissen
Contents
Preface
vii
List of Contributors 1
xiii
History, habitat, variability, nomenclature and phylogenetic position Hansenula polymorphic
1.1 1.2 1.3 1.4 1.5
of
i
Introduction i Isolation and habitat i Assimilation of methanol 2 Nomenclatural problems 3 Phylogenetic position 4
2
Basic genetics of Hansenula polymorphic
2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8 2.9 2.10
Introduction 8 Strains 8 Auxotrophic mutants 9 Morphological mutants 10 Heavy metal resistance/sensitivity 11 Thermostability 12 Methanol non-utilization mutants 12 Genetic mapping, linkage groups and chromosome number 15 Mating and sporulation 16 Concluding remarks 17
8
3
Biochemistry and genetics of nitrate assimilation 21
3.1 3.2 3.3 3.4 3.5 3.6 3.7 3.8
Introduction 21 Genomic organization of the genes involved in nitrate assimilation 22 Gene disruption in H. polymorpha 23 Nitrate transport 24 Nitrate reductase 26 Nitrite reductase 28 Expression levels of YNTi, YNh and YN#i 29 Transcriptional regulation of YNTi, YNh and YNRi genes involved in nitrate assimilation 31
Contents
3.9
3.11
The YNAi and YNA2 gene products activate the transcription of YNTi, YNh and YNRi genes 33 Hansenula polymorpha as a model to study plant genes involved in nitrate assimilation 35 Concluding remarks 36
4
Amino acid biosynthesis 41
4.1 4.2 4.3 4.4 4.5 4.6 4.7
Introduction 42 Making the building blocks for proteins 42 Biosynthesis of amino acids in yeast - a short survey 42 Amino acid auxotrophic mutant strains of Hansenula polymorpha 45 Amino acid biosynthetic genes of Hansenula polymorpha 47 The general control system - lessons learned from bakers' yeast 52 Biotechnological aspects and outlook 55
3.10
5
Methanol metabolism
5.1 5.2 5.3 5.4 5.5 5.6
Introduction 61 Methanol metabolism in methylotrophic yeast 61 Regulation of methanol metabolism 68 Detoxification of toxic compounds during growth on methanol 69 Methylamine as a nitrogen source 72 Other types of peroxisomal metabolism known in methylotrophic yeasts 72
61
6
Hansenula polymorpha: a versatile model organism in peroxisome research
6.1 6.2 6.3 6.4 6.5 6.6 6.7
Introduction 76 Peroxisome function 77 Peroxisome biogenesis and degradation 80 Genes involved in peroxisome biogenesis (PEX genes) 81 Assembly of octameric, FAD-containing AO 86 Biogenesis of the peroxisomal membrane 87 Peroxisome degradation 88
7
Characteristics of the Hansenula polymorpha genome
7.1 7.2 7.3 7.4 7.5 7.6 7.7
Introduction 95 Electrophoretic karyotyping 95 Genome mapping (Chromo Blot) 97 The structure of ribosomal DNA 98 Regulatory elements in the rRNA genes 99 Nucleolar complex 100 Integration of heterologous DNA into rDNA 101
8
95
The expression platform based on H. polymorpha strain RBn and its derivatives - history, status and perspectives 205
8.1 8.2
76
Introduction 205 A toolbox of expression vectors 206
Contents
8.3 8.4 8.5 8.7 8.8 9
Promoters used in H. polymorpha RB11-based expression systems HARSi 113 Co-expression 116 New aspects 119 Conclusive remarks 120
Development of expression systems for the production of recombinant proteins in Hansenula polymorpha DL-i
9.1 9.2 9.3 9.4 9.5 10
224
Introduction 124 Development of host strains 125 Vector systems 128 Optimization of production systems for secretory proteins 138 Concluding remarks 142 Foreign gene expression in Hansenula polymorpha - approaches for "difficult proteins"
10.1 10.2 10.3 10.4 10.5
no
147
Introduction 147 Peroxisomal packaging of labile or toxic proteins 148 Production of membrane proteins 150 Secretion of active oligomeric heterologous proteins 152 Concluding remarks 152
n
Fermentation and primary product recovery 256
11.1 11.2 11.3
I ntr eduction 15 6 Strategic considerations for the fermentation of recombinant strains 157 Parallel small scale fed-batch fermentation in sparged column reactors
11.4 11.5 12
y«
High cell density fermentation in stirred tank bioreactors 163 Primary product recovery 168 Recombinant hepatitis B vaccines - disease characterization and vaccine
production
12.1 12.2 12.3 12.4 12.5
275
Introduction 275 Virus and disease characteristics 276 Recombinant vaccine production 285 The future of hepatitis B vaccination 296 Conclusion 200
13
Production of anticoagulants in Hansenula polymorpha
13.1 13.2 13.3
Introduction 222 Production and characterization of H. polymorpha-derived hirudin 222 Production and characterization of H. polymorpha-derived saratin 220
14
Production of cytokines in Hansenula polymorpha
14.1
Introduction
229
229
211
xii
Contents
14.2 14.3 14.4
Production of IFN oc-2a 230 Strain development for the production of IL-6, IL-8, IL-io, and IFNy 239 Conclusion 248
15
Technical enzyme production and whole-cell biocatalysis: application of Hansenula polymorpha
15.1 15.2 15.3 15.4 15.5 16
255
I ntr oduction 255 Important groups of technical enzymes 258 Pathway engineering and biocatalysis 261 The application of H. polymorpha as an expression system for technical enzymes and as a whole-cell biocatalyst 261 Conclusion 267 Biosafety aspects of genetically engineered Hansenula polymorpha - a case study about non-deliberate environmental releases
16.1 16.2 16.3 16.4 16.5 16.6 16.7 16.8 16.9 16.10
272
The sense and nonsense of monitoring programs with genetically engineered microorganisms 272 Risks of non-deliberate releases of yeasts and bacteria engineered with a recombinant aprotinin-gene: the case study 274 The capacity of H. polymorpha to colonize soil, aquatic habitats or sewage is low 274 Competition experiments indicate a decreased fitness of genetically modified H. polymorpha in soil 275 The aprotinin gene can be used as a tag for monitoring 276 H. polymorpha does not survive in bulk soil, surface water or sewage 277 The FMD promoter is turned off in soil 277 Aprotinin is utilized as a substrate by microorganisms and quickly eliminated from soil 279 Probabilities and risks of a horizontal gene transfer 281 Summary and conclusions 282
17
Methods
17.1 17.2 17.3 17.4 17.5
I ntr oduction 2 85 Classical genetic techniques 285 Transformation of H. polymorpha 291 Genome analysis 293 Generation of mitotically stable H. polymorpha strains harboring multiple copies of expression plasmids 296 Insertional mutagenesis 301 Fermentation 306 Methods for detection of recombinant H. polymorpha in soil 317 Nitrite determination in H. polymorpha cultures 324
17.6 17.7 17.8 17.9
Index
337
285
List of Contributors -Corresponding author Michael O. Agaphonov Institute of Experimental Cardiology Cardiology Research Centre 3rd Cherepkovskaya Str. I5A 121552 Moscow Russia Chapter 9 Sang-Jeom Ahn Greencross Vaccine Corporation 227-3 Kugal-Ri Kiheung-Eup Yongin City Kyunggi-Do 449-900 Korea Chapter 12 Christopher S. Barnes Department of Cardiovascular Research Biomedical Research Merck KgaA Frankfurter Str. 250 0-64271 Darmstadt Germany Chapter 13 Oliver Bartelsen* Rhein Biotech GmbH Eichsfelder Str. n 0-40595 Diisseldorf Germany E-mail:
[email protected] Chapter 13
Gerhard H. Braus* Abt. MolekulareMikrobiologie Institut fur Mikrobiologie & Genetik Georg-August-Universitat Gottingen Grisebachstr. 8 0-37077 Gottingen Germany E-mail:
[email protected] Chapter 4 Eui-Sung Choi Korea Research Institute of Bioscience and Biotechnology PO Box 115 Yusong, Taejon 305-600 Korea Chapter 9 Ulrike Dahlems Rhein Biotech GmbH Eichsfelder Strasse n 0-40595 Diisseldorf Germany Chapter 15 Adelheid Degelmann* Rhein Biotech GmbH Eichsfelder Strasse n 0-40595 Diisseldorf Germany E-mail:
[email protected] Chapters 14 and 17
List of Contributors
Klaas Nico Faber Division of Hepatology and Gastroenterology Groningen University Institute for Drug Exploration (GUIDE) Groningen University Hospital Hanzeplein i, P.O. Box 30001 Groningen The Netherlands Chapter 10 Gerd Gellissen* Rhein Biotech GmbH Eichsfelder Str. n 0-40595 Diisseldorf Germany E -mail: g. gellissen @ rheinbiotech. de Chapters 8, 12, 13, 14 and 15 Cornelis P. Hollenberg* Institut fur Mikrobiologie Heinrich-Heine-Universitat Diisseldorf Universitatsstr. i 0-40225 Diisseldorf Germany E-mail:
[email protected] Chapter 7 Zbigniew A. Janowicz Rhein Biotech GmbH Eichsfelder Str. n 0-40595 Diisseldorf Germany Chapter 12 Volker Jenzelewski* Rhein Biotech GmbH Eichsfelder Str. n 0-40595 Diisseldorf Germany E-mail:
[email protected] Chapters n and 14
Hyun Ah Kang* Korea Research Institute of Bioscience and Biotechnology PO Box 115 Yusong, Taejon 305-600 Korea E-mail:
[email protected] Chapter 9 Nobuo Kato* Division of Applied Life Sciences Graduate School of Agriculture Kyoto University Kitashirakawa-Oiwake, Sakyo-ku Kyoto 606-8502 Japan E-mail:
[email protected] Chapter 5 Jens Klabunde Institut fur Mikrobiologie Heinrich-Heine-Universitat Diisseldorf Universitatsstr. i 0-40225 Diisseldorf Germany Chapter 7 Ida J. van der Klei* Eukaryotic Microbiology Groningen Biomolecular Sciences and Biotechnology Institute Postbus 14 9750 AA Haren The Netherlands E-mail:
[email protected] Chapter 6 Sven Krappmann Abt. Molekulare Mikrobiologie Institut fur Mikrobiologie & Genetik Georg-August-Universitat Gottingen Grisebachstr. 8 0-37077 Gottingen Germany Chapter 4
List of Contributors
Kantcho Lahtchev* Institute of Microbiology Bulgarian Academy of Sciences Acad. G. Bontchev Str. 26 1113 Sofia Bulgaria E-mail:
[email protected] Chapter 2 Wouter J. Middelhoven* Laboratory of Microbiology Wageningen University P.O. Box 8033 6700 EJ Wageningen The Netherlands E-mail:
[email protected]. wau.nl Chapter i
Sang Ki Rhee* Korea Research Institute of Bioscience and Biotechnology PO Box 115 Yusong, Taejon 305-600 Korea E-mail:
[email protected] Chapter 9 Yasuyoshi Sakai Division of Applied Life Sciences Graduate School of Agriculture Kyoto University Kitashirakawa-Oiwake, Sakyo-ku Kyoto 606-8502 Japan Chapter 5
Frank Miiller Rhein Biotech GmbH Eichsfelder Str. n 0-40595 Diisseldorf Germany Chapter 14
Stephan Schaefer* Institut fur Medizinische Virologie Justus-Liebig-Unversitat Frankfurter Str. 107 0-35392 Giessen Germany Chapter 12
Adam Papendieck Rhein Biotech GmbH Eichsfelder Str. n 0-40595 Diisseldorf Germany Chapters 12 and 15
Heike Sieber Rhein Biotech GmbH Eichsfelder Str. n 0-40595 Diisseldorf Germany Chapter 14
Michael Piontek Rhein Biotech GmbH Eichsfelder Str. n 0-40595 Diisseldorf Germany Chapter 12
Jose M. Siverio* Departamento de Bioquimica y Biologia Molecular Universidad de La Laguna £-38206 La Laguna Tenerife, Canarias Spain E-mail:
[email protected] Chapter 3
List of Contributors
Jung-Hoon Sohn Korea Research Institute of Bioscience and Biotechnology PO Box 115 Yusong, Taejon 305-600 Korea Chapter 9 Alexander W.M. Strasser Rhein Biotech GmbH Eichsfelder Str. n 0-40595 Dusseldorf Germany Chapter 14 Manfred Suckow* Rhein Biotech GmbH Eichsfelder Strasse n 0-40595 Dusseldorf Germany E-mail: m.suckow@ Rheinbiotech.de Chapters 7 and 8 Christoph C. Tebbe* Institut fur Agrarokologie Bundesforschungsanstalt fur Landwirtschaft (FAL) Bundesallee 50 0-38116 Braunschweig Germany E-mail:
[email protected] Chapter 16 Michael D. Ter-Avanesyan Institute of Experimental Cardiology Cardiology Research Centre 3rd Cherepkovskaya Str. i5A 121552 Moscow Russia Chapter 9
Anni Tieke Rhein Biotech GmbH Eichsfelder Str. n 0-40595 Dusseldorf Germany Chapter 14 Ivo Timmermanns Rhein Biotech N.V. MECC Office Building Gaetano Martinolaan 95 6229 GS Maastricht The Netherlands Chapter 12 Marten Veenhuis* Eukaryotic Microbiology Groningen Biomolecular Sciences and Biotechnology Institute Postbus 14 9750 AA Haren The Netherlands E-mail:
[email protected] Chapters 6 and 10 Dorothea Waschk Institut fur Mikrobiologie Heinrich-Heine-Universitat Dusseldorf Universitatsstr. i 0-40225 Dusseldorf Germany Chapter 7 Hiroya Yurimoto Division of Applied Life Sciences Graduate School of Agriculture Kyoto University Kitashirakawa-Oiwake, Sakyo-ku Kyoto 606-8502 Japan Chapter 5
1 History, habitat, variability, nomenclature and phylogenetic position of Hansenula polymorpha WouterJ. Middelhoven
l.l Introduction
During the last decades several studies on the yeast Hansenula polymorpha Morais et Maia have been undertaken. Three characters of this yeast, in particular, raised the interest of investigators: • rapid growth at the expense of methanol as the sole source of carbon and energy (Levine and Cooney 1973), • remarkable heat tolerance permitting growth at temperatures up to 49 °C (Teunisson et al. 1960), and • easy interconversion between the haploid and the diploid state (Teunisson et al. 1960). 1.2
Isolation and habitat
Most studies have been done with the type strain of Hansenula polymorpha, CBS 4732. This strain was isolated by Morais and Maia (1959) from soil irrigated with waste water from a distillery in Pernambuco, Brazil. Nevertheless, the history of the species is older. Wickerham (1951) described Hansenula angusta isolated from spoiled concentrated (50% sugar) orange juice from Florida, USA, that had been canned and pasteurized but was fermenting. This species description was not valid, however, for lack of a Latin diagnosis that is prescribed by the International Code of Botanical Nomenclature since January i, 1935. Hence, H. angusta was a "nomen nudum". When Teunisson, Hall and Wickerham (1960) provided a correct description of H. angusta the Brazilian paper already had appeared albeit in a journal of very limited distribution. For this reason the name H. polymorpha was given priority. It is widely accepted since then. Conspecificity of both taxa was deduced from physiological similarity (Wickerham 1970). The epithet "polymorpha"
Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Veriag GmbH, Weinheim ISBN: 3-527-30341-3
1 History, habitat, variability, nomenclature and phylogenetic position of Hansenula polymorpha
reflects the variable colony appearance. The species is commonly isolated in both the haploid and the diploid form from natural habitats. The colonies of the two ploidies differ in intensity of pink color (that is due to the ascospores) when agar plate cultures are about six days old, because diploids sporulate more rapidly than the haploids. Haploid and diploid colonies may be differentiated by color, by the size and arrangement of the cells, and by the conjugated or unconjugated asci they contain, in haploid and in diploid colonies, respectively (Teunisson et al. 1960, Wickerham 1970). H. polymorpha is an excellent species for demonstrating conversion of one ploidy to the other. The epithet "angusta" means narrow. After 1960 more strains of the species have been isolated from fruit flies (Drosophila pseudobscura and other species), from the intestinal tract of a swine, from soil in South Africa, from alpechin (the residue of olives after pressing the oil), from frass of several broad-leaved trees and from larvae that fed upon the kernels of acorns (Wickerham 1970). Phaff (1985) and Starmer et al. (1986) reported H. angusta to be common in rotting Opuntia cacti in the deserts of Arizona and Texas, USA and the desert of Australia. The cactus isolates differ phenotypically by slow, weak growth on methanol but they do grow at elevated temperatures (45-46 °C). Some strains maintained in the CBS yeast collection (Delft, recently moved to Utrecht, The Netherlands) show a lower maximum growth temperature. For CBS 7031 and CBS 8099 this is lower than 42 °C. Strain CBS 5032, isolated by Van der Walt from maize meal in South Africa does not grow on methanol (CBS Yeasts Data Base, http://www.cbs.knaw.nl).
1.3
Assimilation of methanol
The first literature report of a yeast growing at the expense of methanol was that of Ogata et al. (1969). They isolated Kloeckera sp. Nr. 2201, later re-identified as Candida boidinii, from soil. This observation was surprising as until 1969 it was generally believed that yeasts were unable to do so. Therefore, it raised the interest of other investigators who subsequently isolated more methanol-assimilating yeast species, or found them in culture collections. Only 16 species of the approximately 350 yeast species maintained in the CBS Yeast Culture Collection at Delft, The Netherlands, were able to grow on methanol, the type strain of H. polymorpha included (Hazeu et al. 1972). The majority of these methanol-assimilating strains had been isolated from the bark of trees or from insects living on trees. This may be due to the abundance of lignin in these habitats, a polymer compound rich in methoxy groups (Hazeu et al. 1972). Some studies were directed on the production of single-cell protein from this cheap substrate, yeasts generally being considered as safe for foods. Other studies elucidated the cell structure of yeast cultures growing on methanol or dealt with the intermediary metabolism. In this chapter some of the first papers of the research groups are mentioned, the later results being dealt with in other chapters of this book. Tani et al. (1972) and Fujii and Tonomura (1972) demonstrated that a FAD-specific methanol oxidase generating hydrogen peroxide is the first step of the pathway of methanol
1.4 Nomenclatural problems
oxidation. The next steps of methanol catabolism are catalyzed by NAD-specific formaldehyde and formate dehydrogenases (Fujii and Tonomura 1972; Sahm and Wagner 1973). Presence of large catalase activities in methanol-grown yeast cells was demonstrated by Roggenkamp et al. (1974). Van Dijken et al (1975) found that methanol oxidase and catalase are located in the microbodies (peroxisomes) in the type strain of H. polymorpha, grown on methanol, where they form a crystalline structure. Levine and Cooney (1973) had isolated another strain of H. polymorpha from soil by continuous enrichment on methanol medium in a chemostat at relatively high growth temperatures (37-40 °C, maximum growth temperature in the chemostat was 50 °C). Continuous cultivation of yeasts on methanol is hampered by the unusually high oxygen demand. The substrate constant for dissolved oxygen of several methanol-assimilating yeasts, Hansenula spp. included, is 0.5-1.3 mg O2 LT1 when the cells respire methanol, but < 0.15 mg IT1 when respiring on ethanol or on endogeneous substrate (Middelhoven et al. 1976). This implies that the maximum specific growth rate of cultures on methanol can be attained only at oxygen concentrations near half air saturation.
1.4 Nomenclatural problems
The genus Hansenula H. et P. Sydow accommodates ascosporogenic yeast species characterized by spherical, spheroidal, ellipsoidal, oblong, cylindrical or elongated cells. Pseudohyphae and true hyphae may occur. Asci have the shape of vegetative cells. Ascigenic cells are diploid, either proliferating as such or arising from conjugation (mating) of haploid cells that may exhibit different mating types (heterothallism). From one to four ascospores are formed. Ascospores are hatshaped, hemispheroidal, spherical or saturn-shaped. The ring on the latter may be easily seen or may be extremely thin. Ascospores when observed with the light microscope have a smooth surface. Ascospores are usually liberated when mature by rupture of the ascus (Wickerham 1970). In H. polymorpha asci are unconjugated or exhibit conjugation between parent and bud (in diploid colonies or strains) or, less frequently, are formed by conjugation of individual cells (in haploid colonies or strains). H. polymorpha is supposed homothallic. The morphological characteristics of Hansenula species are also shown by species of the genus Pichia Hansen. Both genera differ in nitrate assimilation. Hansenula spp. grow with nitrate as the sole source of nitrogen and Pichia spp. do not. The distinction of both genera was satisfactory until Kurtzman (1984) carried out DNA/DNA reassociations. He found 68% base sequence complementarity between hat-spored H. minuta Wickerham and P. lindneri Henninger et Windisch, and 75% between saturn-spored H. mrakii Wickerham and P. sargentensis Wickerham et Kurtzman. The latter species presently are known as varieties of Williopsis saturnus (Klocker) Zender (Kurtzman 1998). P. lindneri and H. minuta are conspecific and presently known as P. minuta (Wickerham) Kurtzman. The close relationship of Hansenula and Pichia species prompted Kurtzman (1984) to propose merging of both genera. As a
1 History, habitat, variability, nomenclature and phylogenetic position of Hansenula polymorpha
consequence, Hansenula species with hat-shaped ascospores were transferred to Pichia Hansen emend. Kurtzman as the genus Pichia had priority. Both leading yeast monographs (Kurtzman and Fell 1998, Barnett et al. 2000) and some culture collections followed this proposal, but the merging of both genera is still criticized by some yeast taxonomists. However, it was corroborated by Kurtzman and Robnett (1998) who provided a phylogenetic tree in which nitrate-positive and nitrate-negative species of Pichia Hansen emend. Kurtzman clustered. This demonstrates that nitrate assimilation is an unreliable predictor of kinship of these yeasts. Moreover, Williopsis spp. clustered with Pichia spp. As a consequence, H. polymorpha is treated in the yeast monographs and is sold by some culture collections as Pichia angusta (Teunisson et al.) Kurtzman. The epithet "polymorpha" had priority but the binominal P. polymorpha was previously used by Klocker (1912) for a species presently known as Debaryomyces polymorphus (Klocker) Price et Phaff and hence could not be used again. H. polymorpha is also known as Ogataea polymorpha (Morais et Maia) Yamada et al. The genus Ogataea was proposed by Yamada et al. (1994) to accommodate some hat-spored, ascosporogenous, methanol-assimilating yeast species. It was based on partial sequences of i8S and 268 ribosomal RNAs. The name Ogataea polymorpha did not receive wide acceptance. Naumov et al. (1997) published a paper reporting a detailed study of the "Hansenula polymorpha complex". They concluded that the strains studied could be classified in three sibling species, one consisting of the type strain of H. polymorpha (CBS 3742) and the Wickerham (1951) strain of H. angusta (CBS 1976) from canned orange juice, and one species consisting of the type strain of P. angusta (CBS 7073) and other strains isolated from fruit flies. The third sibling species is probably a new taxon associated with Opuntia cacti. The latter is characterized by weak and slow growth on methanol (Phaff 1985, Starmer et al. 1986). Distinction of the three sibling species was made on the basis of genetic hybridization, molecular karyotyping of the chromosomes and UP-PCR (Universally Primed Polymerase Chain Reaction) of the nuclear ribosomal DNA ITSi region. Haploid strains of the three sibling species are able to mate, but their interspecific hybrids are sterile (Naumov et al. 1997). More variety of the H. polymorpha complex could have been demonstrated if the above-mentioned strains unable to grow at 42 °C (CBS 7031 and CBS 8099) or on methanol (CBS 5032) had been included in this study. In contrast to the statement of Wickerham (1970), H. angusta and H. polymorpha are not conspecific and synonymous, though closely related. As a consequence, there are good arguments to maintain the name Hansenula polymorpha at least for the type strain CBS 3742 and for strains and mutants derived thereof. The name Pichia angusta is unlikely to get as much popularity in the laboratories as the nickname "Hans Pol" has since many years.
1.5
Phylogenetic position
The genus Pichia belongs to the family Saccharomycetaceae G. Winter, order Hemiascomycetes, phylum Ascomycota. A phylogenetic tree of all ascomycetous
1.5 Phylogenetic position
yeasts was presented by Kurtzman and Robnett (1998). It was based on partial sequences of the nuclear large subunit (268) of ribosomal DNA. H. polymorpha was represented by the type strain of P. angusta, CBS 7073, isolated from Drosophila sp. As this strain is very closely related to the sibling species H. polymorpha (Naumov et al. 1997), the conclusions regarding P. angusta will also be valid for H. polymorpha. Methanol-assimilating yeasts, with the exception of Pichia pastoris (Guillermond) Phaff that is in another clade of the tree, appear closely related. In the phylogenetic tree P. angusta takes a position in the center of a cluster of 37 species (Kurtzman and Robnett 1998), delimited by P. capsulata (syn. Candida molischiana) and C. boidinii. All these species do assimilate methanol, except for Williopsis salicorniae and a subcluster of 7 species comprising C. llanquihuensis, P. angorophorae and 5 species of Ambrosiozyma (Kurtzman and Robnett 1998).
