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THE ALKALOIDS Chemistry and Pharmacology VOLUME 40

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THE ALKALOIDS Chemistry and Pharmacology Edited by Arnold Brossi National Institutes of Health Bethesda, Maryland

VOLUME 40

Academic Press, Inc. Harcourt Brace Jovanouich, Publishers

San Diego New York Boston London Sydney Tokyo Toronto

This book is printed on acid-free paper.

@

Copyright 0 1991 BY ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press, Inc. San Diego, California 92101 United Kingdom Edition published by ACADEMIC PRESS LIMITED 24-28 Oval Road, London NWl 7DX

Library of Congress Catalog Card Number:

ISBN 0-12-469540-X

(alk. paper)

PRINTED IN THE UNITED STATES OF AMERICA 91

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CONTENTS

CONTRIBUTORS ...................................................................................... PREFACE...............................................................................................

vii ix

Chapter 1. Plant Biotechnology for the Production of Alkaloids: Present Status and Prospects ROBERT VERPOORTE, ROBERTVAN DER HEIJDEN, WALTERM. VANGULIK,AND HENSJ. G . TEN HOOPEN

I. 11. 111. IV. V. VI. VII. VIII. IX. X. XI.

Introduction ....................................... Strategies to and Extracellular Aspects .. Large-Scale Production ................................................................... Nicotine ........................................................... .... Tropane Alkaloids .......................................................................... Isoquinoline Alkaloids ............... Cinchona Alkaloids ........................................................................ Indole Alkaloids ....................,....................................................... Caffeine ............................................. Steroidal Alkaloids ......................................................................... ........................... ,...................................... Concluding Remarks ....... References ............................................................

2 9 20 44 52 72 104 109 154 157 161 163

Chapter 2. Alkaloids from Mushrooms AND WIESJ/AW z.ANTKOWIAK R6ZA ANTKOWIAK

I. Introduction ..................................................................................

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

190 194 225 253 275 28 1 289 305 307 324

CUMULATIVE INDEXOF TITLES ................................................................

34 1

INDEX..................................................................................................

349

11. Physiologically Active Principles of the Genus Arnanita ........................

111. Indole Alkaloids ..... ,......................... ,............. IV. Pyridine Alkaloids ... ................................................... .............................. V. Hydrazine Alkaloids ................................

........................................................ .................. References

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

V


CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors’contributions begin.

R 6 z ANTKOWIAK ~ (189), Faculty of Chemistry, Adam Mickiewicz University, Poznan, Poland WIES~AW Z. ANTKOWIAK (189), Faculty of Chemistry, Adam Mickiewicz University, PoznBn, Poland HENSJ. G. TEN HOOPEN(l), Biotechnology Delft Leiden, Projectgroup Plant Cell Biotechnology, Department of Biochemical Engineering, Delft University of Technology, 2628 BC Delft, The Netherlands ROBERTVAN DER HEIJDEN (l), Biotechnology Delft Leiden, Projectgroup Plant Cell Biotechnology, Center for Bio-Pharmaceutical Sciences, Division of Pharmacognosy, Gorlaeus Laboratories, 2300 RA Leiden, The Netherlands WALTERM. VAN GULIK(l), Biotechnology Delft Leiden, Projectgroup Plant Cell Biotechnology, Department of Biochemical Engineering, Delft University of Technology, 2628 BC Delft, The Netherlands ( l ) , Biotechnology Delft Leiden, Projectgroup Plant ROBERT VERPOORTE Cell Biotechnology, Center for Bio-Pharmaceutical Sciences, Division of Pharmacognosy,Gorlaeus Laboratories, 2300 RA Leiden, The Netherlands

vii


PREFACE

Modern biotechnological methods of producing potentially useful alkaloids, particularly plant cell culturing, is an area of fundamental importance and has been receiving a lot of attention. An analysis of the state of the art and where it is headed is discussed here by a well-known group from The Netherlands in “Plant Biotechnology for the Production of Alkaloids: Present Status and Prospects.” Muscarine from the mushroom, Amanita muscaria, has become a valuable biochemical tool for measuring peripheral effects on cholinergic receptors (see Volume 23 of this series). Each year Phalloidin from the mushroom, Amanita phalloides, causes several deaths in Europe from mushroom poisoning and has been the subject of a book by Theodor Wieland. In this volume, the subjects of alkaloids and mushrooms are brought together in a paper written by a team of experts from Poland in “Alkaloids from Mushrooms.” Arnold Brossi National Institutes of Health