1 History, habitat, variability, nomenclature and phylogenetic position of Hansenula polymorpha
References
Barnett JA, Payne RW, Yarrow D (2000) Yeasts: Characteristics and Identification, 3rd Edn. Cambridge University Press, Cambridge, UK Fujii T, Tonomura K (1972) Oxidation of methanol, formaldehyde and formate by a Candida species. Agric Biol Chem 36: 2297-2306 Hazeu W, de Bruyn JC, Bos P (1972) Methanol assimilation by yeasts. Arch Mikrobiol 87: 185-188 Klocker A (1912) Untersuchungen iiber einige neue PicWa-Arten. Zbl Bakteriol Parasitenknd Abt. II 35: 369-375 Kurtzman CP (1984) Synonymy of the yeast genera Hansenula and Pichia demonstrated through comparisons of deoxyribonucleic acid relatedness. Antonie van Leeuwenhoek 50: 209-217 Kurtzman CP (1998) Williopsis Zender, in: The Yeasts, a Taxonomic Study, 4th Edition (Kurtzman CP, Fell JW, Eds). Elsevier, Amsterdam, The Netherlands, pp. 413-419 Kurtzman CP, Fell JW (Eds) (1998) The Yeasts, a Taxonomic Study, 4th Edn. Elsevier, Amsterdam, The Netherlands Kurtzman CP, Robnett CJ (1998) Identification and phylogeny of ascomycetous yeasts from analysis of nuclear large subunit (26S) ribosomal DNA partial sequences. Antonie van Leeuwenhoek 73: 331-371 Levine DW, Cooney CL (1973) Isolation and characterization of a thermotolerant methanol-utilizing yeast. Appl Microbiol 26: 982-990 Middelhoven WJ, Berends J, van Aert AJM, Bruinsma D (1976) The substrate constant
for dissolved molecular oxygen of methanol-assimilating yeasts. J Gen Microbiol 93: 185-188 Morais JOF, Maia MHD (1959) Estudos de microrganismos encontrados em leitos de despejos de caldas de destilarias de Pernambuco. II. Uma nova especie de Hansenula, H. polymorpha. Anais da Escola Superior de Quimica Universidade do Recife i: 15-20 Naumov GI, Naumova ES, Kondratieva VI, Bulat SA, Mironenko NV, MendoncaHagler LC, Hagler AN (1997) Genetic and molecular delineation of three sibling species in the Hansenula polymorpha complex. Syst Appl Microbiol 20: 50-56 Ogata K, Nishikawa H, Ohsugi M (1969) Yeast capable of utilizing methanol. Agric Biol Chem 33: 1519-1520 Phaff HJ (1985) Biology of yeasts other than Saccharomyces, in: Biology of Industrial Microorganisms (Demain AL, Solomon A, Eds) The Benjamin Cummings Inc., Menlo Park, CA, USA, pp. 537-562 Roggenkamp R, Sahm H, Wagner F (1974) Microbial assimilation of methanol. Induction and function of catalase in Candida boidinii. FEES Lett 41: 283-286 Sahm H, Wagner F (1973) Mikrobielle Verwertung von Methanol. Eigenschaften der Formaldehyddehydrogenase und der Formiatdehydrogenase aus Candida boidinii. Arch Mikrobiol 90: 263-268 Starmer WT, Ganter PF, Phaff HJ (1986) Quantum and continuous evolution of DNA base composition in the yeast genus Pichia. Evolution 40: 1263-1274 Tani Y, Miya T, Nishikawa H, Ogata K (1972) The microbial metabolism of methanol. I.
References
Formation and crystallization of methanoloxidizing enzyme in a methanol-utilizing yeast, Kloeckera sp. No 2201. Agric Biol Chem 36: 68-75 Teunisson DJ, Hall HH, Wickerham LJ (1960) Hansenula angusta, an excellent species for demonstration of the coexistense of haploid and diploid cells in a homomallic yeast. Mycologia 52: 184-188 Van Dijken JP, Veenhuis M, Kreger-van Rij NJW, Harder W (1975) Microbodies in methanol-assimilating yeasts. Arch Microbiol 102: 41-44 Wickerham LJ (1951) Taxonomy of yeasts. Technical Bulletin 1029, US Dept Agric, Washington DC, USA, pp. 1-56
Wickerham LJ (1970) Hansenula H et P Sydow, in: The Yeasts, a Taxonomic Study 2nd Edn (Lodder J, Ed). North Holland, Amsterdam, The Netherlands, pp. 226-315 Yamada Y, Maeda K, Mikata K (1994) The phylogenetic relationships of the hatshaped ascospore-forming, nitrateassimilating Pichia species, formerly classified in the genus Hansenula Sydow et Sydow, based on the partial sequences of i8S and 26S ribosomal RNAs (Saccharomycetaceae): the proposals of three new genera, Ogataea, Kuruaishia and Nakazawaea, Biosci Biotechnol Biochem 58: 1245-1257
2 Basic genetics of Hansenula polymorphic* Kantcho Lahtchev
2.1 Introduction
The methylotrophic yeast Hansenula polymorpha is a favorable model organism for investigation of peroxisome function and biogenesis (see Chapter 6). Also, it has been used to study the genetic control of various aspects of intermediate cellular metabolism as for instance: methanol metabolism (see Chapter 5), nitrate assimilation (see Chapter 3) and resistance to heavy metals, oxidative stress (Mannazzu et al. 2000) and thermostability (Reinders et al. 1999). Furthermore, H. polymorpha has gained increasing interest for use as host for the production of foreign proteins (Gellissen 2000). Despite this, the nature and genetic abilities of the strains used are largely unclear and the genetic control of basic cellular processes such as cell division control, mating and sporulation remain unsolved. 2.2 Strains
So far, most genetic work has been performed on three basic strains designated as H. polymorpha, DL-i, CB84732 and NCYC495, respectively. These strains have independent origins, different features and unclear relationships. The strain DL-i (synonymous to ATCC26oi2, NRRL-Y-756o) was isolated from soil (Levine and Cooney 1973). No data are available about its ability to mate and sporulate. The strain NCYC495 (syn. CBSi976, ATCCi4754, NRRL-Y-I789, VKM-Y-I397) was isolated from spoiled orange juice and described initially as Hansenula angusta (Wickerham 1951). All basic classical genetic techniques like mating, sporulation and random spore analysis were developed using this strain (Gleeson and Sudbery 1988). The NCYC495 strain is homothallic haploid and has good mating and sporulation abilities. Its disadvantage is the poor growth on methanol-containing media. The strain CB84732 (syn. ATCC34438, NRRL-Y-5445, CCY38-22-2) was isolated from soil in Brazil (de
Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Veriag GmbH, Weinheim ISBN: 3-527-30341-3
2.3 Auxotrophic mutants
Morals and Maia 1959). These cells grow well on methanol, and have been demonstrated to perform well in continuous culture (van Dijken et al. 1976). Serious problems were concerned with its semisterility and inability to sporulate (Gleeson et al. 1984). However, isogenic CBS4732 strains with good mating and sporulation abilities have subsequently been developed and are available now (Lahtchev, unpublished data). Two additional genetic lines of H. polymorpha strains with a hybrid origin have been reported. The stock described by Bodunova et al. (1986) has as parental two auxotrophic non-mating mutants, originating from NCYC495 and the ML-3 strain which is probably derived from DL-i. The Veenhuis group (Groningen, The Netherlands) uses strains designated as NCYC495 but in fact they originate from backcrosssing of peroxisome deficient mutants, isolated from the CB84732 wild-type (WT) strain (Gregg et al. 1990) with NCYC495 auxotrophs (Titorenko et al. 1993).
2.3 Auxotrophic mutants
The genetics of H. polymorpha started with the isolation of auxotrophic mutants. These were obtained from each of the strains described above by using either Nmethyl-N'-nitronitroso-guanidine or ethylmethanesulfonate followed by an enrichment step with nystatin. UV irradiation was found to be a powerful mutagen as well, and the mutants isolated had different spectra from those obtained by chemical mutagenesis. The positive selection system based on resistance to 5fluoro-orotic acid has been employed for the isolation of uracil requiring mutants. Two genes designated as odci (= uraj, coding for orotidine- 5'- phosphate decarboxylase) and oppi (=ura$, coding for orotidine- 5'- phosphate pyrophosphorylase) have been identified by this approach (Roggenkamp et al. 1986). Most auxotrophic mutants have been isolated by replica-plating of the colonies, developed after mutagenic treatment, onto corresponding omission media (Sanches and Demain 1977, Gleeson and Sudbery 1988). Several characteristic features of the mutational process in H. polymorpha were evident from these experiments: (1) (2) (3)
Some mutants (e.g. adenine and methionine-requiring) appeared with very high frequencies compared to the others. The distribution of alleles among the mutated genes was non-random as well. Some types of auxotrophic requirements have never been observed, e.g. in the case of tryptophan and tyrosine.
The latter one is probably explained by the fact that mutations in some genes involved in biosynthesis of aromatic amino acids (e.g., HAROj, encoding chorismate mutase; Krappmann et al. 2000) do not allow growth on rich (YPD) media. This specificy of the mutational process indicates that for the isolation of larger mutant spectra it is better to utilize several strains with independent origins as well as different types of mutagens.
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The URAj gene (Merckelbach et al. 1993), the LEU2 gene, encoding (3-isopropyl malate dehydrogenase (Agaphonov et al. 1994) and the PVRj gene, encoding phosphoribosyl-aminoimidazole-succino carboxamide synthetase (Haan et al., unpublished data) have been cloned, sequenced and used as transformation markers. Two other types of auxotrophic mutants have been described. The H. polymorpha rifi mutant is strictly dependent on riboflavin for growth on mineral media and was used to demonstrate that riboflavin limitation had drastic effect on alcohol oxidase (AO) assembly, import into peroxisomes as well as peroxisome proliferation (Evers et al., 1994). The second type involves mutations in the FADi gene, which encodes A-fatty acid desaturase (Anamnart et al. 1998). These mutations are useful for the genetic dissection of the synthesis of poly-unsaturated fatty acids. The genetic control of amino acid biosynthesis in H. polymorpha is poorly investigated. Until now, only one paper dealing with the isolation of mutants of genes involved in leucine biosynthesis is available. Mutants in three genes: LEUicoding for isopropyl malate dehydroganase; LEV2 (see above) and Lfl/j-encoding oc-isopropyl malate synthase have been obtained and studied by complementation and recombination analysis as well as characterized enzymatically (Makhina et al. 1986; see also Chapter 4). It is relevant to note that the number of auxotrophic mutations involved in most employed strains is surprisingly low and the introduction of new markers is desired. Multiple marked strains are easily constructed, but reports on such strains are limited and their use for mapping studies are accidental until now.
2.4 Morphological mutants
When grown on solid media H. polymorpha cells form colonies of characteristic hemispherical shape and a smooth circular outline (not shown). These colonies consist of oval, sometimes spherical yeast cells. Recently, several mutants with irregular colony outline and rough colony surface were obtained. Most of these cells were present in chains of undivided cells (Figure 2.1 A). This mutant phenotype (rough colonies, chains of cells) was designated as Rgh (Lahtchev and Mihailova, 1994). All Rgh mutants contain recessive mutations affecting at least 4 genes: RGHi-RGH4. The gene RGHj appeared to be linked to the LEV2 locus (35.7 cM) (cM: centimorgans; map distance calculated on the basis of recombination frequencies of genetic markers studied). So far, no data are available regarding the ability of H. polymorpha to form pseudomycelia. However, recently a mutant with constitutive formation of pseudohyphal cells was isolated and designated rpmi (Lahtchev, unpublished results) (Figure 2.iB). During prolonged cultivation on YPD plates haploid rpmi cells were found to penetrate into agar by invasive growth, (not shown). This suggests that H. polymorpha cells can indeed undergo filamentous and invasive growth differentiation, which provides new possibilities for investigations of these processes.
2.5 Heavy metal resistance/sensitivity
Fig. 2.1 Morphology of mutant H. polymorpha cells. Phase-contrast images to demonstrate the morphology of glucose-grown rg/i? cells (A, chains of cells) and rpmi cells (B, pseudohyphal cells). The morphology of wild-type cells is shown as control (A, insert). Bar = 10 |im.
2.5 Heavy metal resistance/sensitivity
Recent investigations have shown that H. polymorpha can be successfully used to study resistance to heavy metal ions. H. polymorpha is able to grow in the presence of very high concentrations of different heavy metals that are toxic for other organisms. During growth on vanadate-containing media the cells undergo a significant increase in the levels of vacuolar polyphosphates (Mannazzu et al. 1997). Vacuoles are involved in the activation of an autophagic mechanism which may be required to compensate for nutrient depletion and/or eliminate the aberrant cellular structures induced by this metal ion (Mannazzu et al. 1998). Moreover, the comparison of the cellular response to vanadate and copper has revealed the existence of partially overlapping detoxification pathways for these two metal ions in H. polymorpha (Mannazzu et al. 2000). H. polymorpha cells are very resistant to cadmium ions (Cd 2+ ), compared to S. cerevisiae. This resistance strongly depends on the nature of the carbon source used. Cells are most resistant to Cd2+ when grown in glucose-containing media and very sensitive during growth on media supplemented with methanol as sole carbon and energy source. Mutants with increased Cd2+ sensitivity have been isolated and allocated to 3 complementation groups: cdsi, cds2 and cdsj (Lahtchev, unpublished data). All mutants were unable to grow on methanol-containing media supplemented with 10 mM Cd2+. Moreover, the cds mutants showed enhanced sensitivity to Cd2+ on glucose, compared to WT controls.
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2.6 Thermostability
H. polymorpha cells are well adapted to grow at very high temperatures up to 48 °C (in part depending on the carbon source used for growth). Curiously, this valuable property is still poorly investigated, probably due to the complex multigenic system controlling this phenotype. A region, designated T, responsible for the growth of haploid strains on solid media at 48 °C has been discovered (Bodunova et al. 1990). This region has been found difficult for genetic analysis because of the many deviations on segregation compared to normal monogenic segregation. The MHRi mutant has been isolated by its ability to grow on media with enhanced amounts of methanol (Lahtchev et al. 2000). The growth of WT cells was significantly slower than that of MHRi cells during cultivation on YPD and synthetic glucose media at 46 °C and 48 °C. The mutants cells possessed enhanced total lipid contents and unsaturation levels of phospholipids and triacyl glycerols than the WT strain. Recently, the role of a reserve carbohydrate, trehalose, on the acquisition of thermotolerance and growth at high temperatures has been investigated (Reinders et al. 1999). It has been observed that in H. polymorpha cells trehalose synthesis is part of carbon source starvation and heat shock response. Deletion of the TPSi gene, encoding trehalose6-phosphate synthase did not cause any obvious growth defects on glucosecontaining media, even at elevated temperature. However, Atpsi cells were found to be more sensitive to conditional heat shock, suggesting that trehalose synthesis is an important factor for the acquisition of thermotolerance.
2.7 Methanol non-utilization mutants
Several collections of H. polymorpha mutants affected in their ability to grow on methanol-containing media (Mut~ from methanol non-utilization) have been isolated. Such mutants usually are isolated by negative selection: colonies developed after mutagenic treatment on YPD plates are transferred by replica-plating onto methanol-containing media and those, unable to grow, are selected as Mut~ mutants. Mut~ mutants appeared with very high frequencies after mutagenic treatment suggesting that a high number of genes is involved in methanol metabolism. Most mutants had a recessive phenotype, but dominant alleles appeared also, although at low frequency. Many of the Mut~ mutants were specifically affected in methanol utilization dissimilation/assimilation, others in peroxisome biogenesis. Nevertheless, among the Mut~ mutants, strains unable to grow on media with other carbon sources, like ethanol, glycerol, xylose, etc. were obtained as well. Typically, Mut~ mutants are conditional mutants: they are unable to grow on methanol, but can grow normally on glucose thus allowing genetic analysis. In earlier reports Mut~ mutants were isolated for physiological reasons (de Koning et al. 1987, Bystrykh et al. 1988). Two Mut~ collections were investigated
2.7 Methanol non-utilization mutants
genetically. Gleeson and Sudbery (1988) analyzed 5 Mut~ complementation groups, but only one lacked the activity of dihydroxyacetone synthase (DHAS). Recently, 65 Mut~ strains have been allocated into 12 complementation groups (Vallini et al. 2000). For eight of them a significant decrease in AO activity was detected. Below the different types of mutants will be discussed. 2.7.1
Mutants affected in genes encoding peroxisomal or cytosolic enzymes of methanol metabolism
The AOXi (MOX) gene, coding for the peroxisomal matrix enzyme AO, is one of the best investigated genes of H. polymorpha (Ledeboer et al. 1985). AO is a homooctameric flavoenzyme which catalyzes the first step in methanol metabolism. AOXi is one of the most highly expressed eukaryotic genes and its expression is tightly regulated at the level of transcription. Monomeric AO is synthesized in the cytosol, and assembly into the active octameric enzyme is thought to take place inside the peroxisome. 210 mutants deficient in AO activity (Aox~) were isolated from a Mut~collection by a colony plate activity assay (Titorenko et al. 1995). Complementation tests revealed that over 50% of these were allelic to the AOXi gene. The AOXi gene appeared to be linked with the ADEz locus (39.5 cM). Several mutants, impaired in AO assembly (Ass phenotype) were obtained from a collection of Aox~ strains (van Dijk et al. unpublished data). The protein product of AS Si, pyruvate carboxylase, appeared to possess a dual function. As an enzyme it showed the expected anapleurotic function to fuel the TCA cycle, while the protein (and not the enzyme activity) was essential for AO assembly. During methylotrophic growth, pyruvate carboxylase may facilitate FAD binding to AO monomers prior to import, indicative for an AO-specific chaperone-like function (van Dijk et al. unpublished data). The structural gene of catalase (CAT) has been cloned and sequenced (Didion and Roggenkamp 1992). Various cat -mutants have been identified by a plate activity assay. Complementation tests demonstrated that all of them were affected in the CAT structural gene. Recombination analysis revealed that the CAT gene is linked to the URAy locus (28.8 cM). Mutants lacking dihydroxy acetone synthase (DHAS) activity have been reported by several groups (de Koning et al 1987, Bystrykh et al. 1988, Gleeson and Sudbery 1988). In these mutants, the activities of the other enzymes of the methanol utilization pathway were akin to those in WT cells. The properties of these mutants provide genetic evidence of the participation of DHAS in methanol metabolism. The DHAS gene has been cloned and sequenced (Janowizc et al. 1985). The genes coding for the cytosolic enzymes of Q metabolism have not been studied in such detail. O'Connor and Quayle (1979) reported the isolation of mutants impaired in dihydroxyaceton kinase (DHAK) activity. This type of mutants was used to demonstrate the participation of the xylulose-5-phosphate (Xu5?) pathway for the assimilation of formaldehyde, formed from methanol. Detailed molecular and physiological analyses of strains carrying DHAK deletions revealed
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that prevention of phosphorylation of dihydroxyacetone in a DHAK deletion strain inhibits the function of the Xu5P pathway, thus explaining the Mut~ phenotype of das mutants (van der Klei et al. 1998). Mutants lacking the activity of formaldehyde reductase were selected on medium with glucose and allyl alcohol (Sibirny et al. 1988). Their analyses revealed that formaldehyde reductase is essential for the regulation of formaldehyde levels in H. polymorpha cells. 2.7.2
Mutants with defects in peroxisome biogenesis
In earlier reports dealing with Mut~ mutants it was observed that many of these mutants possess normal activities of the key enzymes of Q metabolism (Gleeson and Sudbery 1988). At that time the reason for this was unclear. Currently, we know that those mutants may be defective in peroxisome biogenesis and that their inability to grow on methanol-containing media is due to mislocalization of peroxisomal matrix enzymes to the cytosol. The first peroxisome-deficient (Pex~) mutants of H. polymorpha were identified within a collection of 260 Mut~ mutants (Gregg et al. 1990). After incubation of the mutants in methanol-containing media, 85 strains were identified by electron microscopy as having one of the following peroxisomal defects: • complete absence of peroxisomes (Pex~ phenotype), • presence of only a few small peroxisomes along with the mislocalization on the bulk of the peroxisomal matrix proteins to the cytosol (Pim~ phenotype), and • aberrations in the peroxisomal sub-structure, i.e. presence of electron dense inclusions in the crystalline peroxisomal matrix (Pss~ phenotype). In addition, various conditional mutants (temperature-or cold-sensitive) have been isolated. PEX mutations were found to be allele-, but not gene-specific: different mutations in one and the same gene can cause different morphological phenotypes. The mutants were genetically analyzed and shown to be determined by single recessive chromosomal mutations. This implies that the absence (or incorrect synthesis) of a single gene product may cause the absence of an entire peroxisome (van der Klei et al. 1995). Extensive complementation analysis of strains carrying different Pex~ mutations revealed many cases of "unlinked non-complementation": diploids, diheterozygous of two different PEX genes, displayed a mutant phenotype which was predominantly observed at lowered temperatures (coldsensitive non-complementation). These results strongly suggest the existence of functional and physical links between PEX proteins (Titorenko et al. 1993; see also Chapter 6). 2.7.3
Regulatory mutants
Many substrates have a strong influence on the synthesis of Q enzymes and/or peroxisome proliferation/degradation. Methanol and its derivatives were shown to
2.8 Genetic mapping, linkage groups and chromosome number
be powerful inducers of peroxisome proliferation and of most, if not all methanolspecific enzymes. Glucose, ethanol and some other carbon compounds strongly repress the synthesis of Q-specific enzymes and peroxisome induction. Several mechanisms like repression, derepression, induction and catabolite inactivation have been established for the regulation of these processes. Earlier studies concerning regulation of the synthesis of enzymes involved in Q metabolism were undertaken using WT cells. Later on, the genetic approach based on the isolation and analysis of mutants with defects in regulation has been employed. Mutants with defects mainly in glucose repression and/or inactivation have been isolated using various selection procedures. The first publications included data on the regulation of peroxisomal and some cytosolic enzymes involved in methanol metabolism. Several papers described the isolation and properties of mutants with defects in the regulation of peroxisome proliferation. Roggenkamp (1988) reported on a mutant displaying constitutive peroxisome synthesis during cultivation of cells on glucose. However, detailed genetic analysis of this mutant is not available and the nature of the corresponding protein is unknown. A similar phenotype has been observed for the mutant GLR2 (Parpinello et al. 1998) and GCRi (Stasyk et al. 2000). The GCRi gene encodes a protein that shares 44% identity and 62% similarity with S. cerevisiae Snfjp, a putative highaffinity glucose sensor. In these mutants, peroxisome degradation seems to be normal, so they are probably affected in the induction of peroxisome synthesis. Recently, mutants affected in a glucose-specific glucokinase and a hexokinase that phosphorylates both glucose and fructose have been isolated (Karp et al. 2000). Analysis of these mutants suggests that the catalytic activity of hexose kinase is critical in sugar repression of maltase- and methanol-specific enzymes. The events of repression, derepression and induction of genes, encoding methanol-specific and peroxisomal enzymes, are highly coordinated and tightly regulated. In H. polymorpha regulation at the level of transcription seems to be the prominent controlling mechanism. Upstream cis-acting regulatory elements responsible for glucose repression of DAS, CAT, FMD have been identified. However, exclusively in the case of the AOXi promoter, a functional analysis for the identification of regions required for glucose repression has been performed (Pereira 1994). In the initial studies the attention has been focussed on catabolic inactivation of major peroxisomal enzymes. Now it is evident that their inactivation is due to a process of selective peroxisome degradation which is described in Chapter 6.
2.8 Genetic mapping, linkage groups and chromosome number
Tetrad analysis is feasible in H. polymorpha, but the small size of the spores makes these analysis slow and tedious, so random spore analysis was generally preferred. In sporulating diploid cultures normal Mendelian segregation was observed for most of the genetic markers employed. The frequencies of irregular tetrads varied
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for the mutations studied, but only in few pairs exceeded 10%. Similar deviations from normal segregation rates have been observed for some markers in random spore analysis. Possibly, some cases were due to aneuploidy events. The average frequency of tetratype formation was estimated at 0.56 and this is lower than the normal tetratype frequency of 0.67 reported for S. cerevisiae. For some pairs of markers a genetic linkage has been found, but the number of linked genes is not high. Unfortunately, so far no centromere-linked markers have been discovered. Several linkage groups have been reported based on the data from random spore analysis (Gleeson and Sudbery 1988, Valini et al. 2000). From mapping data it is evident that many of the PEX genes were clustered into three linkage groups (Titorenko et al. 1993). Pulsed field electrophoresis of chromosomal DNA of H. polymorpha showed 3 to 7 bands, depending of the strain and experimental conditions used. Many examples of chromosome polymorphism have been observed (Mari et al. 1993). Currently, it is generally accepted that H. polymorpha has at least seven chromosomes, some of which are double (Naumov et al. 1992). Recent data for the DNA hybridization of cloned genes with the pulse gel separated chromosomes are included in Chapter 7.
2.9 Mating and sporulation
The factors involved in mating and sporulation of H. polymorpha cells are still largely unresolved. Haploid cells do neither mate on YPD media nor on plain agar plates. Powerful inducers of mating appeared to be maltose, glycerol and sorbitol. Complementation tests of auxotrophic mutants revealed that haploid strains are able to mate in any combination independent from their mating type. Haploid cells fall into four phenotypic groups according to these phenotypes (Lahtchev, unpublished data). The strains belonging to the first two groups were capable of cross-hybridization: very rapid and intensive growth of resulted diploids after one day of cultivation on selective media. The strains belonging to the third group were able to copulate with members of groups i and 2, they were designated plus (+) strains. The strains belonging to the fourth group were able to mate exclusively with the plus strains and were designated as minus (-) strains. The results of genetic analysis suggested that the mating type of H. polymorpha is determined by two unlinked loci named H and P. Each locus has two alleles: dominant (+) and recessive (-). Allele H+ has an epistatic action on the P" allele and, in contrast the P+ allele is epistatic to H". The plus phenotype is a combination of both dominant alleles: H+P+. Both recessive alleles (H~P~) are present in cells of the minus phenotype. The mating type of H. polymorpha is determined by a simple tetrapolar mode which is intermediate between bipolar mating, characteristic for ascomycetous yeasts and tetrapolar mating of basidiomycetous yeasts. Characteristic for H. polymorpha is the fact that in particular haploid cells are able to sporulate. Haploid sporulation can be detected after 8 d of cultivation on media with 3% maltose at lowered temperatures. Under these conditions meiosis is
2.10 Concluding remarks
induced in diploid cells and asci with hat-shaped ascospores can be visualized within 3-4 d. Sporulation is accompanied by the appearance of a light pink color of the diploid colonies. Genetic control of Sporulation has not been studied so far. Probably the mating type loci H and P participate in this process.
2.10
Concluding remarks
It is evident that H. polymorpha is a favorable model system with high potential to provide insights into many research topics. During the last decade remarkable progress has been made, especially towards the investigation of peroxisome biogenesis and the expression of foreign genes. Unfortunately, after the classical paper of Gleeson and Sudbery (1988), no other publications have appeared with new information concerning the basic genetics of H. polymorpha. Obviously such lack of genetic information can hamper the further exploration(s) of this yeast.
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References
Agaphonov MO, Pozznyakovski AI, Bogdanova AI, Ter-Avanesyan MD (1994) Isolation and characterization of the LEU2 gene of Hansenula polymorpha. Yeast 10: 509-513 Anamnart S, Tolstorukov I, Kaneko Y, Harashima S (1998) Fatty acid desuturation in methylotrophic yeast Hansenula polymorpha strain €681976 and unsuaturated fatty acid auxotrophic mutants. J Ferment Bioeng 85: 478-448 Bodunova EN, Donich V, Nesterova GF, Soom YO (1986) Genetic stocks of the yeast Hansenula polymorpha. I. Selection and properties of genetic stocks. Genetika 22: 741-747 Bodunova EN, Nesterova GF, Kapul'tsevich YuG (1990) Genetic stocks of the yeast Hansenula polymorpha. V. Genome structure. Genetika 26: 424-443 Bystrykh LV, Aminova LP, Trotsenko YA (1988) Methanol metabolism in mutants of the methylotrophic yeast Hansenula polymorpha. FEMS Microbiol Lett 51: 89-94 Gregg JM, van der Klei I-J, Suiter GJ, Veenhuis M, Harder W (1990) Peroxisome-deficient mutants of Hansenula poymorpha. Yeast 6: 87-97 Didion T, Roggenkamp R (1992) Targeting signal of the peroxisomal catalase in the methylotrophic yeast Hansenula polymorpha. FEES Lett 303: 113-116 Evers ME, Titorenko VI, van der Klei I-J, Harder W, Veenhuis M (1994) Assembly of alcohol oxidase in peroxisomes of the yeast Hansenula poymorpha requires the co-factor flavin adenine dinucleotide. Mol Biol Cell 5: 829-837
Gellissen G (2000) Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol 54; 741-750 Gleeson MA, Sudbery PE (1988) Genetic analysis in the methylotrophic yeasts Hansenula polymorpha. Yeast 4: 293-303 Gleeson MA, Waites MJ, Sudbery PE (1984) Development of techniques for genetic analysis in the methylotrophic yeast Hansenula polymorpha, in: Microbial growth on Ci compounds (Crawford R.L., Hanson R.S., Eds). Am Soc Microbiol, pp. 228-235 Janowicz Z, Ecjart M, Drewke, Roggenkamp R, Hollenberg CP, Maat J, Ledeboer AM, Visser C, Verrips CT (1985) Cloning and characterization of the DAS gene encoding the major methanol assimilatory enzyme from the methylotrophic yeast Hansenula poymorpha. Nucleic Acids Res 13: 3043-3062 Karp H, Kramarenko T, Laht S, Alamae T (2000) Sugar repression of maltase and methanol-specific enzymes in Hansenula polymorpha. The first Hansenula polymorpha worldwide network (HPWN), University of Duesseldorf, Germany. Koning W de, Gleeson M A G , Harder W, Dijkhuizen L (1987) Regulation of methanol metabolism in the yeast Hansenula polymorpha. Arch Microbiol H7: 375-382 Krappmann S, Pries R, Gellissen G, Hiller M, Braus GH (2000) HARO7 encodes chorismate mutase of the methylotrophic yeast Hansenula polymorpha and is derepressed upon methanol utilization. J Bacteriol 182: 4188-4197 Lahtchev K, Mihailova L (1994) A mutant of
References methylotrophic yeast Hansenula polymorpha with altered morphology of colonies and cells. C R Acad Bulg Sci 48: 53-56 Lahtchev K, Ivanova A, Stefanov K. (2000) lipid content and fatty acid composition of a yeast mutant with increased thermal and alcohol tolerance. C R Acad Bulg Sci 53: 75-78 Ledeboer A M, Edens L, Maat J, Visser C, Boss JW, Verrips CT (1985) Molecular cloning and characterization of a gene coding for methanol oxidase in Hansenula polymorpha. Nucleic Acids Res 13: 3063-3082 Levine DW, Cooney CL (1973) Isolation and characterization of a thermotolerant methanol-utilizing yeasts. Appl Microbiol 26: 982-989 Makhina EN, Nesterova GF, Grishin AV (1986) Genetic control of leucine biosynthesis in the yeast Hansenula polymorpha. Genetika 22: 755-760 Mannazzu I, Guerra E, Strabioli R, Maestrale GB, Masia A, Zoroddu MA, Fatichenri F (1997) Vanadium affects vacuolation and phosphate metabolism in Hansenula polymorpha. FEMS Microbiol Lett 147: 23-28 Mannazzu I, Guerra E, Strabioli R, Pediconi D, Fatichenti F (1998) The vanadate tolerant yeast undergoes cellular reorganization during growth in, and recovery from, the presence of vanadate. Microbiology 144: 2589-2597 Mannazzu I, Guerra E, Ferretty R, Pediconi D, Fatichenti F (2000) Vanadate and copper induce overlapping oxidative stress responses in the vanadate-tolerant yeast Hansenula polymorpha. Biochem Biophys Acta 1475: 151-156 Marri L, Rossolini GM, Satta G (1993) Chromosome polymorphism among strains of Hansenula polymorpha (syn. Pichia angusta). Appl Environ Microbiol 59: 939-941 Merckelbach A, Godecke S, Janowicz ZA, Hollenberg CP (1993) Cloning and sequencing of the URA3 locus of the methylotrophic yeast Hansenula polymorpha and its use for the generation of a deletion by gene replacement. Appl Microbiol Biotechnol 40: 361-364 Morais JOF de, Maia MHD (1959) Estudos de microorganismos encontrados em leitos de despejos de caldas de destilarias de
Pernambuco. II. Una nova especie de Hansenula: H. polymorpha. Anais de Escola Superior de Quimica da Universidade do Recife i: 15-20. Naumov G, Naumova ES, Mendonca-Hagler LC, Hagler AN (1992) Taxogenetics of Pichia angusta and similar methylotrophic yeasts. Ciencia e Cultura 44: 397-400 O'Connor M, Quayle JR (1979) Mutants of Hansenula polymorpha and Candida boidinii impaired in their ability to grow on methanol. J Gen Microbiol 113: 203-208 Parpinello G, Berardi E, Strabbioti (1998) A regulatory mutant of Hansenula polymorpha exhibing methanol utilization metabolism and peroxisome proliferation in glucose. J Bacteriol 180: 2958-2967 Pereira GG (1994) Analysis of the transcriptional regulation of the MOX gene encoding peroxisomal methanol oxidase from Hansenula polymorpha. Thesis, University of Duesseldorf, Germany Reinders A, Romano I, Wiemken A, de Virgilio C (1999) The thermophilic yeast Hansenula polymorpha does not require trehalose for normal acquisition of thermotolerance. J Bacteriol 181: 4665-4668 Roggenkamp R (1988) Constitutive appearance of peroxisomes in a regulatory mutant of the methylotrophic yeast Hansenula polymorpha. Mol Gen Genet 213: 535-540 Roggenkamp R, Hansen H, Eckart N, Janowitcz Z, Hollenberg CP (1986) Transformation of methylotrophic yeast Hansenula polymorpha by autonomous replication and integration vectors. Mol Gen Genet 10: 302-308 Sanchez S, Demain AL (1977) Enrichment of auxotrophic mutants in Hansenula polymorpha. Eur J Appl Microbiol 4: 45-49 Sibirny AA, Titorenko VI, Gonchar MV, Ubiyvovk VM, Ksheminskaya GP, Vitvitskaya OP (1988) Genetic control of methanol utilization in yeasts. J Basic Microbiol 5: 293-319 Stasyk O, Moroz O, Kulachkovsky A, Stasyk O, Gregg J, Sibirny A (2000) Hansenula polymorpha mutant which efficiently express alcohol oxidase promoter-directed own and foreign proteins in glucose medium. The first Hansenula polymorpha
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2 Basic genetics of Hansenula polymorpha worldwide network (HPWN). University of Duesseldorf, Germany Titorenko VI, Waterham HR, Gregg JM, Harder W, Veenhuis M (1993) A complex set of interacting genes controls peroxisome biogenesis in Hansenula polymorpha. Proc Natl Acad Sci USA 90: 7470-7474 Titorenko VI, Keizer I, Harder W, Veenhuis M (1995) Isolation and characterization of mutants impaired in the selective degradation of peroxisomes in the yeast Hansenula polymorpha. } Bacteriol 177: 357-363 Vallini V, Berardi E, Strabbioti R (2000) Mutations affecting the expression of the MOX gene encoding peroxisomal methanol oxidase in Hansenula polymorpha. Curr Genet 38: 163-170 van Dijken, JP, Otto, R, Harder, W (1976) Growth of Hansenula polymorpha in a methanol-limited chemostat. Physiological responses due to the involvement of
methanol oxidase as a key enzyme in methanol metabolism. Arch Microbiol in: 137-144 van der Klei J, Evers ME, Veenhuis M (1995) Biogenesis and function of peroxisomes in Hansenula polymorpha, in: Function and Biogenesis of Peroxisomes in Relation to Human Disease (Wanders, RJA, Schutgens, RBH, Tabak, HF, Eds). Elsevier, Amsterdam, The Netherlands, pp. 125-153 van der Klei J, van der Heide M, Baerends RJS, Rechinger K-B, Nicolay K, Kiel JAKW, Veenhuis M (1998) The Hansenula polymorpha per6 mutant is affected in two adjacent genes which encode dihydroxyacetone kinase and a novel protein, Pakip, involved in peroxisome integrity. Curr Genet 34: i-n Wickerham LJ (1951) Taxonomy of yeasts. Technical Bulletin No 1029, US Dept Agric, Washington, DC, USA, pp. 1-56
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3
Biochemistry and genetics of nitrate assimilation Jose M. Siverio
3.1 Introduction
Nitrate assimilation in yeasts has been a matter of little attention in comparison to the broad and intensive studies carried out in the filamentous fungi Neurospora crassa and Aspergillus nidulans (Marzluf, 1997). This is mainly due to the fact that the classical model yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe do not assimilate nitrate. Furthermore, genetic analysis and molecular biology tools are scarcely developed in those yeasts able to assimilate nitrate as sole nitrogen source. However, the fact that the yeast Hansenula polymorpha is able to use nitrate and nitrite as sole nitrogen sources and that it is amenable to genetic analysis and molecular biological tools has in part modified this situation. The cloning of the nitrate reductase gene (YNRi) (Avila et al. 1995) in H. polymorpha can be considered the starting point of the molecular era of nitrate assimilation in yeast. However, much work concerning physiological studies and biochemical characterization of the elements integrating the nitrate assimilation pathway have been carried out. Although, nitrate assimilation in yeast was reviewed last at the end of the 19805 (Hipkin, 1989), the present review does not intend to consider all the advances achieved in each yeast but is focused on H. polymorpha nitrate assimilation. Nitrate assimilation in those yeasts, able to use nitrate, follows the same pathway as that described for plants and filamentous fungi. Once nitrate enters the cells, it is reduced to ammonium by the consecutive actions of nitrate and nitrite reductase. In nature nitrate is one of the most abundant nitrogen sources. However, nitrate is not a preferred nitrogen source for microorganisms and plants, since nitrate must be reduced to ammonium, a process with a high energetic cost for cells since 8 electrons are involved. Most organisms have evolved complex regulatory systems to adapt their enzymatic machinery in order to use the nitrogen sources available. So, in nitrate assimilatory organisms, genes encoding nitrate transporters and nitrate and nitrite reductase are induced by nitrate and repressed by reduced nitrogen sources such as ammonium or glutamine. In the same way, and as a consequence of transcriptional regulation, nitrate transporter(s) and
Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Verlag GmbH, Weinheim ISBN: 3-527-30341-3
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3 Biochemistry and genetics of nitrate assimilation
nitrite and nitrate reductase are present in cells grown in nitrate and absent in those cells grown in ammonium.
3.2 Genomic organization of the genes involved in nitrate assimilation
The genes involved in nitrate assimilation in H. polymorpha were isolated from a XEMBLj genomic library. Two probes were used: a heterologous probe from Aspergillus nidulans NiR (Johnstone et al. 1990) and a 350 bp homologous probe constructed by PCR using two degenerated oligonucleotides, designed on the basis of protein sequence similarity of NR from N. crassa, A. nidulans and A. niger (Johnstone et al. 1990; Okamoto et al. 1991; Unkles et al. 1992). These probes were used to screen the XEMBLj genomic library, resulting in the isolation of the H. polymorpha nitrate reductase (YNRi, yeast nitrate reductase) and nitrite reductase (YNh, yeast nitrite reductase). Taking into account that the genes involved in nitrate assimilation in A. nidulans were clustered (Johnstone et al. 1990), this possibility was analyzed in H. polymorpha. It was observed that the phages which contained NiR hybridised with the NR probe and vice versa. DNA sequencing in the phages isolated revealed the clustering of the nitrate assimilation pathway genes in H. polymorpha. The cluster contains a nitrate transporter gene (YNTi), nitrite reductase (YNh), a Zn(II) 2 Cys6 transcriptional factor gene involved in nitrate induction (YNAi), the nitrate reductase (YNRi) and a second Zn(II) 2 Cys6 transcriptional factor gene (YNA2) (Figure 3.1). The DNA sequence containing ORFs plus intergenic regions presents 11040 bp of which 10,662 correspond to the 5 ORFs, indicating that in this region about 92.1% is coding DNA. In contrast, in S. cerevisiae ORFs occupy on average 72% of the genome (Dujon, 1996). The clustering of the genes involved in nitrate assimilation has also been found in A. nidulans and in A. niger where at least nitrite and nitrate reductase genes are clustered. A similar situation has also been found in Hansenula anomala (Garcia-Lugo et al. 2000). In the algae Chlamydomonas reinhardtii these genes have also been found clustered. This situation, however, has not been found in N. crassa. An exclusive feature of the H. polymorpha gene cluster
1kb
Fig. 3.1 H. polymorpha genomic DNA region containing the genes involved in nitrate assimilation. The region contains a nitrate transporter (YNTi, 1524 bp), nitrite reductase (YNh, 3132 bp), a Zn(ll) 2 Cys 6 transcriptional factor (YNAi, 1587 bp), nitrate reductase
(YNA1> 2577 bp), a second Zn(ll) 2 Cys 6 transcriptional factor (YNAi, 1842 bp). DNA re ions g between YNTi, YNn, YNh and YNAi, YNAl and YNRl and YNR and YNA2 ^ contains 22 °' 45, "3 and 7 bp, respectively,
3.3 Gene disruption in H. polymorpha
is the presence of YNAi and YNA2 involved in the regulation of the other gene cluster members. Another peculiarity of the H. polymorpha cluster is the fact that nitrite and nitrate reductase genes are convergently transcribed, unlike the divergent transcription of these genes in filamentous fungi (Johnstone et al. 1990). The genes of the cluster are transcribed independently; the transcription start site for each gene was mapped by primer extension. Heterogeneity in the length of the 5' region of the transcript was observed, indicating several transcription start sites, a feature already described in S. cerevisiae (Hahn et al. 1985; McNeil and Smith, 1986; Exley et al. 1993). The YNTi TATA box lies at -58 and the main initiation site is located at -21. YNIi presents four transcription start sites with one nucleotide difference in a region rich in T and A. For YNAi the strongest signal of the primer extension reaction was at -10 and -9; for YNRi three initiation sites were observed at -28, -2i and -20 situated in a CT-rich stretch with the main initiation site in the CAAG sequence.
3,3 Gene disruption in H. polymorpha
Gene disruption represents an invaluable technique to study nitrate assimilation gene function. Gene disruption has mainly been performed by one-step gene disruption (Avila et al. 1995; Avila et al. 1998; Brito et al. 1999; Perez et al. 1997) (Table 3.1). However, contrary to that found in S. cerevisiae where very short target gene sequences as 38-50 bp are enough to produce a very high frequency of disruptants, in H. polymorpha long sequences with an average length of 500 bp give a very low number of disruptants between the transformants obtained due to the high rate of heterologous recombination. This fact makes the use of this approach difficult when the phenotype expected is unknown. We have shown that the frequency at which genes can be disrupted by the one-step method in the yeast H. polymorpha depends mainly on the length of the flanking regions. In all cases studied, the longer the flanking regions, the higher the frequency of the desired
Tab. 3.1
Nitrate assimilation null mutants strain so far obtained in H. polymorpha
N sources
|N0 3 1 NO 2 NH 4 + Urea Proline Glutamate
Ayntr.:URA3
± + + + + +
Aymi::L/flA3
1
;
Aynai::URA3
1
;
(+) indicates growth, (-) no growth, ( + ) reduced growth.
Aynn.vURAj
1
:
&ynai::URA3
1 : 1
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3 Biochemistry and genetics of nitrate assimilation
disruption. The highest frequency observed for the disruption of YNRi was 80% with flanking regions of 1000 bp (Gonzalez et al. 1999) which is still far from the frequencies usually obtained for S. cerevisiae with much shorter flanking regions (Lorenz et al. 1995). Flanking regions of 25-50 bp produce very low disruption frequencies, which hinder the identification of mutants among the transformants, unless a phenotypic selection is available.
3.4
Nitrate transport
YNTi (yeast nitrate transporter) encodes a high-affinity nitrate transporter which is quantitatively the main nitrate transporter in H. polymorpha (Perez et al. 1997). In addition, Yntip also transports nitrite, although one or even more nitrite transport system(s) are involved in nitrite uptake in H. polymorpha (Machin and Siverio, unpublished results). Yntip belongs to the proposed NNP (nitrate nitrite porter) family involved in nitrate and nitrite transport (Forde, 2000). Members of this family have been isolated from prokaryotic and eukaryotic organisms. This family belongs to the Major Facilitator Superfamily (MFS), constituted by proteins with a membrane topology in which 12 membrane helices connect cytosolic N-terminal and C-terminal domains (Pao et al. 1998). Analysis of the a-helical transmembrane domains of Yntip by the Kyte and Doolittle method (Kyte and Doolittle, 1982) showed up some ambiguities with regard to the membrane-spanning domains, while the Eisenberg analysis (Eisenberg et al. 1984) rendered n transmembrane domains. These results could mean that regions of YNTi-encoded protein with a high degree of identity with CRNA (Unkles et al. 1991) and NRT2;i (Quesada et al. 1994) would differ in membrane orientation. Moreover, the YNTi-encoded protein C-terminus would be facing the outer side of the membrane, which is in disagreement to that proposed for the Major Facilitator Superfamily. To attempt to understand these discrepancies concerning the secondary structure of the YNTiproduct, a hydropathy analysis was carried out according to the Kyte and Doolittle method (Kyte and Doolittle, 1982) and compared with that obtained by the same method from the mean index of hydropathy of the residues at each position in the sequence resulting from the alignment of nitrate transporters encoded by YNTi, Nrt2;i and crnA (Perez et al. 1997; Quesada et al. 1994; Unkles et al. 1991). The tentative secondary structure presents 12 transmembrane domains. The regions with high similarity between the proteins compared present the same orientation with respect to the membrane and the putative glycosylation site at position 342, between the VII and VIII membrane-spanning domains, faces the outer side of the membrane (Perez et al. 1997). Thus, Yntip presents similarity in sequence, putative secondary structure and membrane topology with CRNA from Aspergillus (Unkles et al. 1991) and the plant and algae NRT2 group (Daniel-Vedele et al. 1998). However, the shortness of the C-terminal and the length of a central loop between the sixth and the seventh transmembrane domains are characteristic distinctive of Yntip and CRNA with respect to the rest of the NNP family members reported.
3.4 Nitrate transport
The NNP members which have been characterized present high affinity for nitrate and/or nitrite and are induced by nitrate (Crawford and Glass, 1998; DanielVedele et al. 1998; Forde, 2000). The Chlamydomonas rdnhardtii nitrate/nitrite transport is best characterized. So far four systems involved in nitrate and nitrite transport have been characterized. System I corresponds to a bispecific high-affinity nitrate and nitrite transporter encoded by Nrt2;i/Nar2. System II corresponds to a monospecific high-affinity nitrate transporter encoded by Nrt2;2/Nar2. System III is a bispecific high nitrate and low nitrite affinity transporter, probably encoded by Nrt2;j. System IV is a bispecific high-affinity nitrate and nitrite transporter probably encoded by Nrt2/4 (Galvan et al. 1996; Rexach et al. 1999; Navarro et al. 2000). In plants, several NRT2 members are involved in high-affinity nitrate transport (Crawford and Glass, 1998; Daniel-Vedele et al. 1998; Forde, 2000). Another family of transporters, NRT X , includes low-affinity nitrate transporters in plants. No low-affinity nitrate transporters have been cloned so far from lower eukaryotes, although there is evidence for these systems in C. rdnhardtii (Navarro et al. 2000) and H. polymorpha (Machin and Siverio, unpublished results). 3.4.1
Multiple nitrate uptake system
In o.i mM nitrate the Aynti::URAj strain presented reduced YNRi and YNh expression as well as low NR and NiR activity with respect to the WT (wild type). However, it is noteworthy that at 5 mM nitrate YNRi and YNh expression and NiR activity were similar in the WT and the Aymi.vLTRAj strain. On the other hand, NR activity was about 50% less in a nitrite reductase null mutant strain (Perez et al. 1997). The Aynti.vLTRAj strain was slightly less sensitive to chlorate than the WT. However, both were much more sensitive to chlorate than a strain deficient in NR (Aynn.-.'l/RAj). In the Ayntir.URAj strain at 10 mg ml-i in o.i mM nitrate, uptake of nitrate was not significant in 20 min, in contrast to the WT where almost 90% of the nitrate was consumed under the same conditions (Perez et al. 1997). However, after 6-8 h about 50% of the extracellular nitrate was consumed. In addition, the Aynti::URA-$ strain grew in nitrate while mutants lacking Ynnp or Ynaip did not. These results suggest the existence of a nitrate uptake system different to Yntip. The characterization of these transport systems is hindered due to their low activity and to the lack of adequate labels for nitrate. To test the presence of an alternative Yntip nitrate transporter a strain (FM}i) lacking YNTi, YNh, YNAi, and YNRi genes, [A(ynti,yrai, ynai,ynn)::URA^], bearing the fusion MOXi-YNRi was used (Machin 2001). In this strain nitrate entering the cells would be reduced to nitrite by NR in conditions of expression of the MOXi promoter. Since these strain cells lack NiR, nitrite would be excreted to the medium where it could be measured and provide an estimation of nitrate uptake. It was found that the amount of nitrite excreted in a specific time frame was dependent upon the extracellular nitrate concentration. The kinetics shown by the nitrite excretion experiments suggest that the nitrate transport system(s) involved presents low affinity for nitrate, since the highest excretion is reached at high nitrate concentrations. The affinity of the nitrate transporter
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3 Biochemistry and genetics of nitrate assimilation
system(s) measured from the nitrite excretion rates determined in the FMji strain was compared with that present in the strain lacking NiR (Aynir.'.URAj), but expressing the high-affinity nitrate transporter gene YNTi. In this strain nitrite excretion is already maximal at o.i mM nitrate, while in strain FM}! this level was reached only at 10 mM external nitrate. It was remarkable that the nitrite excretion rate in the FMji strain decreased after 20-30 min, an unexpected result considering the high extracellular nitrate concentration. A plausible idea to explain this would be that nitrite excreted could inhibit nitrate uptake, suggesting that this nitrate transporter could also be involved in nitrite transport. Since strain FMji lacks Ynaip involved in nitrate induction (Avila et al. 1998) it may be inferred that the new nitrate uptake system detected is independent of nitrate induction. In addition, nitrite excretion did not increase after previous incubation of cells in nitrate. Using a similar approach, a C. reinhardtii strain lacking the high-affinity nitrate transport system and NiR showed nitrite excretion only from millimolar extracellular nitrate, whereas in a strain bearing high-affinity nitrate transporter systems nitrite excretion was observed at the jiM range. This fact suggests that the system involved in nitrate transport in the strain lacking the high-affinity nitrate transporter systems presents low affinity for nitrate and might correspond to the nitrate/nitrite transport system III (Navarro et al. 2000). In summary, current knowledge on nitrate/nitrite transport in H. polymorpha reveals the presence of an inducible and high-affinity nitrate and nitrite transporter Yntip and Yntip-independent system(s) with, presumably, low affinity for nitrate. Concerning the Yntip-independent nitrate/nitrite transport system(s) several questions remain to be addressed as to whether nitrate and nitrite are transported through the same system, to the number of systems involved in this process, and to the real physiological role of these systems either in transport or signaling or in both.
3.5
Nitrate reductase
Once nitrate is transported into the cell it is reduced to nitrite by assimilatory nitrate reductase (NR). Assimilatory NRs posses three different conserved domains involved in binding of molybdopterin (MoCo), heme-iron and FAD cofactors (Guerrero et al. 1981; Campbell and Kinghorn, 1990). H. polymorpha NR is able to use NADH and NADPH as electron donors (Dujon, 1996). Several genes are responsible for the production of an active NR: • the gene coding the NR apoenzyme, and • several genes involved in the synthesis of the MoCo. Assimilatory NR genes have been isolated from filamentous fungi (Okamoto et al. 1991; Johnstone et al. 1990; Unkles et al. 1992), plants (Cambell, 1999), algae (Fernandez et al. 1989) and the yeasts H. polymorpha (Avila et al. 1995) and H. anomala (Garcia-Lugo et al. 2000).
3.5 Nitrate reductase
The putative NR encoded by YNRi shares a high similarity with other NRs such as that encoded by nitj (N. crassa) (Okamoto et al. 1991; Unkles et al. 1992), niaD (A. nidulans) (Unkles et al. 1992) and niai (tobacco) (Vaucheret et al. 1989). The similarity is especially high in the MoCo, heme and FAD binding regions. These regions have been identified by similarity with the mammalian protein regions of sulfite oxidase (Garrett and Rajagopalan, 1994) cytochrome b5 and NADPH cytochrome b5 reductase (Yoo and Steggles, 1988) proteins that contain the MoCo, heme and FAD domains, respectively. These cofactor binding regions are located in the sequence encoded by YNRi in a linear way, with the MoCo region near the Nterminus followed by the heme binding region and the FAD-NADPH binding region at the C-terminus, in a similar way to that described for other NRs. The Aynn.vlTRAj strain showed no NR activity after incubation in nitrate for 2 h, in contrast to the WT strain. The lack of NR activity in the null mutant strain as well as the Southern blot analysis of the WT strain indicate that H. polymorpha contains only one YNRi copy. As will be discussed below the Aynnr.URAj mutant appears to be an interesting tool to express NR-encoding genes to study structure-function relationships of NR by site-directed mutagenesis. 3.5.1
Postransductional regulation of NR
To examine the importance of the postranscriptional regulation on NR, YNRi was expressed in the strain Aynn.vlTRAj under the control of H. polymorpha MOXi gene promoter to bypass the transcriptional regulation of YNRi by nitrogen sources. MOXi is derepressed by glycerol, induced by methanol and repressed by glucose (Hansen and Hollenberg, 1996). In the strain bearing the fusion MOXi-YNRi, YNRi was induced by methanol with ammonium or nitrate as nitrogen source, observing that NR activity appeared as much in ammonium as in nitrate. In addition, the levels of NR protein are correlated with the activity, while both protein and activity are 2-3 times higher in ammonium than in nitrate medium (Perdomo and Siverio, unpublished results). Nitrate metabolism products such as nitrite, ammonium and further reduced nitrogen compounds could play a role in NR activity levels. These levels were followed in WT cells incubated at different extracellular nitrate concentrations from 0.5-5 mM. NR protein and NRiNADH activity were similar at the nitrate concentrations used. These results are in agreement with the same experiments carried out in the strain bearing the YNRi-lacZ fusion. Again, these results indicate that NR does not undergo postransductional regulation. Summarizing, NR encoded by YNRi presents high similarity with NR from fungi and in a smaller extent with that present in plants. In H. polymorpha NR is present in cells grown or incubated in nitrate, but not in cells grown in ammonium or incubated in nitrogen-free medium. In addition, NR activity is not affected by reduced nitrogen sources, since when YNRi was expressed under the control of MOXi in a strain Aynn.vL/RAj the presence of ammonium did not affect NR activity.
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3 Biochemistry and genetics of nitrate assimilation
3.6 Nitrite reductase
Nitrate is reduced to nitrite by NR and nitrite to ammonium by nitrite reductase (NiR). Regarding the electron donor two types of assimilatory NiR have been reported (Guerrero et al. 1981): a ferredoxin-NiR, characteristic of plants and algae and a NAD(P)H-NiR found in fungi and bacteria. Assimilatory NiR possesses two prosthetic groups, an iron-sulfur center and a heme group termed siroheme (Lancaster et al. 1979; Vega and Kamin, 1977; Campbell and Kinghorn, 1990). In addition to siroheme and iron-sulfur prosthetic groups, NAD(P)H-NiR from fungi and bacteria possesses FAD (Prodouz and Garrett, 1981). Independent of the electron donor both enzymes catalyze the stoichiometric reduction of nitrite to ammonia, by transferring 6 electrons to nitrite. NiR-encoding genes have been isolated from several organisms from fungi to plants including the yeasts H. polymorpha and H. anomala (Luque et al. 1993; Johnstone et al. 1990; Brito et al. 1996; Garcia-Lugo et al. 2000; Quesada et al. 1998; Vaucheret et al. 1992). The H. polymorpha NiR gene, termed YNh encodes a NiR with a calculated molecular mass of 116.6 kDa and high identity with NiRs from other fungi. Similarity searches with the FASTA program analysis (Pearson, 1990) of the predicted NiR encoded by YNIi showed an identity of 44.7%, and 56.0% in the 1,029 and 591 amino acid overlap with NiRs from A. nidulans (Johnstone et al. 1990) and N. crassa (Okamoto et al. 1991), respectively. Significant identity was also found with bacterial NiRs from Klebsiella pneumonias (Lin et al. 1994) and Escherichia coli (Peakman et al. 1990). Among the 40 proteins with the best score from the FASTA analysis no plant NiR appeared. The putative iron-sulfur center and siroheme domains were localized in the predicted H. polymorpha NiR sequence by comparison with fungal NiRs and the corresponding domain of the sulfite reductase from E. coli (Ostrowski et al. 1989). This region possesses the consensus sequence CXXXXXCXnGCXXXC (Campbell and Kinghorn, 1990) where the cysteine has been proposed to be involved in the binding of the tetranuclear ironsulfur center and siroheme to the NiR (Siegel and Wilkerson, 1989). The region around the cysteine consensus sequence is highly conserved between NiRs and poorly between NiRs and sulfite reductase. Likewise, comparison of the cysteinecontaining sequence from plants with those from fungi shows low identity. Concerning the FAD-NAD(P)H domains, analysis of the structure and the amino acid sequence of 6 proteins interacting with FAD, NAD or NADP revealed a common fold £a£,(£ = £ sheet and oc = oc helix) with a motif sequence GXGXXG (Wierenga et al. 1985). This motif has been found twice in the N-terminus of NiR from A. nidulans and N. crassa as well as in H. polymorpha. These two sequences could be involved in the FAD and NAD(P)H binding domains of NiR. The lack of NiR activity in the null mutant (kynhr.URAj) along with the Southern blot analysis confirm that H. polymorpha YNh is present in a single copy. As a direct consequence the null mutant strain is unable to grow in nitrate and nitrite. Another feature of the NiR null mutant is the increase of nitrite excretion when the cells are incubated in nitrate, in a similar way to that reported in A.
3.7 Expression levels ofYNTl, YNI1 and YNR1
nidulans (Cove, 1979). This capacity to extrude nitrite could be involved in maintaining nitrite levels under lethal concentrations. Nitrite has been described to be toxic in the yeast S. cerevisiae because it decreases the levels of ATP (Hinze and Holzer, 1986). The viability of Aynii::URA} and WT strains was not affected by incubation at the concentrations of nitrite (up to i mM) and nitrate (5 mM) tested. The study of the effect of the Aynir.iURAj null mutation on YNRi expression and NR activity revealed that the mutant (Ayrai.vl/RAj) possesses about 50% of the NR activity in comparison with the WT strain. However, Northern blot analysis showed that the expression of YNRi is at the same level in both strains. The decrease of NR activity in the mutant could be due to the inactivation of NR by nitrite as described in H. anomala (Gonzalez et al. 1994). However, in this case decrease in NR activity could also be due to a decrease in the translation of YNRi transcript. Although this was not observed, an increase of YNRi expression could also be expected in the A.ynii::URA^ null mutant because of the release of YNRi repression exerted by reduced nitrogen sources, which are absent in a Ayniiy.URAj null mutant with nitrate as nitrogen source, as described in plants (Vaucheret et al. 1992). In the Ayrai.vlTRAj null mutant YNRi expression shows the same pattern as in the WT. On the contrary, it has been reported that mutations affecting NR-encoding genes modify the expression patterns of nitrate assimilation genes (Fu and Marzluf, 1988; Hawker et al. 1992). As will be discussed in the following section transcriptional modulation of YNTi, YNh and YNRi in response to nitrogen sources is the main long-term regulation of the nitrate assimilation pathway. However, H. polymorpha NiR undergoes activity changes in response to nitrate induction under different concentrations. In this way, low nitrate concentration (o.i mM) leads to NiR activities 4-5 times higher than those carried out at high concentrations (Navarro and Siverio, unpublished results). Although the mechanism of regulation has not been studied, it could be speculated that dephosphorylation/phosphorylation could be involved. In this respect it has been shown that Candida utilis NiR is a heterodimer consisting of two monomers of 58 kDa and 66 kDa which in vivo are differentially phosphorylated in response to nitrate and ammonium. Under nitrate induction conditions the enzyme presents maximum activity, and the 66 kDa subunit is highly phosphorylated while the 58 kDa subunit is not phosphorylated. In addition, in vitro dephosphorylation of NiR produced by alkaline phosphate and NiR activity decrease are well correlated. In ammonium-grown cells NiR activity was 20% of those cells grown in nitrate, and the degree of phosphorylation decreased, but was present in both subunits (Sengupta et al. 1997).
3.7
Expression levels of YNTI, YNH and YNR1
The expression levels of YNTi, YNh, YNRi determined by Northern blot in cells grown in ammonia and transferred to different nitrogen sources for 2 h, showed that these genes are coordinately expressed in nitrate and nitrite, scarcely expressed
29
30
3 Biochemistry and genetics of nitrate assimilation
in nitrate plus ammonium while no or very scarce expression was detected in nitrogen-free medium or ammonium (Figure 3.2). These data concerning the transcriptional modulation of the genes involved in nitrate assimilation reveal that long-term transcriptional regulation is the main regulation mechanism of the nitrate assimilation pathway. To study the strength of YNTi, YNh and YNRi gene promoters and how they are affected by the nitrogen sources, the 5' regions of the H. polymorpha nitrate reductase (YNRi), nitrite reductase (YNh) and nitrate transporter (YNTi) genes were fused to lacZ gene using pHPI plasmids (Brito et al. 1999). No (3-galactosidase activity was found in the cells grown in ammonium nor in those strains bearing the plasmid without any promoter fused to lacZ. In the strains transferred to nitrate, the highest activity was found in strain YNRgal followed by YNIgal and YNTgal (Brito et al. 1999). The effect of nitrite on the expression of lacZ was also studied. In this case, the medium was buffered with
Fig. 3.2 Expression of YNTi, YNRi and YNh genes in H. polymorpha. Expression levels were determined by Northern blot. Cells grown in ammonium were transferred to the nitrogen sources indicated, each at 5 mM.
YNTI
YNRI
YNH
till Tab. 3.2 (3-Galactosidase activity levels in strains expressing lacZ under YNRi, YNh and YNTi gene promoters
\
Strains
YNRgal YNIgal YNTgal
Induction conditions
1 0.5 mM NO3 380 + 20 14.2 ±1.3 0.68 ±0.01
| 5 mM NO3 500 ±45 40±3 0.39 ±0.05
The strains YNRgal, YNIgal and YNTgal, containing the 5' noncoding region corresponding to YNRi, YNh and YNTi genes, respectively, were used.
J ImM N02 | 314 + 23 13±2 3.21 + 0.9
3.8 Transcriptional regulation ofYNTl, YNT1 and YNR1 genes involved in nitrate assimilation 31
50 mM Tris-HCl pH 7, as nitrite may act as an uneoupler at acid pH (Gonzalez et al. 1994), resulting in a similar induction as when using nitrate. The results obtained led us to conclude that, under the conditions used, the strongest promoter is that corresponding to YNRi Mowed by YNh and YNTi (Table 3.2). With regard to the strength of the promoters studied, the low expression obtained for the YNh gene promoter in comparison to YNRi is very surprising, since it is generally assumed that the NiR levels are highest in the nitrate assimilation pathway followed by NR (Guerrero et al. 1981). Although, there is no direct correlation between the level of expression of a gene and the activity shown by the corresponding enzyme it encodes, the low level of NiR detected in H. polymorpha (Medina and Siverio, unpublished results) is consistent with the low level of expression detected in the YNIgal strain. A further explanation for the low level of expression shown in the YNIgal strain could be the fact that the fusion used contains the first 156 amino acids of the NiR and this could destabilize the resulting hybrid protein.
3.8 Transcriptional regulation of YNTI, YNTI and YNRI genes involved in nitrate assimilation
Current knowledge on the mechanisms underlying nitrate induction and nitrogen catabolite repression has mainly been derived from filamentous fungi, where two transcriptional factors have been intensely studied: nit-4 and nit-2 in N. crassa and their counterparts nirA and areA in A. nidulans. Nit-4 ansr-^ O j ™
g ^* X . ^1 J ^ C J r t r t
1 jc>o^;u
p^^;
H^-O^^
-H
S
-rH
S
CJ)
Jj
V3
S 3 2 'S '§ « ^ LO i>5 to > nn > j
••••••i
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rt o
U
Ic
Q. O
_O _^" QJ
E c
O
c
1
QJ
OJ
7T
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t+, able to grow on
QJ
t~, unable to grow
to to ns
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5.2 Methanol metabolism in methylotrophic yeasts
alcohol oxidase (AOD in C. boidinii and AOX in Pichia pastoris), localized in a membrane-bound organelle, the peroxisome (see Chapter 7) (Figure 5.1, (i)). Both products of this reaction, namely formaldehyde and H 2 O 2 , are highly toxic to living cells. Formaldehyde is a central intermediate of methanol metabolism, which enters into both the cytosolic dissimilatory pathway and the assimilatory pathway. Recently, several cellular mechanisms to avoid toxicity of these compounds have been revealed with C. boidinii (see Sect. 5.4).
5.2.1.1
Alcohol oxidase
Alcohol oxidase (AOD, EC 1.1.2.13), a flavoprotein .containing FAD as a prosthetic group, catalyzes the oxidation of methanol, using molecular O2 as an electron acceptor to yield formaldehyde and H 2 O 2 (Figure 5.1, (i)). CH3OH
O2 -> HCHO + H 2 O 2
AOD is synthesized in the cytosol in an inactive monomeric form (approximately 72kDa) and is posttranslationally imported into peroxisomes where the active homooctameric FAD-containing enzyme is assembled (Goodman et al. 1984). Each FAD-containing subunit is arranged in an octad aggregate composed of two tetragons face-to-face (Kato et al. 1976). During methylotrophic growth, the synthesis of AOD is greatly enhanced, and the protein reaches up to 20-30% of the total soluble protein. Due to this high inducibility, the gene promoters of AOD are commonly used for highlevel heterologous gene expression in methylotrophic yeast systems (Gellissen 2000). CH3OH
Cytosol
r
Peroxisome — O2 + H2O 2 .
(6)
»- r*u /- CO2
RCOOH,
Fig. 5.1 Dissimilation pathway of methanol metabolism in methylotrophic yeasts. Enzymes: (i) alcohol oxidase, (2) catalase, (3) glutathione-dependent formaldehyde dehydrogenase, (4) S-formylglutathione hydrolase, (5) formate dehydrogenase,
(6) methyl formate synthase, (7) Pmp2O, Abbreviations: S-HMG, S-hydroxymethylglutathione; S-FG, S-formylglutathione; GSH, a reduced form of glutathione; GS-SG, an oxidized form of glutathione.
63
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5 Methanol metabolism
The genes encoding AOD, together with their promoter regions, were cloned from H. polymorpha (MOX) (Ledeboer et al. 1985), P. pastoris (AOXi, AOX2) (Gregg et al. 1989), C. boidinii (AODi) (Sakai and Tani 1992), and P. methanolica (AUGi, AUG2 also known as MODi, MOD2, respectively) (Nakagawa et al. 1999, Raymond et al. 1998). The primary structures of these AOD are similar, with amino acid sequence similarities of approximately 80%. AOD belongs to a CMC oxidoreductase family, which includes glucose oxidase, choline dehydrogenase, glucose dehydrogenase, and cholesterol oxidase (Cavener 1992). Members of the CMC oxidoreductase family share a number of regions of sequence similarity (CMC oxidoreductase signatures), one of which is located in the N-terminal section and corresponds to the FAD-ADP-binding domain. The C-terminal tripeptides of AODs are -ARF (MOX, AOXi, AOX2, MODi), -ARY (AODi), and -GRF (MOD2), which are consensus sequences for a typei peroxisomal targeting signal (PTSi), [STAGCN]-[RKH]-[LIVMAFY] (Gould et al. 1990, Hansen et al. 1992). P. pastoris and P. methanolica possess two sets of AOD genes. In these strains AOXi and AUGi are expressed at significantly higher levels than AOX2 and AUG2 (Gregg et al. 1989, Raymond et al. 1998). Disruption of the AOXi or AUGi genes caused a severe growth defect on methanol as the sole carbon source, while growth of AOX2- and A If G2-disruptants was similar to that of the wild-type strains. Cellfree extracts of methanol-grown cells of P. methanolica showed nine multiple AOD bands on active staining in native polyacrylamide gel electrophoresis. These multiple bands represent two homooctamers and seven heterooctamers of Modip and Mod2p. Modip was found to be preferably induced at low methanol concentrations because of its higher affinity to methanol than Mod2p (Gruzman et al. 1996, Nakagawa et al. 1999). Therefore, AOD activity in P. methanolica is finely tuned by a combination of two factors: the rate of catalytic activity and the amount of each subunit protein (see Sects. 5.3 and 5.4). 5.2.2 Dissimilatory pathway
In the dissimilatory methanol oxidation pathway, formaldehyde is oxidized to CO2 via formate by two subsequent cytosolic dehydrogenases, glutathione-dependent formaldehyde dehydrogenase (FLD) and formate dehydrogenase (FDH). Formaldehyde reacts spontaneously with reduced glutathione (GSH) to form Shydroxymethylglutathione (S-HMG). FLD uses S-HMG as a substrate and generates S-formylglutathione (S-FG) (Figure 5.1, (3)). S-FG is hydrolyzed by Sformylglutathione hydrolase to generate GSH and formate (Figure 5.1, (4)). Finally, formate is oxidized to CO2 by FDH (Figure 5.1, (5)). NADH, generated during these two dehydrogenase reactions, provides energy for the cell.
5.2.2.1
Clutathione-dependent formaldehyde dehydrogenase
Formaldehyde reacts non-enzymatically with glutathione (GSH) to form Shydroxymethylglutathione (S-HMG). NAD-linked and glutathione-dependent for-
5.2 Methanol metabolism in methylotrophic yeasts
maldehyde dehydrogenase (FLD, EC 1.2.1.1) uses S-HMG as a substrate to yield Sformylglutathione (S-FG) and NADH (Figure 5.1, (3)). HCHO + GSH + NAD + -> S-FG + NADH + H + Until recently, the formation of S-HMG was thought to occur in the cytosol. However, a reduced form of GSH has now been found to function as a reductant for Pmpzo (see Sect. 5.4.2), and a significant level of GSH was found to be present within peroxisomes. Formaldehyde generated by peroxisomal AOD would, therefore, react with GSH in peroxisomes and S-HMG may be exported to the cytosol by a specific transport mechanism. FLD is a dimeric enzyme and the molecular weight of each subunit is about 4okDa (Kato 19903). FLD catalyzes the oxidation of formaldehyde and methylglyoxal, but does not use other aliphatic or aromatic aldehydes as substrates. The gene encoding FLD was cloned from P. pastoris (Shen et al. 1998) and C. boidinii (Sakai, unpublished results). The deduced amino acid sequences include zinc-containing alcohol dehydrogenase signatures (Sun and Plapp 1992). The promoter of the FLDi gene from P. pastoris is strongly and independently induced by either methanol as a carbon source or methylamine as a nitrogen source, and the level of expression under the FLDi promoter is comparable to that obtained with the AOXi gene promoter (Shen et al. 1998). The physiological role of FLD has two aspects in methanol metabolism. One is that NADH produced in the reaction serves as a primary source of energy during methylotrophic growth. Another is that FLD protects the cells from the toxic effects of formaldehyde. A mutant of H. polymorpha deficient in FLD was able to grow on methanol as the sole carbon and energy source under chemostat cultivation. It was speculated that in this mutant, most energy necessary for growth came from tricarboxylic acid cycle reactions (Sibirny et al. 1990). On the other hand, a mutant of P. pastoris deficient in FLD was defective in terms of its ability to grow on methanol specifically (Shen et al. 1998). Recently, a gene disruptant strain of C. boidinii (fldiA) was obtained (Sakai, unpublished results). ThefldiA strain could not grow on methanol as the sole carbon and energy source under either batch culture or chemostat culture conditions. Furthermore, the addition of formaldehyde inhibited the growth of this strain. It has been confirmed that FLD is essential for methylotrophic growth and that the physiological function of FLD is not only the detoxification of formaldehyde, but also energy generation.
5.2.2.2
S-Formylglutathione hydrolase
S-Formylglutathione (S-FG) is hydrolyzed to formate and glutathione (GSH) by Sformylglutathione hydrolase (S-FGH, EC 3.1.2.12) (Figure 5.1, (4)). S-FG + H20 -> HCOOH + GSH S-FGH was purified from two strains of C. boidinii (Kato et al. 1980, Neben et al. 1980). Although the genes encoding S-FGH have not been cloned from methylotrophic yeasts, the fghA gene encoding S-FGH was identified from the
65
66
5 Methanol metabolism
methylotrophic bacterium, Paracoccus denitrificans (Harms et al. 1996). The deduced amino acid sequence of fghA showed a significant similarity to human esterase D, and orthologs of these proteins were found in Escherichia coli and Saccharomyces cerevisiae. S-FGH, together with FLD and FDH, may be ubiquitously present in nature from lower to higher organisms as a general formaldehyde detoxification pathway.
5.2.2.3
Formate dehydrogenase
NAD-linked formate dehydrogenase (FDH, EC 1.2.1.2) is the last enzyme involved in the methanol dissimilatory pathway. Formate is oxidized to CO2 and NADH is generated through the following reaction (Figure 5.1, (5)). HCOOH + NAD + -> CO2 + NADH + H+ FDH is a dimeric enzyme and the molecular weight of each subunit is about 40 kDa (Kato 199013). FDH from methylotrophic yeasts is the most commonly used enzyme for regenerating NADH from NAD + in many bioreactor reactions. The gene encoding FDH was cloned from H. polymorpha, C. methylica and C. boidinii (Sakai et al. 1997). Since FDH is induced by methanol and FDH reaches up to 20% of total soluble protein, the FDH promoter has been applied in high-level expression vectors in H. polymorpha and C. boidinii (Gellissen et al. 1991, Komeda et al. 1999). Induction of the FDH-encoding gene was not repressed by glucose and could be induced by methanol, methylamine, and formate (Sakai et al. 1997) (see Sect. 5.5). An FDH-negative strain of H. polymorpha did not differ from the wild-type strain in the growth rate on methanol as the sole carbon source, but showed a lower cell yield (Sibirny et al. 1990). On the other hand, a strain of C. boidinii (fdhiA) in which the FDH-gene had been disrupted showed a low growth rate. The growth yield of thefdhiA strain was only about 25% of that of the wild-type strain under methanollimited chemostat conditions (Sakai et al. 1997), although formate was not detected in the medium. NADH generated through an FDH-catalyzed reaction, therefore, significantly contributes to energy generation during methylotrophic growth. 5.2.3 Assimilatory pathway
Since methanol has no carbon-to-carbon bonds, cells growing on methanol have to form these bonds in order to synthesize cell constituents. In the assimilatory metabolic pathway of the methylotrophic yeast, the Q unit is condensed with a C5 sugar to form two C3 compounds. The initial reaction is catalyzed by dihydroxyacetone synthase (DHAS), localized in peroxisomes, generating dihydroxyacetone (DHA) and glyceraldehyde 3-phosphate (GAP) through a transketolase reaction between formaldehyde and xylulose 5-phosphate (Xu5?) (Figure 5.2, (2)). DHA and GAP are further assimilated within the cytosol. DHA is phosphorylated by dihydroxyacetone kinase (DHAK) (Figure 5.2, (3)), and
5.2 Methanol metabolism in methylotrophic yeasts
subsequently, dihydroxyacetone phosphate (DHAP) and GAP form fructose 1,6bisphosphate by fructose bisphosphatase (Figure 5.2, (4)). Xu5P is regenerated through rearrangement reactions in the pentose phosphate cycle. One third of the DHAP molecules generated are used for gluconeogenesis.
5.2.3.1
Dihydroxyacetone synthase
Dihydroxyacetone synthase (DHAS, EC 2.2.1.3) ^s tne ^rst enzyme in the formaldehyde assimilation pathway. DHAS catalyzes the thiamine pyrophosphate (TPP)-dependent transfer of the C2 unit of xylulose 5-phosphate (Xu5?) to formaldehyde yielding dihydroxyacetone (DHA) and glyceraldehyde 3-phosphate (GAP) (Figure 5.2, (2)). HCHO + Xu5? -> DHA + GAP This can be considered a kind of transketolase reaction, however, DHAS differs from transketolase with respect to both substrate specificity and its subcellular localization in peroxisomes (Douma et al. 1985, Goodman 1985). Like other transketolases, DHAS requires Mg2+ ions and can use other substrates such as hydroxypyruvate and fructose-6-phosphate. DHAS, purified from C. boidinii, is a dimeric enzyme and the molecular weight of each subunit is approximately ySkDa (Kato et al. 1982, Sakai et al. 1998). The genes coding for DHAS were cloned from H. polymorpha and C. boidinii (Janowicz CH3OH Cytosol
Peroxisome \
CH3OH 02 (i)
H202
r
HCHO Xu5P-
.1 DHA + GAP
ATP
—GAP 3 GAP
*- Rearrangement reactions
ADP
Fig. 5.2 Assimilation pathway of methanol metabolism in methylotrophic yeasts. Enzymes: (i) alcohol oxidase, (2) dihydroxyacetone synthase, (3) dihydroxyacetone kinase, (4) fructose bisphosphatase. Abbreviations: DHA, dihydroxyacetone; GAP, glyceraldehyde 3-phosphate; DHAP, dihydroxyacetone phosphate; Xu5P, xylulose 5-phosphate.
67
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5 Methanol metabolism
et al. 1985, Sakai et al. 1998). The deduced amino acid sequences of DHAS from these two strains showed similarities and contained transketolase signatures and a possible TPP binding domain (Schenk et al. 1997). The C-terminal tripeptides of DHAS from H. polymorpha and C. boidinii are -NKL and -NHL, respectively, which belong to a consensus sequence for PTSi (Hansen et al. 1992, Sakai et al. 1998). Expression analysis of the DHAS-encoding gene, DASi, in C. boidinii revealed that DHAS is induced by methanol or formaldehyde and that induction of DHAS is susceptible to glucose repression. The regulation of DASi is more similar to that of AODi than that of FLDi or FDHi. The DA Si-disrupted strain (dasiA) completely loses the ability to grow on methanol (Sakai et al. 1998). A peroxisomal membrane protein, Pmp47, of C. boidinii is involved in the formation of active DHAS (Nakagawa et al. 2000, Sakai et al. 1996). In the pmp4jA strain, the DHAS protein was found to aggregate in the cytosol as an inclusion body. Since Pmp47 belongs to a family of mitochondrial solute transporters and ATP can bind to Pmp47 (Nakagawa et al. 2000), ATP transported by Pmp47 seems to be necessary for the folding or transport process of DHAS.
5.2.3.2
Dihydroxyacetone kinase
Dihydroxyacetone (DHA) is phosphorylated by dihydroxyacetone kinase (DHAK, EC 2.7.1.29) to yield dihydroxyacetone phosphate (DHAP) and ADP (Figure 5.2, (3)). DHA + ATP -> DHAP + ADP Purified DHAK from H. polymorpha is a homodimer composed of two subunits of 72kDa (Kato et al. 1988). DHAK was reported to catalyze the phosphorylation of DHA as well as D-and L-glyceraldehydes, with ATP serving as a donor. The affinity and activity of DHAK towards DHA is much higher than towards D-glyceraldehyde, and on this basis DHAK is distinguishable from triokinase (EC 2.7.1.28). The genes encoding DHAK were cloned from P. pastoris and H. polymorpha (Liiers et al. 1998, van der Klei et al. 1998). In these two strains, disruption of the DHAK gene conferred an inability to grow on methanol. Although DHAK from P. pastoris contained a C-terminal tripeptide sequence, TKL, which could act as a consensus sequence for PTSi, the enzyme was shown to be cytosolic (Liiers et al. 1998).
5.3
Regulation of methanol metabolism
The synthesis of dissimilatory enzymes was regulated in a derepression/repression manner rather than in an induction/repression manner. During chemostat growth under glucose limitation, the dissimilatory enzymes increase with decreasing dilution rates (Egli et al. 1980). Except for AOD, the extent to which catabolite repression of the dissimilatory enzymes is relieved at low dilution rates is similar in H. polymorpha and C. boidinii. However, the levels of AOD gradually increased with decreasing dilution rate, whereas in C. boidinii the derepression of AOD was more
5.4 Detoxification of toxic compounds during growth on methanol
reduced than in H. polymorpha. The analysis of the promoter region of the AODencoding gene revealed that in H. polymorpha the expression of the MOX gene is regulated by a derepression/repression mechanism whereas in C. boidinii regulation of the AODi gene is achieved by an induction/repression mechanism (Roggenkamp et al. 1984, Sakai and Tani 1992). Sequences responsible for transcriptional regulation of the MOX gene have been identified in H. polymorpha (Godecke et al. 1994). Using a heterologous reporter gene system it was shown in S. cerevisiae that Adrip, a transcription factor involved in derepression of the alcohol dehydrogenase gene (ADH2), regulates derepression of the MOX gene (Pereira and Hollenberg 1996). In P. pastoris, the positive and negative as-acting elements for methanol regulation of the AOX2 gene have been identified (Ohi et al. 1994). However, transcriptional factors involved in the regulation of AOD have not been described in any methylotrophic yeast strains. We have analyzed five methanol-inducible promoters (AODi, DASi, FDHi, PM?47, PMPzo) in C. boidinii using acid phosphatase as a reporter (Yurimoto et al. 2ooob). Of the five promoters, the DASi promoter was the most potent, giving an approximately 1.5 times higher expression than the AODi promoter. The AODi promoter showed a maximum level of expression in cells grown on methanol, a derepressed level of expression in cells grown on glycerol or oleate, and was repressed in the cells grown on glucose or ethanol. In contrast, the DASi promoter did not show a derepressed level of expression in any of the carbon sources. Similar results were reported for the DAS gene in H. polymorpha and P. pastoris (Roggenkamp et al. 1984, Tschopp et al. 1987).
5.4
Detoxification of toxic compounds during growth on methanol
Two toxic compounds, formaldehyde and H2O2, are generated by AOD, which is the first reaction of methanol metabolism. How do cells overcome the toxicity of these compounds? 5.4.1 Formaldehyde toxicity
The major pathway for eliminating the toxicity of formaldehyde may be its cytosolic oxidation by FLD, because an FLD gene disruptant strain (fldiA) of C. boidinii was unable to grow on methanol and accumulated formaldehyde in its culture medium (Sakai, unpublished results). Recently, an alternative pathway for formaldehyde oxidation was also proposed (Murdanoto et al. 1997, Sakai et al. 1995). Thus, in various methylotrophic yeast strains, a significant amount of methyl formate was found to accumulate during methylotrophic growth, and this accumulation was stimulated by the addition of formaldehyde to the culture medium. The formation of methyl formate was catalyzed by a NAD+-dependent dehydrogenase reaction of the hemiacetal adduct of methanol and formaldehyde (Figure 5.1, (6)).
69
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5 Methanol metabolism
Methylotrophic yeasts have a cytosolic formaldehyde detoxification pathway. However, since the generation of formaldehyde takes place within peroxisomes, mechanisms for the prevention of a sudden increase in concentration of formaldehyde in peroxisomes are likely to exist. In this respect, one interesting observation was that induction of DAS preceded that of AOD during the early stages of methanol induction in C. boidinii (Sakai et al. 1996). If AOD was induced strongly by methanol at an earlier stage than DHAS, a large amount of formaldehyde would accumulate in the peroxisomes. Indeed, the growth of the dasi A strain was greatly inhibited in medium containing both methanol and glycerol where formaldehyde generated by AOD could not be metabolized via a dissimilation pathway (Sakai et al. 1998). To avoid this problem, C. boidinii is thought to minimize formaldehyde toxicity by regulating the timing of AOD and DAS induction during methanol induction, although the molecular basis for this regulatory process is unknown. 5.4.2 Reactive oxygen species
Another toxic compound generated in methanol metabolism is H 2 O 2 , which is broken down by a peroxisomal catalase (CTA, EC i.n.i.6) (Figure 5.1, (2)). 2H 2 O 2 -» O2 + 2H 2 O CTA is a tetrameric enzyme and the molecular weight of each subunit is about 6okDa. CTA is used as a marker enzyme for peroxisomes. The C-terminal tripeptide of this enzyme from H. polymorpha, SKI, constitutes a PTSi consensus sequence and was shown to be necessary and sufficient for peroxisome targeting (Didion and Roggenkamp 1992). CTA-negative mutants of H. polymorpha were unable to grow on methanol as the sole carbon source, but were able to utilize methanol in the presence of glucose when these mutants were grown in carbon-limited chemostat cultures (Verduyn et al. 1988). In C. boidinii, a CTA-depleted strain (ctaiA) was able to grow on methanol as the sole carbon source although its growth rate was much lower than that of the wildtype strain (Horiguchi et al. 2001). It is thought that in CTA-deficient strains H2O2 is degraded by other systems, e.g. cytochrome c peroxidase (Verduyn et al. 1988). Recently, a 2okDa peroxisomal peripheral membrane protein of C. boidinii (CbPmp2o) was identified as peroxiredoxine, an anti-oxidant enzyme necessary for methylotrophic growth (Horiguchi et al. 2001). CbPmp2o showed glutathione peroxidase activity (Figure 5.1, (7)), reactive against alkyl hydroperoxides and H2O2. R-COOOH + 2GSH -> R-COOH + GS-SG + H 2 O Interestingly, the pmp2oA strain had a more severe growth defect than the ctaiA strain. During incubation of these strains in methanol medium the ctaiA strain accumulated H2O2, while the pmpzoA strain did not. Therefore, the main function of Pmp2o is thought to be to degrade reactive oxygen species generated at the peroxisomal membrane surface, e.g., lipid hydroperoxides, rather than to degrade H2O2.
5.6 Other types of peroxisomal metabolism known in methylotrophic yeasts
5.5
Methylamine as a nitrogen source
Methylotrophic yeasts are able to utilize methylamine as a nitrogen source (Zwart and Harder 1983). Methylamine is oxidized by a peroxisomal copper/carbonyl type amine oxidase (AMO, EC. 1.4.3.6) to form formaldehyde, ammonia, and H2O2. CH 3 NH 3 + 02 + H 2 0 -> HCHO + NH 3 + H 2 O 2 AMO from H. polymorpha contains a type 2 peroxisome targeting signal (PTS2) at its N-terminus (Faber et al. 1995). Although most yeast strains can use methylamine as a nitrogen source, they are unable to use it as the sole carbon and energy source. During growth on glucose with methylamine as a nitrogen source, peroxisomal AMO and CTA are induced together with cytosolic FLD and FDH (Sakai et al. 1997, Zwart et al. 1980). DHAS is also induced during growth on glycerol with methylamine as a nitrogen source (Sakai et al. 1998). These results suggest that in methylotrophic yeasts formaldehyde generated by AMO-catalyzed reactions can be oxidized by the cytosolic dissimilatory pathway and assimilated by the dihydroxyacetone cycle. Nevertheless, methylamine cannot be utilized by any methylotrophic yeast strains as a single carbon and energy source.
5.6
Other types of peroxisomal metabolism known in methylotrophic yeasts
Since methylotrophic yeasts grow on several compounds concomitant with peroxisomal proliferation, they have been used as model organisms to study peroxisome biogenesis and metabolism (see Chapter 7). In addition to methanol and methylamine metabolism, peroxisomes in methylotrophic yeasts are known to contain acyl-CoA oxidase for (3-oxidation of fatty acids, o-amino acid oxidase for Damino acid utilization (Yurimoto et al. 2oooa), and acetylspermidine oxidase for polyamine metabolism (Nishikawa et al. 2000).
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5 Methanol metabolism
References
Anthony C (1982) The Biochemistry of Methylotrophs. Academic Press, London Cavener DR (1992) CMC oxidoreductases. A newly defined family of homologous proteins with diverse catalytic activities. J Mol Biol 223: 811-814 Gregg JM (1993) Recent advances in the expression of foreign genes in Pichia pastoris. Bio/Technology n: 905-910 Gregg JM, Madden KR, Barringer KJ, Thill GP, Stillman CA (1989) Functional characterization of the two alcohol oxidase genes from the yeast Pichia pastoris. Mol Cell Biol 9: 1316-1323 de Koning W, Gleeson MAG, Harder W, Dijkhuizen L (1987) Regulation of methanol metabolism in the yeast Hansenula polymorpha. Isolation and characterization of mutants blocked in methanol assimilatory enzymes. Arch Microbiol 147: 375-382 Didion T, Roggenkamp R (1992) Targeting signal of the peroxisomal catalase in the methylotrophic yeast Hansenula polymorpha. FEES Lett 303: 113-116 Douma AC, Veenhuis M, de Koning W, Evers M, Harder W (1985) Dihydroxyacetone synthase is localized in the peroxisomal matrix of methanol-grown Hansenula polymorpha. Arch Microbiol 143: 237-243 Egli T, van Dijken JP, Veenhuis M, Harder W, Fiechter A (1980) Methanol metabolism in yeasts: Regulation of the synthesis of catabolic enzymes. Arch Microbiol 124: 115-122 Faber KN, Keizer-Gunnink I, Pluim D, Harder W, Ab G, Veenhuis M (1995) The N-terminus of amine oxidase of Hansenula polymorpha contains a peroxisomal targeting signal. FEES Lett 357: 115-120
Gellissen G (2000) Heterologous protein production in methylotrophic yeasts. Appl Microbiol Biotechnol 54: 741-750 Gellissen G, Janowicz ZA, Merckelbach A, Piontek M, Keup P, Weydemann U, Hollenberg CP (1991) Heterologous gene expression in Hansenula polymorpha: efficient secretion of glucoamylase. Bio/ Technology 9: 291-295 Godecke S, Eckart M, Janowicz ZA, Hollenberg CP (1994) Identification of sequences responsible or transcriptional regulation of the strongly expressed methanol oxidase-encoding gene in Hansenula polymorpha. Gene 139: 35-42 Goodman JM (1985) Dihydroxyacetone synthase is an abundant constituent of the methanol-induced peroxisome of Candida boidinii. J Biol Chem 260: 7108-7113 Goodman JM, Scott CW, Donahue PN, Atherton JP (1984) Alcohol oxidase assembles post-translationally into the peroxisome of Candida boidinii. J Biol Chem 259: 8485-8493 Gould SJ, Keller GA, Schneider M, Howell SH, Garrard LJ, Goodman JM, Distel B, Tabak H, Subramani S (1990) Peroxisomal protein import is conserved between yeast, plants, insects and mammals. EMBO J 9: 85-90 Gruzman MB, Titorenko VI, Ashin W, Lusta KA, Trotsenko YA (1996) Multiple molecular forms of alcohol oxidase from the methylotrophic yeast Pichia methanolica. Biochemistry (Moscow) 61: I 537'I544 Hansen H, Didion T, Thiemann A, Veenhuis M, Roggenkamp R (1992) Targeting sequences of the two major peroxisomal
References proteins in the methylotrophic yeast Hansenula polymorpha. Mol Gen Genet 235: 269-278 Harms N, Ras }, Reijnders WNM, van Spanning RJM, Southamer AH (1996) SFormylglutathione hydrolase of Paracoccus denitrificans is homologous to human esterase D: a universal pathway for formaldehyde detoxification? J Bacteriol 178: 6296-6299 Horiguchi H, Yurimoto H, Kato N, Sakai Y (2001) Antioxidant system within yeast peroxisome: Biochemical and physiological characterization of CbPmp20 in the methylotrophic yeast Candida boidinii. } Biol Chem 276: 1427914288 Janowicz Z, Eckart M, Drewke C, Roggenkamp R, Hollenberg CP, Maat J, Ledeboer AM, Visser C, Verrips CT (1985) Cloning and characterization of the DAS gene encoding the major methanol assimilatory enzyme from the methylotrophic yeast Hansenula polymorpha. Nucleic Acids Res 13: 30433062 Kato N (i99oa) Formaldehyde dehydrogenase from methylotrophic yeasts. Methods Enzymol 188: 455-459 Kato N (i99ob) Formate dehydrogenase from methylotrophic yeasts. Methods Enzymol 188: 459-462 Kato N, Omori Y, Tani Y, Ogata K (1976) Alcohol oxidase of Kloeckera sp. and Hansenula polymorpha. Catalytic properties and subunit structures. Eur J Biochem 64: 341-350 Kato N, Sakazawa C, Nishizawa T, Tani Y, Yamada H (1980) Purification and characterization of S-formylglutathione hydrolase from a methylotrophic yeast, Kloeckera sp. No.22Oi. Biochim Biophys Acta 611: 323-332 Kato N, Higuchi T, Sakazawa C, Nishizawa T, Tani Y, Yamada H (1982) Purification and properties of a transketolase responsible for formaldehyde fixation in methanolutilizing yeast, Candida boidinii (Kloeckera sp.) No. 2201. Biochim Biophys Acta 715: 143-450 Kato N, Yoshikawa H, Tanaka K, Shimano M, Sakazawa C (1988) Dihydroxyacetone kinase from a methylotrophic yeast, Hansenula polymorpha CBS 4732: purification,
characterization and physiological role. Arch Microbiol 150: 155-159 Komeda T, Sakai Y, Kato N, Kondo K (1999) cis-Acting element for regulation of the FDHi gene in the methylotrophic yeast, Candida boidinii. Curr Genet 35: 309 Large PJ, Bamforth CW (1988) Methylotrophy and Biotechnology. Longman Scientific & Technical, Harlow Ledeboer AM, Edens L, Maat J, Visser C, Bos JW, Verrips CT, Janowicz Z, Eckart M, Roggenkamp R, Hollenberg CP (1985) Molecular cloning and characterization of a gene coding for methanol oxidase in Hansenula polymorpha. Nucleic Acids Res 13: 3063-3082 Luers GH, Advani R, Wenzel T, Subramani S (1998) The Pichia pastoris dihydroxyacetone kinase is a PTS-i containing, but cytosolic, protein that is essential for growth on methanol. Yeast 14: 759-771 Murdanoto AP, Sakai Y, Konishi T, Yasuda F, Tani Y, Kato N (1997) Purification and properties of methyl formate synthase, a mitochondrial alcohol dehydrogenase, participating in formaldehyde oxidation in methylotrophic yeasts. Appl Environ Microbiol 63: 1715-1720 Murrell JC, Dalton H (1992) Methane and Methanol Utilizers. Plenum Press, New York Nakagawa T, Mukaiyama H, Yurimoto H, Sakai Y, Kato N (1999) Alcohol oxidase hybrid oligomers formed in vivo and in vitro. Yeast 15: 1223-1230 Nakagawa T, Imanaka T, Morita M, Ishiguro K, Yurimoto H, Yamashita A, Kato N, Sakai Y (2000) Peroxisomal membrane protein Pmp47 is essential in the metabolism of middle-chain fatty acid in yeast peroxisomes and is associated with peroxisome proliferation. J Biol Chem 275: 3455-3461 Neben I, Sahm H, Kura M-R (1980) Studies on an enzyme, S-formylglutathione hydrolase, of the dissimilatory pathway of methanol in Candida boidinii. Biochim Biophys Acta 614: 81-91 Nishikawa M, Hagishita T, Yurimoto H, Kato N, Sakai Y, Hatanaka T (2000) Primary structure and expression of peroxisomal acetylspermidine oxidase in the methylotrophic yeast Candida boidinii. FEES Lett 476: 150-154
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5 Methanol metabolism Ogata K, Nishikawa H, Ohsugi M (1969) A yeast capable of utilizing methanol. Agric Biol Chem 33: 1519-1520 Ohi H, Miura M, Hiramatsu R, Ohmura T (1994) The positive and negative as-acting elements for methanol regulation in the Pichia pastoris AOX2 gene. Mol Gen Genet 243: 489-499 Pereira GG, Hollenberg CP (1996) Conserved regulation of the Hansenula polymorpha MOX promoter in Saccharomyces cerevisiae reveals insights in the transcriptional activation by Adrip. Eur J Biochem 238: 181-191 Raymond CK, Bukowski T, Holderman SD, Ching AFT, Vanaja E, Stamm MR (1998) Development of the methylotrophic yeast, Pichia methanolica, for the expression of the 65-kilodalton isoform of human glutamate decarboxylase. Yeast 14: 11-23 Roggenkamp R, Janowicz Z, Stanikowski B, Hollenberg CP (1984) Biosynthesis and regulation of the peroxisomal methanol oxidase from the methylotrophic yeast Hansenula polymorpha. Mol Gen Genet 194: 489-493 Sakai Y, Tani Y (1992) Cloning and sequencing of the alcohol oxidaseencoding gene (AODi) from the formaldehyde-producing asporogenous methylotrophic yeast, Candida boidinii 82. Gene 114: 67-73 Sakai Y, Murdanoto AP, Sembiring L, Tani Y, Kato N (1995) A novel formaldehyde oxidation pathway in methylotrophic yeasts: methylformate as a possible intermediate. FEMS Microbiol Lett 127: 229-234 Sakai Y, Saiganji A, Yurimoto H, Takabe K, Saiki H, Kato N (1996) The absence of Pmp47, a putative yeast peroxisomal transporter, causes a defect in transport and folding of a specific matrix enzyme. J Cell Biol 134: 37-51 Sakai Y, Murdanoto AP, Konishi T, Iwamatsu A, Kato N (1997) Regulation of the formate dehydrogenase gene, FDHi, in the methylotrophic yeast Candida boidinii and growth characteristics of an FDHidisrupted strain on methanol, methylamine, and choline. J Bacteriol 179: 4480-4485 Sakai Y, Nakagawa T, Shimase M, Kato N (1998) Regulation and physiological role
of the DASi gene, encoding dihydroxyacetone synthase, in the methylotrophic yeast Candida boidinii. } Bacteriol 180: 5885-5890 Sakai Y, Tani Y, Kato N (1999) Biotechnological application of cellular functions of the methylotrophic yeast. J Mol Catal B: Enzymatic 6: 161-173 Schenk G, Layfield R, Candy }M, Duggleby RG, Nixon PF (1997) Molecular evolutionary analysis of the thiaminediphosphate-dependent enzyme, transketolase. J Mol Evol 44: 552-572 Shen S, Suiter G, Jeffries TW, Gregg JM (1998) A strong nitrogen source-regulated promoter for controlled expression of foreign genes in the yeast Pichia pastoris. Gene 216: 93-102 Sibirny AA, Ubiyvovk VM, Gonchar MV, Titorenko VI, Voronovsky AY, Kapultsevich YG, Bliznik KM (1990) Reactions of direct formaldehyde oxidation to CO2 are non-essential for energy supply of yeast methylotrophic growth. Arch Microbiol 154: 566-575 Sun HW, Plapp BV (1992) Progressive sequence alignment and molecular evolution of the Zn-containing alcohol dehydrogenase family. J Mol Evol 34: 522535 Tani Y (1984) Microbiology and biochemistry of methylotrophic yeasts, in: Methylotrophs: Microbiology, Biochemistry, and Genetics (Hou CT, Ed). CRC Press, Boca Raton, FL, USA, pp. 5586 Tschopp JF, Brust PF, Gregg JM, Stillman CA, Gingeras TR (1987) Expression of the lacZ gene from two methanol-regulated promoters in Pichia pastoris. Nucleic Acids Res 15: 3859-3876 van der Klei IJ, van der Heide M, Baerends RJ, Rechinger KB, Nicolay K, Kiel JA, Veenhuis M (1998) The Hansenula polymorpha per6 mutant is affected in two adjacent genes which encode dihydroxyacetone kinase and a novel protein, Pakip, involved in peroxisome integrity. Curr Genet 34: i-n Veenhuis M, van Dijken JP, Harder W (1983) The significance of peroxisomes in the metabolism of one-carbon compounds in yeasts. Adv Microb Physiol 24: 1-82 Verduyn C, Giuseppin MLF, Scheffers WA,
References
van Dijken JP (1989) Hydrogen peroxide metabolism in yeasts. Appl Environ Microbiol 54: 2086-2090 Yurimoto H, Hasegawa T, Sakai Y, Kato N (20ooa) Physiological role of the D-amino acid oxidase gene, DAOi, in carbon and nitrogen metabolism in the methylotrophic yeast Candida boidinii. Yeast 16: 1217-1227 Yurimoto H, Komeda T, Lim CR, Nakagawa T, Kondo K, Kato N, Sakai Y (2Ooob) Regulation and evaluation of five methanol-inducible promoters in the
methylotrophic yeast Candida boidinii. Biochim Biophys Acta 1493: 56-63 Zwart KB, Harder W (1983) Regulation of the metabolism of some alkylated amines in the yeasts Candida utilis and Hansenula polymorpha. } Gen Microbiol 129: 31573169 Zwart K, Veenhuis M, van Dijken JP, Harder W (1980) Development of amine oxidase containing peroxisomes in yeasts during growth of glucose in the presence of methylamine as the nitrogen source. Arch Microbiol 126: 117-126
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6 Hansenula polymorpha: a versatile model organism in peroxisome research Ida J. van der Kiel, Marten Veenhuis
6.1 Introduction
Peroxisomes are morphologically simple cell organelles that consist of a proteinaceous matrix surrounded by a single membrane. By definition, they contain one or more H2O2-producing oxidases together with catalase as the H 2 O 2 scavenging system. In Hansenula polymorpha peroxisomes are essential for growth of cells on methanol as sole source of carbon and energy. Under these conditions the organelles harbor the key enzymes of methanol metabolism: alcohol oxidase (AO), dihydroxyacetone synthase (DHAS) and catalase (CAT). In H. polymorpha the organelles also play a role in the metabolism of ethanol and in the oxidation of several organic nitrogen sources (e.g., primary amines, D-amino acids and purins). During the last 10 years much progress has been made in the understanding of peroxisome function and homeostasis (biogenesis and degradation). In these studies methylotrophic yeasts, such as H. polymorpha, turned out to be model organisms par excellence. First, compared to other yeasts a high number of peroxisome-associated metabolic pathways exist. Secondly, peroxisome proliferation and degradation can easily be controlled by manipulating the growth conditions. Finally, yeast cells that lack intact peroxisome (peoc mutants) are viable and able to grow on rich growth media. This latter property opened the way to study the principles of peroxisome function and homeostasis in H. polymorpha at the molecular level. So far, these studies have led to the identification of 13 different H. polymorpha PEX genes involved in peroxisome biogenesis. In addition, 22 complementation groups of H. polymorpha mutants defective in selective peroxisome degradation have been isolated (pdd mutants) and used to clone the corresponding genes (PDD). Detailed analysis of the various H. polymorpha PEX and PDD genes and their translation products have provided first insight into the molecular mechanisms of peroxisome homeostasis. In this chapter we present an overview of our current knowledge on H. polymorpha peroxisomes, thereby highlighting recent achievements with this intriguing organelle.
Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Veriag GmbH, Weinheim ISBN: 3-527-30341-3
6.2 Peroxisome junction
6.2 Peroxisome function 6.2.1
Peroxisome composition
Peroxisomes are morphologically simple organelles: a single membrane encloses the matrix filled with enzyme molecules. A characteristic of the peroxisomal matrix is that it is extremely high in protein content. In methanol-grown H. polymorpha cells the high concentration of AO molecules in peroxisomes leads to the formation of AO protein crystalloids that are responsible for the typical cubic shape of the organelles (Figure 6.1). In contrast to the matrix, the peroxisomal membrane has a very low protein content. So far, no enzymes have been found to be associated with the peroxisomal membrane of H. polymorpha. Instead, all known peroxisomal membrane proteins of H. polymorpha are involved in the formation of the organelle (peroxins) or in solute transport (for a recent review, see Baerends et al. 2oooa). 6.2.2 Peroxisome-bound metabolic pathways in H. polymorpha
In methanol-grown cells, peroxisomes contain AO, CAT and DHAS, key enzymes of methylotrophic growth (see Chapter 5). AO catalyses the oxidation of methanol into formaldehyde and hydrogen peroxide. Hydrogen peroxide is decomposed by CAT into water and O2, whereas formaldehyde is either assimilated by peroxisomal DHAS or dissimilated in the cytosol to generate energy. DHAS is an enzyme of the
ifo
Fig. 6.1 (A) shows a thin section of a methanol-grown H. polymorpha WT cell, characterized by several large cubic peroxisomes (KMnO4 fixation). Immunolabeling of such cells using antibodies against catalase and gold-conjugated goat-anti-rabbit antibodies (GAR-gold) revealed that catalase is predominantly located at the periphery of peroxisomes between the AO crystalloid and the peroxisomal membrane (B; glutaraldehyde fixation).
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6 Hansenula polymorpha: a versatile model organism in peroxisome research
xylulose-5-phosphate (Xu5?) pathway. In this pathway only DHAS is peroxisomal; the other enzymes are located in the cytosol. DHAS converts formaldehyde and Xu5? into glyceraldehyde-3-phosphate (GAP) and dihydroxyacetone (DHA). Both molecules are released into the cytosol and converted by cytosolic enzymes (phosphorylation of DHA by DHA kinase and rearrangement reactions) into i Xu5? molecule and 1/3 GAP (see Chapter 5). The Xu5? molecule is available for another cycle of assimilation reactions, whereas GAP is used for the biosynthesis of cell constituents. Only a portion of the formaldehyde generated from methanol is assimilated. The remaining is released into the cytosol, where it is dissimilated to CO2 by two NAD-dependent dehydrogenases. Due to the high glutathione concentration in the cytosol, formaldehyde is immediately converted into its hemimercaptal - S-hydroxymethyl glutathione - upon release in the cytosol. This compound is the substrate for formaldehyde dehydrogenase and converted into Sformylglutathione, which is further oxidized into CO2 by formate dehydrogenase (Van Dijken et al. 1976; see Chapter 5). These reactions yield 2 NADH molecules, which can be used to generate ATP in mitochondria via oxidative phosphorylation. The advantage of compartmentalizing specific enzymes in peroxisomes is apparently to control specific metabolic routes. Segregation of sets of enzymes in peroxisomes can favor certain pathways and prevent others that are undesirable. This is particularly evident in the case of methanol metabolism. At high energy levels, no formaldehyde generated from methanol will leave the organelle, but will be handed over to peroxisomal DHAS for assimilation. However, the Xu5? pathway requires energy, and thus when the utilization equilibrium of formaldehyde is not appropriate for dissimilation, the intracellular ATP levels will drop. This causes a reduction in the formation of Xu5P, and therefore in assimilation, allowing the formaldehyde to enter the cytosol for dissimilation. This then enhances the ATP levels, and assimilation occurs. In this way the peroxisomes play a role in the proper balancing of the formaldehyde over the assimilatory and dissimilatory pathways according to the needs of the cell. In H. polymorpha several other hydrogen peroxide-producing oxidases have been detected in peroxisomes (Table 6.1), which are predominantly involved in the oxidation of organic nitrogen sources. Peroxisomal oxidases are invariably accompanied by CAT to detoxify the hydrogen peroxide produced. Co-localization of oxidases with peroxisomal CAT prevents escape of hydrogen peroxide from the organelle into the cytosol. Hence, other hydrogen peroxide consuming enzymes that are present at other locations in the cell cannot compete with peroxisomal catalase for the substrate, even when their affinities for the hydrogen peroxide substrate are much higher. Thus, compartmentalization of specific enzymes in peroxisomes can fully prevent certain undesired reactions. The significance of peroxisomal CAT is illustrated by the fact that CAT-deficient mutants of H. polymorpha are unable to grow on methanol. In these mutants the hydrogen peroxide produced is most likely decomposed by mitochondrial cytochrome c peroxidase (CCP; Verduyn et al. 1988). This alternative pathway is energetically unfavorable, because oxidation of cytochrome c by CCP interferes with mitochondrial oxidative phosphorylation leading to a reduction in the ATP
6.2 Peroxisome function Tab. 6.1 Growth substrates that induce peroxisomes in H. polymorpha, the corresponding peroxisomal key enzymes and volume fraction Growth Substrates \ Ethanol Methanol D-amino acids D-amino acids Primary amines Uric acid a) b)
c)
C/N
|c C C N N N
Key Enzymes 1 Isocitrate lyase, malate synthase Alcohol oxidase, catalase, dihydroxy acetone synthase D-amino acid oxidase, catalase D-amino acid oxidase, catalase Amine oxidase, catalase Urate oxidase, catalase
1
Volume Fraction10 1.0 19.9 (48.4C)
1
4.1 1.4 2.3 1.8
Growth substrate used as carbon (C) or nitrogen (N) source. The volume fraction was determined using thin sections of exponentially grown batch cells. The values are expressed as percentage of the cytoplasmic volume. Cells taken from a methanol-limited chemostat, D 0.03 h"1.
yield. During methanol metabolism in a CAT-deficient strain this loss of energy is obviously very high, since for each methanol molecule oxidized one molecule of hydrogen peroxide is produced, whereas maximally only 2 NADH molecules can be generated. This explains why CAT-deficient H. polymorpha strains fail to grow on methanol: the energy requirement for hydrogen peroxide decomposition apparently exceeds the energy yielded from dissimilation. It is, therefore, essential for the cells that H 2 O 2 is decomposed by catalase in order to prevent undesired energy loss. This can efficiently be solved by compartmentalizing H 2 O 2 generation and decomposition in one organelle as in H. polymorpha peroxisomes. The opposite process, that the H 2 O 2 metabolism would occur in the cytosol, is much less efficient. This is because the affinity of CCP for H 2 O 2 is much higher than that of CAT (at least 3 orders of magnitude higher), moreover CCP is extremely active. Thus, upon entering the cytosol H 2 O 2 would be metabolized via CCP at the expense of energy instead of being decomposed by catalase. This is a major reason why pex mutants cannot grow on methanol (see below). Immunolabeling experiments revealed that in methanol-grown H. polymorpha CAT is positioned at the periphery of the organelle around the AO crystalloid (Figure 6.1). In this way CAT forms a perfect barrier to prevent leakage of any hydrogen peroxide produced by AO into the cytosol. Finally, enzymes of the glyoxylate pathway (malate synthase and isocitrate lyase) have been shown to be located in peroxisomes of H. polymorpha. The reason for segregation of these enzymes in peroxisomes is so far unknown. 6.2.3 Peroxisome-bound metabolic pathways in H. polymorpha pex mutants
In H. polymorpha mutants lacking intact peroxisomes (pex mutants), peroxisomal enzymes normally are synthesized and active, but mislocated in the cytosol (Suiter
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6 Hansenula polymorpha: a versatile model organism in peroxisome research
et al. 1990; Van der Kiel et al. 19913). As argued above, when such mutants are exposed to methanol, cytosolic CAT cannot compete with CCP for the H 2 O 2 substrate, which is now formed by AO in the cytosol leading to serious energetical disadvantages (Van der Klei et al. I99ib). A second disadvantage is that partitioning of formaldehyde over the assimilatory and dissimilatory pathways is disturbed in pex mutants, because the bulk of the cytosolically produced formaldehyde is converted into S-hydroxymethyl glutathione, which is not a substrate for DHAS (Bystrykh et al. 1981) and, therefore, also assimilation is severely hampered in these cells. However, pex mutants are capable to utilize organic nitrogen sources that are metabolized by peroxisomal oxidases. This can be explained by the fact that under these conditions the rate of hydrogen peroxide production is relatively low compared to methylotrophic growth conditions (Suiter et al. 1990). In addition, H. polymorpha pex mutants can still grow on ethanol, although the growth yield is somewhat reduced compared to WT cells (Suiter et al. 1991). Hence, compartmentalization of the glyoxylate cycle enzymes in peroxisomes is not essential for C2 metabolism.
6.3 Peroxisome biogenesis and degradation
In H. polymorpha highest peroxisome induction is obtained in cells grown in methanol-limited continuous cultures at low dilution rates. Under these conditions peroxisomes may occupy up to 80 % of the cytoplasmic volume. In contrast, peroxisome proliferation is fully repressed in glucose-grown cells. In such cells characteristically only one or few small peroxisomes are present. Upon transfer of glucose-grown cells into fresh methanol-containing media, these small organelles serve as targets for the newly induced peroxisomal enzymes synthesized in the cytosol. Together with the incorporation of lipids and insertion of membrane proteins into the organellar membrane, this results in growth of the peroxisomes. This process proceeds until the organelles have reached a certain, mature size. Subsequently, one or a few new organelles are formed by fission from the mature organelle, which in turn start to grow in prolonged cultivation. Apparently, these small organelles have "inherited" the capacity to grow from the mature parent organelle, which is no longer capable of growing, but remains able to metabolize the growth substrate (as "enzyme bag"). After multiple rounds of growth and division, cells may contain over 20 microbodies. When methanol-grown cells are exposed to conditions in which the peroxisomal enzymes have become redundant for growth (e.g., upon a shift to glucose- or ethanol-containing media), the organelles are selectively degraded. Ultrastructural studies in H. polymorpha have suggested that the mature organelles are degraded in particular, leaving at least one small, import-competent organelle per cell unaffected. In this way the cells can rapidly adapt to new growth substrates that may require other peroxisomal enzymes.
6.4 Genes involved in peroxisome biogenesis (PEX genes)
6.4 Genes involved in peroxisome biogenesis (PEX genes)
Molecular genetic approaches to identify proteins involved in peroxisome biogenesis (peroxins) came within reach in the early 19905 with the discovery that mutations causing defects in peroxisome biogenesis or function (pex mutants: see Distel et al. 1996 for the unified nomenclature) are not lethal in yeasts. H. polymorpha pex mutants (originally designated per mutants) were first described in 1991 (Gregg et al. 1990). These mutants were selected from methanol utilizationdeficient strains (Mut~) that were obtained upon chemical mutagenesis. The Mut~phenotype of pex mutants enabled cloning of the corresponding genes (PEX genes) by transforming the mutants with DNA libraries followed by selection of those transformants that have re-gained the capacity to grow on methanol. More recently, an alternative approach was used, which is based on gene tagging by RAndom integration of Linear DNA Fragments (RALF; Van Dijk et al. 2ooia). Also, H. polymorpha orthologs of PEX genes first identified in other organisms have been cloned by PCR approaches using primers that were based on conserved regions in these genes. These approaches have resulted now in the identification of 13 H. polymorpha PEX genes (PEXi, 2, 3, 4, 5, 6, 7, 8, w, 12, 13, 14 and 19; see Table 6.2). The majority of the known H. polymorpha PEX genes encode proteins that function in matrix protein import. Cells lacking these peroxins still synthesize peroxisomal membranes, but are hampered in matrix protein import (Figure 6.2). Other peroxins are thought to be essential for the formation of the peroxisomal
Fig. 6.2 shows a H. polymorpha pex mutant that is specifically defective in matrix protein import. In these cells the matrix proteins are mislocated in the cytosol (* cytosolic AO crystalloid). PMPs are correctly inserted in peroxisomal membrane remnants ("ghosts"; immunolabeling using antiPexiop antibodies and GAR-gold, glutaraldehyde fixation). P : peroxisome, N : nucleus. The bar represents 0.5 urn.
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6 Hansenula polymorpha: a versatile model organism in peroxisome research
Tab. 6.2
Hansenula polymorpha peroxins
Calculated MW [kDa]
Peroxin I Pexlp
Pex2p
Pex3p
Pex4p
PexSp
PexGp Pex7p
PexSp
PexlOp
Pexl2p
Pexl3p
Pexl4p
Pexl9p
I
Properties/Function of the Protein
Reference
I Kiel et al. 1999a I 120 I AAA protein, involved in peroxisomal matrix protein import not published 42 Integral peroxisomal membrane protein, zinc-finger protein, involved in matrix protein import Baerends et al. 1996 52 Associated with outer surface of peroxisomal membrane, involved in biogenesis and maintenance of the peroxisomal membrane 22 Ubiquitin conjugating enzyme, Van der Kiel et al. 1998 involved in PTS1 protein import, important for recycling of PexSp from the matrix to the cytosol 64 TPR domains in carboxy terminal Van der Klei et al. 1995 half of the protein, involved in PTS1 protein import, receptor of PTS1 signals 126 AAA protein, involved in matrix Kiel et al. 1999a protein import 41 WD-40 protein, involved in PTS2 not published import, receptor of the PTS2 signal 74 Contains PTS1 and PTS2, Waterham et al. 1994 associated with inner surface of the peroxisomal membrane, involved in matrix protein import 34 Integral peroxisomal membrane Tan et al. 1995 protein, zinc-finger protein, involved in matrix protein import 46 Integral peroxisomal membrane not published protein, zinc-finger protein, involved in matrix protein import 42 SH3 protein, associated with not published peroxisomal membrane, involved in matrix protein import 39 Associated with peroxisomal Komori et al. 1997, 1999 membrane, involved in matrix protein import, can be phosphorylated 33 Farnesylated peroxisomal not published membrane protein, involved in membrane biogenesis
6.4 Genes involved in peroxisome biogenesis (PEX genes)
membrane (Pex3p and Pexi9p). This view is primarily based on the finding that cells unable to produce these proteins completely lack peroxisomal membrane structures (also designated "ghost" or peroxisomal membrane remnants). 6.4.1
Matrix protein import
Peroxisomal matrix proteins are encoded by nuclear genes and synthesized in the cytosol on free ribosomes (Lazarow and Fujiki 1985). So far, two Peroxisomal Targeting Signals (PTSi and PTS2) have been characterized that are essential for sorting the protein to the proper organelle (De Hoop and AB 1992; Rachubinski and Subramani 1995; Subramani 1998). The PTSi signal consists of a tripeptide that is located at the extreme C-terminus of the protein and is the most common peroxisomal targeting signal. The consensus sequence is -S-K-L, but various (conserved) variants of this motif are allowed (Gould et al. 1989). H. polymorpha PTSi proteins include AO, CAT and DHAS. The PTS2 is present at the N-terminus
Out
Fig. 6.3 Schematic representation of the extended shuttle model of the PTSi protein import pathway. A PTSi protein is synthesized in the cytosol on free ribosomes. Next, the PTSi at the extreme carboxy terminus is recognized by its receptor, Pex5p, in the cytosol. The receptor-cargo complex is recognized by a putative docking complex at the peroxisomal membrane containing Pexi3p, Pexi4p and Pexiyp. Subsequently, the Pex5pcargo complex is translocated across the peroxisomal membrane, a process that may involve the Zn-binding proteins Pex2p, Pexiop and Pexi2p. In the organellar matrix the cargo dissociates from Pex5p, which may be mediated by PexSp. Subsequently, Pexsp is exported to the cytosol. The peroxins Pex4p, Pex22p and possibly also Pexip and Pex6p are predicted to be required for efficient recycling.
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6 Hansenula polymorpha: a versatile model organism in peroxisome research
of peroxisomal matrix proteins and consists of a nonapeptide with the consensus (R/K)-(L/V/I)-X 5 -(H/Q)-(L/A). In H. polymorpha the two PTS2-containing enzymes known so far are amine oxidase (AMO; Faber et al. 1995) and thiolase. Remarkably, the peroxin PexSp contains both, a PTSi and a PTS2 (Waterham et al. 1994). H. polymorpha malate synthase contains neither a PTSi nor a PTS2 (Bruinenberg et al. 1990). However, this enzyme most likely uses the PTSi import pathway, as import is prohibited in pex$ mutant cells that are specifically affected in PTSi protein import (Van der Klei et al. 1995). PEXj and PEXj encode the PTSi and PTS2 receptors, respectively. In the absence of Pex5p PTSi protein import is fully impaired, whereas import of PTS2-proteins is unaffected. The opposite is observed in cells lacking a functional Pexyp. 6.4.2
PTSI protein import
In WT H. polymorpha Pex5p is predominantly located in the cytosol, whereas a minor fraction is associated with peroxisomes. The amount of Pex5p associated with the peroxisomal membrane is invariably low and below the limit of detection (Van der Klei et al. 1995, 1998). However both, immunolabeling and cell fractionation experiments indicated that a portion of Pex5p is located in the peroxisomal matrix. Based on this observation we proposed that Pex5p functions as a cycling receptor between the cytosol and the peroxisomal matrix (Figure 6.3; Van der Klei and Veenhuis 1996). According to this model, the first step in matrix protein import is the recruitment of a newly synthesized PTSi protein by Pex5p in the cytosol. Subsequently, the Pex5p-cargo complex is recognized by a Pex5p docking site at the peroxisomal membrane, followed by translocation of the receptor/cargo complex into the peroxisomal matrix. Upon dissociation of the PTSi protein, Pex5p is recycled to the cytosol where it can mediate another round of import (Figure 6.3). Based on studies in other yeasts it has been proposed that Pexi3p, Pexi4p and Pexiyp (reviewed by Erdmann et al. 1997; Subramani 1998) are components of this docking site. Although it is generally accepted that Pexi4p plays a central role in Pex5p docking, H. polymorpha Pexi4p (Komori et al. 1997) is not essential for this process, because overproduction of Pex5p causes an almost complete restoration of the PTSi import defect in a H. polymorpha PEXi4 null mutant (Salomons et al. 2000). Under these conditions Pex5p accumulated at the outer surface of the peroxisomal membrane. Hence, HpPexi4p is not essential for Pex5p docking, but may function at a later stage, where it is important for the efficiency of the import process. Two-hybrid studies have led to the identification of several additional interacting partners of Pex5p, including PexSp, Pexiop and Pexi2p. This suggests that the function of Pex5p involves a cascade of protein binding and dissociation events. Pex2p, Pexiop and Pexi2p are integral membrane proteins that contain Zn-binding domains. These three proteins may form a complex in the peroxisomal membrane that functions in a stage after initial docking of Pex5p. PexSp is a peroxisomal
6.4 Genes involved in peroxisome biogenesis (PEX genes)
matrix protein associated with the inner surface of the peroxisomal membrane and essential for matrix protein import (Waterham et al. 1994). The finding that in S. cerevisiae PexSp physically interacts with Pex5p supports the view that Pex5p enters the peroxisomal matrix, according to the extended shuttle model (Rehling et al. 2000). Recent data on mammalian Pex5p have experimentally proven that Pex5p is imported into the matrix and subsequently exported again to the cytosol during one round of matrix protein import (Dammai and Subramani, 2001). Consequently, peroxisomes also must contain a protein export site. Whether this export site is the same as the import site (as for instance in the ER) remains to be elucidated. HpPex4p is an ubiquitin-conjugating enzyme that functions at a very late stage in Pex5p-dependent import, namely export/recycling of Pex5p (Van der Klei et al. 1998). Because the target protein of this enzyme is not known yet, the molecular function of Pex4p is still an enigma. In H. polymorpha Pex4p is specifically involved in PTSi import, but interaction between Pex4p and Pex5p has not been demonstrated yet. In the related yeast Pichia pastoris Pex4p was shown to be associated with the peroxisomal membrane, bound to the integral peroxisomal membrane protein Pex22p and facing the cytosol (Roller et al. 1999). We have not been able so far to confirm this location in H. polymorpha due to the very low levels of Pex4p in this organism. Like in H. polymorpha Apexi^, overproduction of Pex5p suppresses the PTSi import defect in H. polymorpha kpex^. In contrast to Apexi^ cells that overproduce Pex5p, in Apex^ cells increased amounts of Pex5p are found at the inner surface of the peroxisomal membrane (Van der Klei et al. 1998). Consistent with the extended Pex5p recycling model, this observation implies that export of Pex5p to the cytosol is blocked in the absence of Pex4p. As a result, Pex5p accumulates inside peroxisomes thereby exhausting the cytosolic Pex5p pool. By overproduction of Pex5p the cytosolic pool can be replenished, explaining the restoration of the PTSi protein import defect in pex^. null mutants upon over expression of P-HXj. Studies in P. pastoris suggested that in addition to Pex4p and Pex22p, Pexip and Pex6p are also involved in terminal steps of the PTSi protein import pathway that occur after the actual matrix protein import process. Hence, these two peroxins may play a role in Pex5p recycling (Collins et al. 2000). 6.4.3
PTS2 import
The intracellular location of the PTS2 receptor, Pexyp, has been described for several organisms, but is still controversial. For H. polymorpha the location of Pexyp has not been established yet, mainly because the expression levels of PEXj in H. polymorpha are extremely low (Koek, Van der Klei and Veenhuis, unpublished results). It is tempting to speculate that Pexyp follows a similar pathway to that of Pex5p, thus explaining why the protein in other organisms has been reported to be located in the cytosol, on the peroxisomal membrane or in the organellar matrix. Two-hybrid studies in bakers' yeast revealed that Pexyp, like Pex5p, interacts with Pexi3p and Pexi4p. Hence, the initial steps in PTS2 protein import may be the
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6 Hansenula polymorpha: a versatile model organism in peroxisome research
same as in PTSi import. We found that in H. polymorpha, Pex4p is not required for PTS2 import, because pe*4 mutants still import AMO (van der Klei et al. 1998). Hence, recycling of Pexyp, if any, may occur by another mechanism. 6.5
Assembly of octameric, FAD-containing AO
At present little is known about the mechanisms involved in the assembly of peroxisomal matrix proteins. In fact, even the subcellular site where matrix proteins are assembled and oligomerized is still a matter of debate. It has been suggested that some proteins can be imported as oligomers into the peroxisomal matrix, but it is unlikely that this represents a general mechanism. The import and assembly of H. polymorpha AO has been a topic of investigation for over 10 years. AO is an oligomeric enzyme that consists of 8 identical subunits each of which contains an FAD molecule non-covalently bound. In WT cells the activity of this enzyme is confined to the peroxisomal matrix, and several lines of evidence have lent support to the view that octamerization occurs inside the organelle upon import of inactive monomers. Two independent approaches have revealed that octameric AO cannot be transported across the peroxisomal membrane. First, upon introduction of octameric AO into the cytosol of H. polymorpha protoplasts by fusion with AOcontaining liposomes, the protein remained located in the cytosol (Douma et al. 1990). Also, experiments using a temperature-sensitive pex mutant revealed that octameric AO, which had accumulated at the restrictive temperature in the cytosol, was not imported into peroxisomes formed upon a shift of cells to permissive temperatures (Waterham et al. 1993). Recently, we showed that under the same experimental conditions enzymatically active dimeric DHAS and folded, monomeric green fluorescent protein (GFP) that contained a PTSi are imported into newly formed organelles upon the shift to permissive temperatures (Faber, Van Dijk and Veenhuis, unpublished results). This indicated that, like other organisms, H. polymorpha peroxisomes are capable of taking up folded, oligomeric proteins. Possibly, import of AO is an exception to the rule of oligomeric protein import. As a consequence, specific proteins may be involved in AO import/activation that are not required for other H. polymorpha PTSi proteins. In order to identify such proteins H. polymorpha mutants were isolated that are specifically blocked in AO import and activation. The predicted phenotype of these mutants is a strongly reduced AO activity (screened by an AO activity plate assay) and, as a consequence, failure to grow on methanol. Complementation analysis of the mutants available so far revealed the presence of 10 different complementation groups (Van Dijk et al. 2OOib). One of these groups - complementation group 3 - has been studied in detail. These mutants are characterized by accumulation of inactive, FAD-lacking monomeric AO in the cytosol while other peroxisomal matrix proteins are normally activated and sorted to peroxisomes. The gene that functionally complemented the AO assembly-defective phenotype in this group of mutants encodes the enzyme pyruvate carboxylase (HpPycip). Pyruvate carboxylase is an anapleurotic enzyme,
6.6 Biogenesis of the peroxisomal membrane
localized in the cytosol, replenishing the tricarboxylic acid cycle by the synthesis of oxaloacetate. Mutational analyses revealed that it was not HpPycip enzyme activity, but protein that was essential to functionally complement the AO assembly defect in these mutants. Hence, HpPycip fulfils a dual role in the organism. Besides its wellcharacterized metabolic function as an anapleurotic enzyme, the protein plays a specific role in AO sorting and assembly. Because FAD-lacking AO monomers accumulate in the absence of HpPyci, it is tempting to speculate that HpPycip mediates FAD binding to AO monomers in the cytosol. Previous studies using a H. polymorpha riboflavin-deficient mutant (rifi) already indicated that FAD binding is essential to allow efficient import and octamerization of AO (Evers et al. 1994,1996). Most likely newly synthesized AO monomers first bind FAD, mediated by HpPycip, followed by binding to the PTSi receptor Pex5p. Then, FAD-containing monomers bound to Pex5p are taken up by the organelles followed by dissociation of Pex5p. This allows the FAD-containing monomers to oligomerize into the enzymatically active octamers - a process that most likely occurs spontaneously (Evers et al. 1995). 6.6 Biogenesis of the peroxisomal membrane
When peroxisomes take up newly synthesised matrix proteins from the cytosol, the organellar membrane increases in size by recruitment of lipids and insertion of membrane proteins (for review, see Baerends et al. 200oa). At present, it has not been established what the origin of these lipids is and how they are incorporated into the peroxisomal membrane. Several experimental data point towards a role of the endoplasmatic reticulum (ER) and ER-derived vesicles in the biogenesis of the peroxisomal membrane (reviewed by Titorenko and Rachubinski 19983; Kunau and Erdmann 1998). Other studies, however, suggest that these data could also be interpreted in another way or that the observations made are not generally valid for all eukaryotes. For instance, studies in Saccharomyces cerevisiae revealed that upon overexpression of the peroxisomal membrane protein (PMP) Pexi5p, this protein was transported to the ER where it became O-glycosylated (Elgersma et al. 1997). Also, the PMPs Pex2p and PexiGp in Yarrowia lipolytica were implicated to reach the peroxisomal membrane via the ER (Titorenko and Rachubinski 1998^. Hence, these proteins may first be targeted to the ER followed by incorporation into vesicles that fuse with the peroxisomal membrane. However, for Pexi5p this interpretation has been questioned, because the ER membrane may readily take up hydrophobic proteins that do not reach their normal membrane in time, i.e., under artificial conditions in mutant strains (Stroobants et al. 1999). Another indication that vesicle trafficking and fusion may play a role in peroxisome biogenesis was the finding that two interacting peroxins, Pexip and Pex6p - both members of the AAA-protein family - are homologous to proteins involved in vesicle fusion processes (e.g., S. cerevisiae SeciSp and Cdoj-Sp; for a review, see Confalonieri and Duguet 1995). Indeed, in P. pastoris these proteins were found to be associated with small membranous structures which were not peroxisomal in nature (Faber et
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6 Hansenula polymorpha: a versatile model organism in peroxisome research
al. 1998). Moreover, using an in vitro approach Titorenko and Rachubinski (2000) showed that Pexip and Pex6p can mediate fusion of small peroxisomal membrane structures. In contrast, however, we found that in H. polymorpha Pexip and Pex6p are associated with the outer surface of the peroxisomal membrane and most likely involved in matrix protein import (Kiel et al. 19993). This view is based on the finding that H. polymorpha strains lacking either Pexip or Pex6p still contain peroxisomal membranes in which PMPs are normally inserted. Moreover, these structures contain a low amount of matrix proteins, which suggests that Pexip and Pex6p are not essential for the initial steps of the import pathway. This finding is in line with recent observations in P. pastoris that suggest that Pexip and Pex6p act at a late stage in PTSi matrix protein import, possibly in recycling of Pex5p (Collins et al. 2000). In H. polymorpha we observed that brefeldin A, a fungal toxin that interferes with the formation of ER-derived vesicles, inhibited peroxisome biogenesis and resulted in the accumulation of peroxisomal membrane and matrix proteins at the ER (Salomons et al. 1997). However, in humans no indications were obtained that BFA or other inhibitors of CO PI or COPII -dependent vesicle formation affected peroxisome formation (South et al. 2000). In summary, the possible role of the ER in the formation of the peroxisomal membrane is still a matter of debate and requires additional investigations. In contrast to the wealth of indirect evidence that has been reported so far, firm and direct proof for the role of the ER in peroxisome formation is still lacking. Sorting of peroxisomal membrane proteins does not proceed via the PTSi/PTS2 system, because yeast mutants defective in PTSi and/or PTS2 import still properly sort and insert PMPs in the peroxisomal membrane (Figure 6.1). In some cases, the targeting sequence for peroxisomal membrane proteins (called mPTSs) have been identified (reviewed by Baerends et al. 2000). However, unlike PTSi and PTS2, no common amino acid motifs or features could yet be identified in the mPTSs. In H. polymorpha we have analyzed the targeting information of Pex}p in detail. This PMP contains targeting information in the first 37 amino terminal amino acids (Baerends et al. 1996). This region contains a stretch of positively charged amino acids (amino acids 11-15: RHKKK) that is conserved in all known Pex3ps. Mutational analysis revealed that changing i of the 4 basic amino acids in an uncharged or negatively charged one had no effect on Pex3p targeting. Only after changing 3 or 4 of these residues into negative ones sorting of Pex3p was strongly affected (Baerends et al. 2Ooob). Recently, it has been shown that certain PMPs have 2 independent, non-overlapping sets of targeting information, which both are sufficient for insertion into the peroxisomal membrane (Jones et al. 2001). Hence, it cannot be excluded that HpPex3p also contains additional peroxisomal targeting information. 6.7 Peroxisome degradation
In H. polymorpha peroxisome degradation has been observed under various experimental conditions. Upon nitrogen starvation peroxisomes are degraded by
6.7 Peroxisome degradation
autophagy together with other cytoplasmic constituents (Bellu et al. 2001). During this process the vacuolar membrane engulfs major portions of the cytoplasm followed by homotypic fusion of the vacuolar membrane, resulting in the uptake of cytoplasmic components in the vacuolar lumen followed by degradation of the organellar components by vacuolar hydrolases (generally designated as microautophagy). Upon a shift of methanol-grown cells to glucose or ethanol media the organelles are degraded by a selective mechanism (designated macroautophagy). This type of degradation has also been observed in H. polymorpha when the organelles became non-functional due to treatment of whole cells with specific chemicals that affect peroxisomal matrix enzymes (e.g., KCN, Van der Klei et al. 1989) or the peroxisomal membrane (e.g., toxin T-5I4; Sepulveda Saavedra et al. 1992). In H. polymorpha macroautophagy of peroxisomes includes three distinct steps: • sequestration of the organelle to be degraded by, most likely, endoplasmic reticulum (ER)-derived membranous layers, • heterotypic fusion of the sequestered compartment with the vacuole, and • degradation of the organellar contents in the autophagic vacuole. H. polymorpha mutants defective in this process have been isolated using a colony assay based on the visualization of the activity of AO. Mutagenized cells were first grown on methanol plates to induce AO-containing peroxisomes, followed by transfer of the colonies to glucose or ethanol containing plates. Upon incubation for a few hours, the colonies were overlayed with an AO activity assay mixture, which allowed to select those mutants that had maintained AO activity and hence were potential peroxisome degradation-deficient mutants (Titorenko et al. 1995). H. polymorpha mutants defective in peroxisome degradation are designated pdd (peroxisome degradation-deficient; Titorenko et al. 1995). The H. polymorpha pdd mutants so far isolated belong to 22 complementation groups (Titorenko et al. 1995; Bellu, Kiel, Komduur, Monastyrska and Veenhuis, unpublished results). Electron microscopical analysis revealed that all of them are blocked at initial stages of the peroxisome degradation process (sequestration of the individual organelles or fusion of enwrapped organelles with the vacuole). Moreover, the pdd mutants are invariably defective in both glucose- and ethanol-induced degradation, suggesting that these morphologically similar processes required the same genes. Although in H. polymorpha peroxisomes are degraded by a mechanistically distinct process under nitrogen starvation, some of the isolated pdd mutants are defective in this process as well (e.g., pddi, pddy). This suggests that both processes have overlapping steps requiring common genes. However, other pdd mutants are specifically defective in glucose- and ethanol-induced selective peroxisome degradation and still capable of degrading these organelles under nitrogen starvation conditions (Bellu et al. 2001). Hence, glucose- or ethanol-induced peroxisome degradation in H. polymorpha requires unique genes that do not play a role in general, non-selective autophagy in this organism. Mutants belonging to the pddi complementation group are affected in an early stage of selective peroxisome degradation, namely the sequestration of individual organelles from the cytosol by membrane layers. The corresponding gene, PDDi
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6 Hansenula polymorpha: a versatile model organism in peroxisome research
(Kiel et al. 1999!)), is similar to S. cerevisiae VPSj4 - a gene involved in vacuolar protein sorting and endocytosis. The translation product of VPS34 is activated by another Vps-protein, Vpsi5. Also VPSi5 has recently been shown to be essential for peroxisome degradation in P. pastoris (Stasyk et al. 1999) and H. polymorpha (Kiel and Veenhuis, unpublished results). Vpsi5p belongs to the serine/threonine family of protein kinases, whereas VpS34p is a phosphatidyl inositol 3-kinase. Vpsi5p recruits Vps34p to a yet unidentified intracellular membrane, where Vps34p phosphorylates phosphatidyl inositol (Ptdlns) molecules in the lipid bilayer. As a result patches of PtdIns-3-P are formed, which are thought to be important for binding of other effector molecules. Because VpS34p/Vpsi5p play a role in several vacuolar delivery pathways (vacuolar protein sorting, endocytosis, selective peroxisome degradation) different effector molecules may be involved in these processes. PDDy encodes a gene homologous to APGi, a gene implicated in autophagy in S. cerevisiae (Komduur and Veenhuis, unpublished results; Matsuura et al., 1997). The other PDD genes are currently being investigated.
Acknowledgement
I.J. van der Klei holds a PIONIER grant (NWO).
References
References
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References
Salomons FA, Kiel JAKW, Faber KN, Veenhuis M, Van der Kiel IJ (2000) Overproduction of Pex5p stimulates import of alcohol oxidase and dihydroxyacetone synthase in a Hansenula polymorpha pexi4 null mutant. J Biol Chem 275: 12603-12611 Sapulveda Saveedra J, Van der Klei IJ, Keizer I, Lopez AP, Harder W, Veenhuis M (1992) Studies on the effect of toxin 1-514 on the integrity of peroxisomes in methylotrophic yeasts. FEMS Microbiol Lett 91: 207-212 South ST, Sacksteder KA, Li X, Liu Y, Gould SJ (2000) Inhibitors of COPI and COPII do not block P£Xj mediated peroxisome synthesis. J Cell Biol 149: 1345-1359 Stasyk OV, Van der Klei IJ, Bellu AR, Kiel JAKW, Cregg JM, Veenhuis M (1999) A Pichia pastoris VPS 15 homologue is required in selective peroxisome autophagy. Curr Genet 36: 262-269 Stroobants AK, Hettema EH, Van den Berg M, Tabak HF (1999) Enlargement of the endoplasmic reticulum membrane in Saccharomyces cerevisiae is not necessarily linked to the unfolded protein response via Ireip. FEES Lett 453: 210-214 Subramani S (1998) Components involved in peroxisome import, biogenesis, proliferation, turnover, and movement. Physiol Rev 78: 171-188 Suiter GJ, Van der Klei IJ, Harder W, Veenhuis M (1990) Assembly of amine oxidase and o-amino acid oxidase in the cytosol of peroxisome-deficient mutants of the yeast Hansenula polymorpha during growth of cells on glucose in the presence of primary amines or o-alanine as the sole nitrogen source. Yeast 6: 501-509 Suiter GJ., Van der Klei IJ, Schanstra J, Harder W, Veenhuis M (1991) Ethanolmetabolism in a peroxisome-deficient mutant of the yeast Hansenula polymorpha. FEMS Microbiol Lett 82: 297-302 Tan X, Waterham HR, Veenhuis M, Cregg JM (1995) The Hansenula polymorpha PER8 gene encodes a novel peroxisomal integral membrane protein involved in proliferation. J Cell Biol 128: 307-319 Titorenko VI, Rachubinski RA(i998a) The endoplasmic reticulum plays an essential role in peroxisome biogenesis. Trends Biochem Sci 23: 231-233
Titorenko VI, Rachubinski RA Mutants of the yeast Yarrowia lipolytica defective in protein exit from the endoplasmic reticulum are also defective in peroxisome biogenesis. Mol Cell Biol. 18: 2789-2803. Titorenko VI, Rachubinski RA (2000) Peroxisomal membrane fusion requires two AAA family ATPases, Pexip and Pex6p. J Cell Biol 150: 881-886 Titorenko VI, Keizer I, Harder W, Veenhuis M (1995) Isolation of peroxisomedegradation-deficient mutants of Hansenula polymorpha. J Bacteriol 177: 357363 Van der Klei IJ, Veenhuis M (1996) A molecular analysis of peroxisome biogenesis and function in Hansenula polymorpha: a structural and functional analysis. Ann NY Acad Sci 9-2: 47-59 Van der Klei IJ, Veenhuis M (1997) Yeast peroxisomes: function and biogenesis of a versatile cell organelle. Trends Microbiol 5: 502-509 Van der Klei IJ, Veenhuis M, Nicolay K, Harder W (1989) In vivo inactivation of peroxisomal alcohol oxidase in Hansenula polymorpha by KCN is an irreversible process. Arch Microbiol 151: 26-33 Van der Klei IJ, Suiter GJ, Harder W, Veenhuis M (i99ia) Assembly of alcohol oxidase in the cytosol of a peroxisomedeficient mutant of Hansenula polymorpha: properties of the protein and architecture of the crystals. Yeast 7: 15-24 Van der Klei IJ, Harder W, Veenhuis M (i99ib) Methanol metabolism in a peroxisome-deficient mutant of Hansenula polymorpha: a physiological study. Arch Microbiol 156: 15-23 Van der Klei IJ, Hilbrands RE, Swaving GJ, Waterham HR, Vrieling EG, Titorenko VI, Cregg JM, Harder W, Veenhuis M (1995) The Hansenula polymorpha PER3 gene is essential for the import of PTSi proteins into the peroxisomal matrix. J Biol Chem 270: 17229-17236 Van der Klei IJ, Hilbrands RE, Kiel JAKW, Rasmussen SW, Cregg JM, Veenhuis M (1998) The ubiquitin-conjugating enzyme Pex4p of Hansenula polymorpha is required for efficient functioning of the PTSi import machinery. EMBO J 17: 3608-3618
93
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6 Hansenula polymorpha: a versatile model organism in peroxisome research
Van Dijk R, Faber KN, Hammond AT, Click B, Veenhuis M, Kiel JAKW (20013) Tagging Hansenula polymorpha genes by random integration of linear DNA fragments (RALF). Mol Gen Genet, in press Van Dijk R, Lahchev KL, Kram AM, Van der Klei IJ, Veenhuis M (2ooib) Isolation of mutants defective in the assembly of octameric alcohol oxidase of Hansenula polymorpha. FEMS Yeast Res, in press 9-2 Van Dijken JP, Oostra-Demkes GJ, Otto R, Harder W (1976) S-formylglutathione: the substrate for formate dehydrogenase in methanol-utilizing yeasts. Arch Microbiol in: 77-83 Verduyn C, Giuseppin MLF, Scheffers AL, Van Dijken JP (1988) Hydrogen peroxide
metabolism in yeasts. Appl Environ Microbiol 54: 2086-2090. Waterham HR, Titorenko VI, Swaving GJ, Harder W, Veenhuis M (1993) Peroxisomes in the methylotrophic yeast Hansenula polymorpha do not necessarily derive from pre-existing organelles. EM BO J 12: 4785-4794 Waterham HR, Titorenko VI, Haima P, Gregg JM, Harder W, Veenhuis M. (1994) The Hansenula polymorpha PERi gene is essential for peroxisome biogenesis and encodes a peroxisomal matrix protein with both carboxy-and amino-terminal targeting signals. J Cell Biol 127: 737-749
95
7 Characteristics of the Hansenula polymorphic* genome
Dorothea Waschk, Jens Klabunde, Manfred Suckow, Cornells P. Hollenberg
7.1 Introduction
Besides the metabolism of methanol and the associated process of peroxisomal biogenesis, Hansenula polymorpha has a number of other interesting features, some of which will be discussed in this chapter. First we deal with the analysis of the organization of the genome and the location of integrated genes in recombinant H. polymorpha strains. Next we present an analysis of the rDNA genes and results obtained with a vector system based on sequences of the gene encoding the i8S rRNA, which generates stable transformants and allows for the simultaneous insertion of multiple genes. In a recent study seven French laboratories have performed a comparative DNA sequence analysis of 13 yeast species, based on a partial random sequencing approach. The data for H. polymorpha (Pichia angusta) cover 0.5 genome equivalents, and this fraction was found to contain 2,000 genes - complete or in part - having significant similarity with Saccharomyces cerevisiae protein-encoding genes (Blandin et al. 2000). The authors assume that more than 50% of all H. polymorpha genes have been identified. However, because the sequence data cover only part of the genome and have a low level of fidelity, gene identification is subject to some uncertainty. 7.2
Electrophoretic karyotyping
Yeast chromosomes range in size from several hundred to several thousand kilobases, and thus cannot be separated by conventional gel electrophoresis. To separate large DNA molecules Schwartz and Cantor (1984) introduced pulsed-field gel electrophoresis (PFGE). This technique has made possible the resolution of DNA molecules up to several million base pairs in length.
Hansenula polymorpha: Biology and Applications. Edited by G. Gellissen Copyright © 2002 WILEY-VCH Veriag GmbH, Weinheim ISBN: 3-527-30341-3
96
7 Characteristics of the Hansenula polymorpha genome
Electrophoretic karyotyping refers to the separation of intact chromosomal DNA according to size on an agarose gel. Depending on the number and size of the chromosomes present in a strain, a specific banding pattern will be obtained, and the total genome size can be estimated. In order to reach this goal, two demands must be met: First, it is important to prepare the DNA without degradation by mechanical stress or by DNases. Second, depending on the range of sizes encountered, a procedure for the electrophoretic separation of the extremely large molecules must be developed. Preparation of the DNA is usually carried out by lysing cells or protoplasts which have been encapsulated in agarose, thus preventing mechanical shearing. The agarose matrix keeps larger DNA molecules intact, while permitting free diffusion of the buffers and enzymes. A high concentration of ethylendiamine tetraacetic acid (Na 2 -EDTA) is maintained to reduce nuclease activity to levels where no doublestranded breaks can be detected. Since the theory of PFGE is rather complex, a short and simplified explanation will be given. (For a detailed description of PFGE technique and theory, see Gemmill 1991 and Chu 1991.) The DNA is electrophoresed through an agarose gel, under the influence of electrical fields which are turned on alternatively. These fields are orientated towards each other at an angle of more than 90°, so that the DNA will be forced to reorientate after each change in field direction. The time for which a field is applied in one direction before being switched abruptly to another is called the pulse time. Hence, when the field direction is altered molecules of increasing size must spend a larger portion of time reorienting before they again begin to migrate through the gel. This accentuates the difference in mobility between longer and shorter molecules that is the basis of conventional gel electrophoresis, with longer molecules moving more slowly than shorter ones.
A
B
3.1 2.7 2.3
"""" B^I^B [^^^H — 9^1^
^'8* 7*1 Electrophoretic karyotype. - Chromosome pattern of H. polymorpha separated by PFGE using the Pulsaphor apparatus (Pharmacia). The §e' (0.8% in 0.5 x TBE) was run at 140 V for 70 h. The pulse time varies from 140 to 200 s with a linear ramp during the first 35 h, then it was held at 200 s for the following 35 h. The temperature of the buffer was maintained at 9-10° C. Chromosome bands I - VI are shown on the right side and molecular weight markers (Mbp) are shown on the left side. B. Chromo Blot. The chromosome pattern was transferred to a Nylon membrane and hybridized with a probe generated from a cloned URA^ fragment. A signal was obtained exclusively with chromosome I.
A
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WKi^H V ~~ JHliiiH IV
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7.3 Genome mapping (Chromo Blot)
Pulsed-field gel electrophoresis of H. polymorpha RBu chromosomes revealed six chromosomal bands (Figure 7-iA). The sizes of chromosomes were estimated by calibrating the pulsed-field gels with chromosomes of known sizes from S. cerevisiae and Hansenula wingei. Figure 7.1 shows that the size of the smallest chromosome of H. polymorpha, designated chromosome I, is in the order of 900 kb. The other chromosomal DNA bands, which were designated chromosomes II-VI, are larger than i Mb, ranging in size from approximately 1.2-2.2 Mb. The sum of the molecular weights suggests a genome size of about 9-10 Mb, which is significantly smaller than the genome size of other yeasts (S. cerevisiae, 13 Mb; Schizosaccharomyces pombe, 14 Mb; H. wingei, 14 Mb). Marri et al, (1993) have shown that the chromosomal pattern of wild-type strains can vary considerably. With the genomic sequences available, it will be possible to determine reasons for such variations. 7.3 Genome mapping (Chromo Blot)
After a gene has been cloned, it can be assigned to electrophoretically separated chromosomes by Southern blotting and hybridization. Specific hybridization of separated and immobilized chromosomes of H. polymorpha yield positive Tab. 7.1 Chromosomal localization of several cloned genes or sequences in H. polymorpha. Chromosomes were separated by pulsed-field gel electrophoresis
Cloned gene/sequence used as specific probe
Function
\ URA1
\ Orotidine-S'-phosphate decarboxylase Carboxypeptidase Y CPY Glycerinaldehyd-3-phosphate GAP dehydrogenase rDNA (5.8 S, 18 S, 26 S) Ribosomal DNA Autonomously replicating HARSi sequence 1 Trehalose-6-phosphate TPSi synthase /3-Isopropylmala HLEU2 dehydrogenase Methanol oxidase MOX
FMD 1)
2) 3)
Formate dehydrogenase
Reference
Chromosone no.
Merckelbach I et al. 1993 unpublished data1* I unpublished data2) I Klabunde3) Ledeboer et al. 1986 Reinders et al. 1999 Agaphonov et al. 1994 Ledeboer et al. 1985 Hollenberg and Janowicz 1989
Bae JH, Kim HY, Sohn JH, Choi ES and Rhee, SK. Accession number 1167174 Sohn JH, Choi ES and Rhee SK. Accession number 1)95625 Klabunde J, personal communication
II III IV IV V VI
1
97
98
7 Characteristics of the Hansenula polymorpha genome Tab. 7.2 Comparison of rDNA cluster size in a set of yeast species (Maleszka and Clark-Walker 1993)
Yeast
Cluster length (kb)
Hansenula polymorpha I? Chromosome II 450 Ashbya gossypii 1100/200 Candida glabrata (two loci) 580 Kluyveromyces lactis 1500 Saccharomyces cerevisiae Chromosome XII 620 Kluyveromyces wickerhamii Torulaspora 980 delbrueckii Schizosaccharomyces 900/340 pombe (two loci)
Unit length (kb)
Copy number
Genome size (kb)
% Genome
8.2 11
28 100/18
13500
9.6
8.5 9.1
68 150
12300 12267
4.7 8.05
8.5
72
12500
4.9
8.5
115
13000
7.5
10.5
85/32
13900
8.9
,
>
Data for A. gossypii from Wendland et al. 1999. Data for K. lactis from Philippsen et al. 1991.
hybridization signals to various chromosomes listed in Table 7.1. For example, Figure 7.16 shows the localization of the L/RAj gene on chromosome I, after hybridization of immobilized chromosomes to a digoxygenin-labeled URA^ probe. In a similar way, for each of the six chromosomes a "marker gene" could be detected. 7.4 The structure of ribosomal DMA
Ribosomes are composed of a variety of polypeptides and several RNA species, the ribosomal RNAs (rRNAs). The genes encoding the rRNAs are typically clustered in high copy numbers as head-to-tail tandem arrays of identical rDNA units (Philippsen et al. 1991). The number and length of these rDNA repeats can vary considerably between species (Table 7.2). In H. polymorpha about 50 copies of an 8.1 kb unit are localized at a single locus on chromosome II (D. Waschk, personal communication). During the S phase, the rDNA repeats aggregate to form the nucleolus, a morphologically distinct sub-structure of the nucleus. In the case of bakers' yeast, the primary transcript 358 molecule is transcribed within the nucleolus by RNA polymerase I. The transcriptional starting point is located Soobp upstream of the i8S rRNA. This precursor is subsequently processed to form the 258, i8S, and 5.88 rRNA species (Udem and Warner 1972). Simultaneously, the 58 rDNA
7.5 Regulatory elements in the rRNA genes
genes, as well as the genes encoding the ribosomal polypeptides, are transcribed by RNA polymerases III and II, respectively. In H. polymorpha we have found that the organization of the rDNA repeat is very similar to that in S. cerevisiae. The coding region for the 58 rRNA is located between the two non-transcribed spacers (NTSi/2). In other fungi, such as Schizosaccharomyces, Aspergillus and Neurospora, and in higher eukaryotes, the genes for the 58 rRNA are arranged in tandem repeats elsewhere in the genome (Garber et al. 1988). The intergenic spacer regions (ETS, ITS and NTS) between the rDNA genes harbor regulatory elements for functions in transcription, mitotic replication and processing of the precursor rRNA (Figure 7.2). The coding regions of the rDNA genes are highly conserved between different species. The degree of sequence homology can be used to determine taxonomic relationships. Table 7.3 lists the percentage of homology between the rDNA genes of Hansenula polymorpha, Ashbya gossypii and the well known yeast species Saccharomyces cerevisiae. 7.5 Regulatory elements in the rRNA genes
In S. cerevisiae the functions of some regulatory elements in the intergenic spacer region (NTS) have been described. Several cis-acting elements and trans-acting E. coll (dispersed unit)1 RNA polymerase
30SpreRNA
Leader 16S
23S
5S
H. polymorpha (tandem units) RNA polymerase I
35S preRNA
i.5kb) is regulated by the presence of certain carbon sources. On glucose, the MOX promoter is repressed, while on MeOH induction is observed (Egli et al. 1980). In parallel to MOX other genes encoding key enzymes of the MeOH catabolism such as FMD, DAS and CAT are induced. This induction is accompanied by a massive peroxisomal proliferation (for an overview, see Veenhuis et al. 1983; Chapter 6). With certain low concentrations of glycerol as a sole carbon source, a "derepression" is observed, in which the MOX promoter can elicit expression rates comparable to those obtained by induction. Three regulative elements (UASi, UAS2 and URSi) have been mapped by in vitro DNase-I footprinting experiments and corresponding transcriptional analyses in vivo. The occupation of the activating sequences UASi and UAS2 by transcription factors was found to correlate with MeOH induction of the MOX promoter (Godecke et al. 1994). In gel retardation assays using DNA fragments harboring the UASi element of MOX, shifts could be observed if cell extracts of MeOH-grown H. polymorpha were added (Godecke et al. 1994; Pereira and Hollenberg 1996). However, the corresponding binding factors
8.3 Promoters used in H. polymorpha RBll-based expression systems
have not yet been isolated. Indirect evidence of the nature of these transcription factor(s) was obtained from the assessment of S. cerevisiae-denved Adrip. The UASi of the MOX promoter significantly resembles the UASi of the S. cerevisiae ADH2 promoter (Cheng et al. 1994), activated by binding of the zink finger protein Adrip (Thukral et al. 1991). It could be demonstrated that Adrip also binds to the UASi element of MOX. This makes it likely that MeOH induction of the MOX promoter may be conferred by a H. polymorpha zink finger protein homologous to the S. cerevisiae-derived Adrip (Pereira and Hollenberg 1996). The other promoters of the genes involved in MeOH utilization, FMD, DAS and CAT, are regulated similarly to the MOX promoter as mentioned above. However, except for the CAT promoter, studies of their regulation at the molecular level have not yet been performed. The sequence of the CAT promoter includes an element with a high homology to the UASi of the MOX promoter. It could be demonstrated in gel retardation experiments that the UASi elements of MOX and CAT promoters are recognized by the same H. polymorpha transcription factor (Godecke et al. 1994). Until now no respective data were available for the FMD and DAS promoters. Besides MOX the FMD promoter is the only control element derived from methanol-inducible genes employed in H. polymorpha RBn-based expression systems increasingly replacing the MOX promoter in biotechnological applications. Although the maximal strength of MOX and FMD promoters have never been directly compared, a series of indirect comparisons revealed advantages of the FMD over the MOX promoter, especially under industrially relevant fermentation conditions. For example, H. polymorpha RBn-derivatives expressing a phytase gene under control of the FMD promoter provide maximal yields in fermentations under conditions of glucose starvation (Mayer et al. 1999). Also, derepression of FMD promoter-controlled foreign genes under glycerol conditions typically led to product yields higher than those obtained in corresponding MOX promoter-based systems (unpublished data). A recent example of an efficient FMD promoter-based expression strain is that used for the production of saratin (Barnes et al. 2001; Chapter 13). All promoters of the MeOH-utilization pathway genes described above are carbon source-regulated in a similar way. It thus seemed desirable to recruit other strong, differently regulated promoters which are not derived from this pathway for heterologous gene expression in H. polymorpha RBn. In this regard, genes were assessed which are expressed upon temperature stress. In S. cerevisiae, a particular group of heat shock proteins belonging to the family of Hspyo proteins has been described that are encoded by SSA genes (Werner-Washburne et al. 1987). The conserved H. polymorpha homologs of the S. cerevisiae SSAi and SSA2 genes could be isolated (Titorenko et al. 1996; Diesel 1997), and their short promoters ( 95%. Purified HL-2O hirudin was chemically coupled with PEG using an activated PEGylation reagent based on a ]?-nitrophenyl carbonate derivate (Figure 13.4) (Avgerinos et al. 2001). The predominant reaction product was PEG2-hirudin, coupled with lysine at positions 27 and 33. Incomplete PEGylation led to PEG r hirudin, and PEGylation of the hirudin HL-2O N-terminus led to PEG3-hirudin. The reaction conditions were adjusted to minimize those side reactions. Remaining side products and reactants were resolved via subsequent steps of anion exchange chromatography, hydrophobic interaction chromatography and a combination of ultrafiltration and diafiltration (Figure 13.3). 13.2.4 Potential therapeutic applications of PECylated hirudin
Purification and filling of bulk PEG2-hirudin, final release testing and process characterization have to meet the strict regulatory requirements for the development of recombinant protein therapeutics to be used in clinical trials. Most recent results from preclinical studies using PEG-hirudin and clinical data from placebocontrolled clinical trials were summarized by Avgerinos et al. (2001). Different animal models were analyzed to investigate the effect of PEGylated hirudin on various acute coronary syndromes, e.g., restenosis after resolution of carotid artery thrombosis using tissue plasminogen activator (Ruebsamen and Hornberger, 1996), stenosis of injured coronary arteries (Ruebsamen and Kirchengast, 1998), coronary angioplasty and stenting (Buchwald et al. 1996; Unterberg et al. 1997), and hemodialytic treatment (Hoppenstaedt et al. 2000). Comparisons to appropriate controls using unfractionated heparin and/or
217
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13 Production of anticoagulants in Hansenula polymorpha
0 • CH3-0-PEG-0-CH2CH2-OH
Activation of PEG CH3-0-PEG-0-CH2CH2-0-C-N H2 (CHJ, LYS
HO- 92% was analyzed for biological (viral protection) activity in "Wish" cells challenged with vesicular stomatitis virus (VSV). The biological activity was determined to be 2.5 x io8 IU mg~ : thus exceeding the initial expectations (according to the respective Pharmacopeia an activity of not less than 1.4 x io8 IU mg~ x is required).
14.3 Strain development for the production of IL-6, IL-8, IL-10, and IFNy
In the following section the current, early status of strain development for the production of IL-6, IL-8, IL-io, and IFNy is described. The general approach to generating such strains was identical in each case. Gene sequences encoding the
120,0
80,0
0,0 5,0
10,0
15,0
20,0
25,0
30,0
35,0
40,0
45,0
t(h)
Fig. 14.5 Fermentation of a IFNoc-2a production strain on a 1.5 L scale in complex media. The fermentation was started with 3% (w/v) glycerol at the beginning and a pO2 statglycerol feeding mode during the initial growth phase of the culture. After 15 h (arrow) a constant low feed is initiating limited cell growth. At these low glycerol concentrations
the MOX promoter is derepressed resulting in IFN production. Timed fermentation samples were taken and inspected for secreted IFN and other fermentation parameters. After 42 h the culture broth was harvested and IFN was purified from the supernatant. A IFNfoc-2a [mg L"1], • OD6oo, a. growth phase at pH 6.5, b. derepression phase pO2 stat
239
240
14 Production ofcytokines in Hansenula polymorpha Tab. 14.2
Purification of IFNa-2a
Process step
1 Fermentation Centrifugation Ion exchange chromatography RP chromatography Ultrafiltration Total recovery
MW
1
/FNa-20
Volumes [mL]
/™g/ 1 118 118 101.5 60.9 57.8 57.8
\ 1300 1160 50 58 58 58
8
Recovery
r/oj
|ioo
1
100 86 60 95 49.0
MW
Fig. 14.6 SDS-PAGE analysis of IFNa-2a samples. Aliquots of the various purification steps summarized in Table 14.2 were separated through 4-20% gradient gels and visualized by silver staining. Lane i, clarified culture supernatant (dilution 1:5); lane 2, acetone-precipitated proteins (dilution 1:5); lane 3, pooled fractions 3-6, C4-matrix; lane 4, pooled fractions 1-8, biopolymer matrix; lane 5, fraction i, IEC (ion exchange chromatography); lane 5, fraction 2, IEC (dilution 1:2); lane 7, fraction 3, IEC (dilution 1:2); lane 8, E. co//-derived standard (400 ng); MW size marker (6-200 kDa).
mature forms of the various cytokines were amplified by PCR from commercially available cDNA from cytokine-producing cells using specific primer pairs for amplification designed according to published gene sequences. Furthermore, the design of the amplificates allowed an in-frame fusion to an MFai leader segment contained in the two basic expression vectors pFPMTi2i-MFai and pTPSMT-MFoci.
14.3 Strain development for the production of IL-6, IL-S, IL-10, and IFNj
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a
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. =
0)
Q ^
bb § -£ iZ _o ~D
241
242
14 Production ofcytokines in Hansenula polymorpha
The two vectors differ in that they harbor the FMD promoter (Gellissen et al. 1991) and the TPSi promoter (Amuel et al. 2000), respectively, as control elements for heterologous gene expression. The basic design of the resulting cytokine expression vectors is shown in Figure 14.7. 14.3.1
Strain development for the production of IL-6
Interleukin-6 (IL-6) is produced by both lymphoid and non-lymphoid cells and has multifunctional properties in the regulation of immune responses, acute-phase reactions and hematopoiesis (Le and Vilcek 1989; Hirano 1992, 1998; see also Figures 14.1 and 14.2). Similar to the IL-i and IL-2 examples mentioned in the introduction, IL-6 came to stand for previously separate cytokine activities and designations (Le and Vilcek 1989; Heinrich et al. 1990; Hirano 1998), such as interferon-p2 (IFN-fte) (May et al. 1986), B cell differentiation factor (BCDF) (Okada et al. 1983), hepatocyte stimulating factor (HSF) (Andus et al. 1987), and monocyte-granulocyte inducer (MGI-2) (Shabo et al. 1988). IL-6 and IL-3 synergistically induce the proliferation of murine hematopoietic progenitors in vitro. IL-6 most likely triggers the entry of dormant progenitor cells into the cell division cycle, whereas IL-3 supports the continuous proliferation of such progenitor cells (Ogawa 1992). Bone marrow cells cultured with these two cytokines for 6d were able to rescue lethally irradiated mice. Thus IL-6 (in combination with IL-3) could potentially be applied in bone marrow transplantation (Bodine et al. 1989; Okano et al. 1989). Another important potential application of IL-6 is the treatment of thrombocytopenia. Especially polyvinylpyrrolidoneconjugated (Tsunoda et al. 2000) and PEGylated (Tsunoda et al. 2001) forms of IL-6 exhibit a particularly high selective thrombopoietic activity in a mouse model. IL-6 is a protein of 184 amino acids glycosylated at positions 73 and 172. It contains two disulfide bonds (Cys45-Cys5i and Cys74-Cys84). The first disulfide bond is not required for biological activity (Snouwaert et al. 1991). It is processed from a precursor polypeptide of 212 amino acids, and the amino acid sequence of the mature protein commences with a Pro residue (Ibelgaufts 1995). Analysis of hIL-6 muteins produced in E. coli showed that the biological activity was unaffected by the deletion of amino acids 1-28 (Brakenhoff et al. 1989). Another report postulates a role of the N-terminus in receptor binding when assessing human/mouse interleukin-6 hybrid molecules in vitro (Fiorillo et al. 1992). In contrast, internal deletions of amino acids 29-42 differentially affect biological activity (Arcone et al. 1991). The integrity of the C-terminus was found to be crucial for biological activity. Argi79 is essential for active site formation (Fontaine et al. 1993). The modification of three periodic leucine residues (Leui68, 175 and 182) causes a dramatic decrease of receptor binding and Ig-induction activities (Nishimura et al. 1992). Recombinant expression systems for IL-6 have been established based on E. coli (Tonouchi et al. 1988), and Saccharomyces cerevisiae (Guisez et al. 1991). The drawbacks of the E. coli-based process are similar to those already described for the production of IFNoc-2a and b: The molecule is produced as an insoluble inclusion
14.3 Strain development for the production of IL-6, IL-8, 11-10, and IFNy
body, and an improved denaturation/renaturation process (17% yield of the initial product concentration) might be appropriate for commercial-scale production (Ejima et al, 1999). Recombinant human IL-6 was found to undergo selective Nterminal degradation when produced in E. coli. Cleavage by a thiol protease yielded two new N-termini at Arg9 and Hisi5 (Proudfoot et al. 1993). Approaches have been undertaken to remove the N-terminal methionine by aminopeptidase P both in vitro (Yaseuda et al. 1990) and in vivo by coexpression of the respective gene (Yaseuda et al, 1991). Alternatively, the recombinant product was directed to the periplasmic space and processed from a ompA/IL-6 fusion. The secretion process was only efficient when the N-terminus of the processed IL-6 was not proline (Barthelemy et al. 1993). In the S. cerevisiae system it was found that the endogenous pKex2 protease was unable to cleave the prepro-Lys-Arg-Pro-IL-6 sequence of the engineered MFoci/IL-6 precursor, and that unspecific cleavage of the precursor molecule occurred, leading to a molecule with N-terminal extensions (Guisez et al. 1991). The modified sequence prepro-Lys-Arg-Ala-Pro-IL6 was correctly recognized by the pKex2 protease and the emerging N-terminal Ala-Pro dipeptide was subsequently removed by a pStei3 protease. The processed IL-6 was secreted into the medium (up to 30 mg L"1 on a 2 L scale) (Guisez et al. 1991). H. polymorpha strains were constructed using the two types of vectors for transformation described in Figure 14.7, differing in the inclusion of either the TPSi or the FMD promoter for expression control. The expressed sequence codes for an MFoa/IL6 fusion retaining the Pro residue in position i. In case of the first construction line (FMD constructs) 72 recombinant strains were analyzed. The copy number of the integrated plasmids was found to vary between 20 and 30 in the analyzed strains, and the productivity was found to be approximately 5omg L"1 in the best strains on a 3mL screening scale. The majority of the secreted IL-6 was found to comigrate in SDS-PAGE with an E. coli-derived standard, indicating the secretion of correctly processed IL-6. In addition, degradation products and molecules of higher molecular weight were present, probably due to a small proportion of overglycosylated and incorrectly processed proteins. In the second construction line (TPSi constructs) 60 transformants were analyzed. The copy number was found to be approximately 40 in the analyzed strains, and the productivity 50 mg L"1 on a 3 mL screening scale. From this expression level, a productivity of several grams per liter can be predicted for optimized fermentations on a multiliter scale. The extent of overglycosylation, misprocessing and degradation was dramatically reduced when compared to the FMD constructs (see also Figure 14.8), and some of the identified strains (lanes 9 and 10 in Figure 14.8) promise to be appropriate for further development. 14.3.2 Strain development for the production of IL-8
Interleukin-8 (IL-8) belongs to a family of related cytokines with chemotactic activities (chemokines) for certain types of leukocytes (Wuyts et al. 1998). IL-8 is now a term that designates cytokines formerly referred to as NAF (neutrophilactivating factor), monocyte-derived neutrophil activating factor (MONAP),
243
244
14 Production ofcytokines in Hansenula polymorpha 1
2
3
4
5
6
7
8
91 0
kDa
64 50 36 30
16
Fig. 14.8 Analysis of IL-6 secreted by recombinant H. polymorpha strains. Recombinant strains were generated using a vector of type 2 (TPSi promoter; see Figure 14.7) for transformation. The generated strains were cultured at a 3 ml screening scale under standard conditions. The supernatants were inspected for IL-6 content by Western blot analysis. For other details, see text. Lane i, MW standard; lane 2, E. co//-derived IL-6 standard; lane 3, (negative) control supernatant derived from a mock-transformant; lanes 4-10, IL-6-secreting transformants. IL-6-derived polypeptides: (a) overglycosylated (?) IL-6, (b) misprocessed IL-6; (c) correctly processed IL-6, (d) degradation products of undetermined structure.
chemotactic factor (MDNCF) (Walz et al. 1987; Yoshimura et al. 1987) or as a T cell chemotactic peptide produced by peripheral blood mononuclear cells (Larsen et al. 1989). Chemokines are small basic proteins with 4 conserved cystein residues forming 2 disulfide bridges (bonds between the first and third and between the second and fourth cysteins) necessary for the tertiary structure of the protein. Chemokines can be divided in CXC, CC, and CX3C subfamilies depending on the number of amino acids separating the first and second cysteine (Bazan et al. 1997). In neutrophil-attracting chemokines the CXC sequence is preceded by a Glu-LeuArg (ELR)-tripeptide. IL-8 is a member of this group (Wuyts et al. 1998). A 77 amino acid protein is processed from a 99 amino acid precursor (Schmid and Weismann 1987). Truncated analogs may arise by further N-terminal processing depending on the producer cell and culture conditions, yielding 72-, 71-, 70-, and 69 amino acid forms (van Damme et al. 1990) with the 72 form as the majority product. In vivo both major forms (the 77 and the 72 amino acid protein) are equipotent (Noursharg et al. 1992). IL-8 is a non-glycosylated dimer of two identical subunits (Clore and Gronenborn 1991). For a summary of protein characteristics the reader is referred to Ibelgaufts (1995) and Wuyts et al. (1998). The N-terminal ELR motif (residues 4-6) is essential for binding to the IL-8 A receptor on leukocytes (Hebert et al. 1991; Clark-Lewis et al. 1994). Tyri3 and Lysi5
14.3 Strain development for the production of IL-6, IL-8, IL-10, and IFNj
(Schraufstatter et al. 1995) and Pheiy, Phezi, Ile22 and Leu43 (Williams et al. 1996) are further arnino acids identified to be involved in the process. Formation of both disulfide bridges are essential for biological function (Clark-Lewis et al. 1994). IL-8 has activities in many cell types other than leukocytes and a wide range of effects are described, for example by Wuyts et al. (1998). IL-8 may be of clinical relevance for the treatment of patients with myelodysplastic syndrome. The lesions responsible for defective neutrophil function in these patients could be restored by IL-8 without stimulating myeloid progenitor cells (see Figure 14.1), thus reducing the risk of lethal infection without the potential risk of stimulating leukemic clones (Ibelgaufts 1995). Recombinant expression systems for the production of human IL-8 have been established based on E. coli (Furuta et al. 1989; Jin et al. 1993; Miller et al. 1995) and the baculoviral system (Kang et al. 1992), and a design for a scale-up process has been worked out for the former (Koltermann et al. 1997). For expression in H. polymorpha a construction strategy was executed according to that already described for IL-6. A sequence encoding the yyaa form of IL-8 was amplified by PCR from human kidney cDNA with gene-specific primers and fused to the MFoti leader of the two basic vectors (Figure 14.7). 72 transformants were analyzed for the presence of the FMD promoter construct. The analyzed strains harbored up to 50 copies of the expression cassette integrated into the genome, and the productivity of selected strains was found to be 30 mg L"1 at a 3mL scale. SDS analysis of the secreted IL-8 revealed an Mr of 8.9kDa, comparable to that of an E. coli-derived standard and indicating the correct processing of the precursor protein. 60 transformants of the TPSi promoter construct were analyzed. More than 50 copies were found to be integrated in the analyzed strains. The productivity was found to be 40 mg L"1 on a 3mL screening scale. Again, the IL8 was found to be correctly processed. In addition, a minor IL-8 component of unknown structure could be detected. These molecules of higher molecular weight could represent dimers or incorrectly processed precursor molecules. A representative analysis of IL-8 secreted by recombinant H. polymorpha strains of this series is documented in Figure 14.9. 14.3.3 Strain development for the production of IL-10
Interleukin-io (IL-io) is a cytokine produced in a variety of cell types including B and T cells, bronchial epithelial cells (Bonfield et al. 1995), and by melanomas (Dummer et al. 1996) or carcinomas (Kim et al. 1995). Alternative names are B cellderived T cell growth factor (BTCGF), cytokine synthesis inhibitory factor (CSIF) or T cell growth inhibitory factor (TGIF) and macrophage deactivating factor (Fiorentino et al. 1989; Bogdan et al. 1991; Moore et al. 1993; Ibelgaufts 1995; de Waal Malefyt and Moore 1998). The amino acid sequence of IL-io has been deduced from the respective cDNA sequence (Vieira et al. 1991). The human IL-io is a non-covalent homodimer of two interpenetrating subunits of 160 amino acids (18,6 kDa). The non-
245
246
14 Production ofcytokines in Hansenula polymorpha
kDa
^^^^^^^^^^
36 30 I I
16
Fig. 14.9 Analysis of IL-8 secreted by recombinant H. polymorpha strains. Recombinant strains were generated using a vector of type 2 (TPSi-promoter; see Figure 14.7) for transformation. Analytical conditions as described in Figure 14.8. Lane i, MW standard; lane 2, £ co//-derived standard; control supernatant from a mock-transformant, lanes 3-8, IL-8 secreting transformants
glycosylated subunit chain contains two disulfide bridges which are necessary for biological activity (Windsor et al. 1993). IL-io is a potential therapeutic agent for acute and chronic, systemic and localized inflammatory reactions. It controls inflammatory responses by preventing activation of monocytes and macrophages (Chernoff et al. 1995; Huhn et al. 1996). IL-io was shown to prevent and to reverse cartilage degradation in rheumatoid arthritis (van Roon et al. 1996). Treatment with recombinant human IL-io may have therapeutic potential for psoriatic patients (Asadullah et al. 1999). In a mouse model with cutaneous inflammatory lesions intramuscularly injected IL-io expression vectors were tested with promising results for a potential gene therapeutical approach (Chun et al. 1999). Recombinant H. polymorpha strains for the production of IL-io were generated as described for the other cytokines. 72 transformants of the FMD promoter construct were characterized and inspected for IL-io secretion on a 3 mL screening scale. The analyzed strains were found to harbor some 40 copies of the expression plasmid and found to secrete the IL-io at a productivity of 10 mg mL"1. 60 transformants of the TPSi construct were analyzed in the same way. The analyzed strains harbored up to 50 copies of the heterologous DNA and were found to secrete 15 mg L"1 of the reecombinant IL-io. Two polypeptides of similar size appeared to be the majority products of the secreted IL-io, the larger comigrating with a bacterial standard. The structure of the truncated form is unclear. In addition to these two polypeptides, faint amounts of smaller peptides are present probably due to proteolytic degradation. A representative analysis of the IL-io secreted by strains of the TPSi promoter type is shown in Figure 14.10.
14.3 Strain development for the production of IL-6, 1L-S, 11-10, and IFNy I 247
14.3.4 Strain development for the production of IFNy
IFNy is produced by CD4 and CDS positive T cells and NK (natural killer) cells. The mature protein consists of 146 amino acids processed from a precursor with a leader of additional 23 amino acids. Two stretches of basic amino acids (LysLysLysArg in positions 86-90 and LysArgLysArg in positions 128-132, respectively) cause instability of the protein under acid conditions (de Maeyer and de Meyer-Gruignard 1988,1998; Ibelgaufts 1995). Two active cytokine forms of 20 kDa and 25 kDa exist, differing in the extent of N-glycosylation. The smaller protein is glycosylated at Asn25, the bigger is additionally glycosylated at Asn97 (Rinderknecht et al. 1992). Glycosylation is not required for biological activity (Arakawa et al. 1986), but glycosylation at position 25 was found to improve proteolytic stability (Sareneva et al. 1995). The protein contains two cysteine residues, but no disulfide bridges are present. The protein has been produced in E. coll (Jay et al. 1984; Simons et al. 1984; Nishi et al. 1985) and in mammalian cells (Riske et al. 1991). In mammalian cells the protein reveals a heterogeneous glycosylation pattern depending on the cell type and on culture conditions. IFNy is a potent antiviral and antiparasitic agent. It has been assessed for treatment of opportunistic infections in AIDS patients, for treatment of eosinophilia in severe atopic dermatitis and for the treatment of osteopetrosis (Key et al. 1992, 1995). A recombinant IFNy (Actimmune®; Intermune Pharmaceuticals) is applied for treatment of chronic granulomatous disease. Application of this compound for
kDa
64
8 36 jfj 30 1
Fig. 14.10 Analysis of IL-io secreted by recombinant H. polymorpha strains. Recombinant strains were generated using a vector of type 2 (TPSi promoter; see Figure 14.7) for transformation. Analytical conditions are as described in Figure 14.8. Lane i, MW standard; lane 2, E. co//-derived standard; lane 3-9, IL-io secreting transformants; lane 10, supernatant derived from a mock transformant.
248
14 Production ofcytokines in Hansenula polymorpha
treatment of idiopathic pulmonary fibrosis are at present in phase I/I I evaluation, and application for tuberculosis are in phase III (see Table 14.1; Wetzel 2001). Again, two basic H. polymorpha strains were constructed as described in the other cytokine examples. For both collections of transformants, strains were identified secreting 5-10 mg mL" 1 of the interferon on a 3 mL screening scale. Selected strains of the two types were found to harbor 40 copies of the expression cassette. A representative analysis of the secreted compound is documented in Figure 14.11. Several strains of the TPSi promoter type constructs were analyzed by SDS-PAGE for IFN production. The secreted products appear as heterogeneous smears indicative of hyperglycosylation (Figure 14.11 A). Treatment with N-glycosidase F results in a single protein band comigrating in SDS-PAGE with an E. coZi-derived standard (ly.ikDa). The glycosylation is currently being analyzed in more detail and mutant host strains that provide a modified glycosylation pattern are under assessment.
14.4 Conclusion
This chapter has demonstrated the capabilities of the H. polymorpha system for the production ofcytokines. In case of IFNoc-2a a production strain has been identified and the steps for a purification procedure have been defined. The purified product exceeds all expectations with respect to the biological activity. The process is thus ready for industrial scale-up.
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Fig. 14.11 Analysis of IFNy secreted by recombinant H. polymorpha strains. Recombinant strains were generated using a vector of type 2 (TPSi) promoter; see Figure 14.7) for transformation. Analytical conditions are as described in Figure 8. Undeglycosylated (A) and deglycosylated (PNGaseF-treated) (B) IFN samples are analyzed. (A) Analysis of undeglycosylated IFNy. Lane i, MW marker; lane 2, £ co//-derived standard; lane 3, supernatant derived from a mock transformant; lanes 4-10 IFNy-secreting transformants. (B) Analysis of deglycosylated IFNy. Lane i, MW marker, lane 2, E. co//-derived standard; lanes 5)9.13, H. polymorpha-derived IFNy after deglycosylation
14.4 Conclusion Tab, 14.3
Summary of cytokine expression in Hansenula polymorpha
Construct
Transformants screened
• FMD-MFIL6 ' 72
Best production strains
Product in supernatant (3 mL scale)
|l8-8 48-8
' ~25mg L4
Copy number
-20
TPS-MFIL6
60
21-9 36-7
-25 mg L4
-40 >40
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10-9 69-9
-30 mg L4
-50 -50
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60
29-9 35-6
-40 mg L4
>50 >50
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72
6-5 67-5
-10 mg L4
-40 >40
TPS-MFIL10
60
36-3 38-1
-15 mg L4
-50 >50
FMD-MFIFNy 72
23-2 52-2
5-10 mg L4
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