ix


-CHAPTER

1-

PLANT BIOTECHNOLOGY FOR THE PRODUCTION OF ALKALOIDS: PRESENT STATUS AND PROSPECTS* ROBERTVERPOORTE AND ROBERT VAN

DER

HEIJDEN

Biotechnology Devt Leiden Projectgroup Plant Cell Biotechnology Center f o r Bio-Pharmaceutical Sciences Division of Pharmacognosy Gorlaeus Laboratories 2300 R A Leiden. The Netherlands

WALTERM.

VAN

GULIK,AND HENSJ. G. TEN HOOPEN

Biotechnology Devt Leiden Projectgroup Plant Cell Biotechnology Department of Biochemical Engineering Devt University of Technology 2628 BC Delft, The Netherlands

I. Introduction ......................................... A. Production by Means of Genetically Engineered Microo ................ B. Production by Means of Plant Cell Cultures C. Bioconversion of Available Precursors. ... D. Production by Genetically Engineered Plan E. Production of Novel Compounds.. 11. Strategies to Improve Product Yield: Cellular and Extracellular Aspects.. .. A. Screening and Selection. .......................... B. Culture Conditions ............................... C. Alkaloid Storage Compartments .......................... D. Elicitation.. ..................................... E. Feeding of Precursors and Bioconversions. .......................... F. Immobilization. ........................ .................... G. Permeabilization ................................. H. Differentiation and Culture Type ................................... I. Genetic Approaches and Genetic Modification ................... J. Combination of Treatments: Toward High Productivity ...............

2 6 6 7 8 9 9 10

12 14 16

16 17 17 18 19 19

* The following abbreviations are used in the text: ABA: abscisic acid; BAP: 6-benzylaminopurine; DW: dry weight; 2,4-D: 2,4-dichlorophenoxyaceticacid; FW: fresh weight; IAA: indole-3-acetic acid; IBA: indole-3-butyric acid; MS: Murashige and Skoog medium; NAA: naphtaleneacetic acid. 1

THE ALKALOIDS, VOL. 40 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

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ROBERT VERPOORTE ET AL.

111. Large-Scale Production. . . . . . . . . . . .

A. Economic Feasibility . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Technological Aspects ................ IV. Nicotine ......... . ....... . . ........ ....... . ..... . ..... . .... . . . . A. Secondary Metabolites in Cell Cultures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Screening, Selection, and Stability C. Effects of Growth Conditions. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Large-Scale Suspension Cultures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Root Cultures.. . . . . . . . . . . . . . . . . . . . F. Conclusion.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Tropane Alkaloids ........................ A. Plant Biotechnology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Production of Tropane Alkaloids by Cell Cultures . . . . . . . . . . . . . . . . . . . . VI. Isoquinoline Alkaloids. . . . . . . . . . . . . . . . . . . A. Ipecacuanha Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Colchicine ........................ C. Cephalotaxus s . ............................ D. Bisbenzylisoquinoline Alkaloids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Opium Alkaloids F. Sanguinarine. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

VII. Cinchona

............................................ ...................... logy ..............................................

C. Bioconversions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Conclusions .......................... VIII. Indole Alkaloids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Catharanthus Alkaloids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Rauuolfia Alkaloids C. Miscellaneous Indol ........... IX. Caffeine ...... . ..... . . .... . .... . . ..... . . . .... . . ... . . .... . .... .. . .... X. Steroidal Alkaloids ............... B. Miscellaneous Steroid Alkaloids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

20 22 25 44 44 47 48 48 50 51 52 52 65 72 72 74 74 75 77 85 94 104 104 105 108 108 109 109 142 149 154 157 158 161 161 163

I. Introduction Plants produce a variety of alkaloids. Table I presents some of the major classes and an estimation of the number of representatives (N. R. Farnsworth, personal communication). About 30 alkaloids have great commercial interest, mostly because of their use as medicines, flavorings, or poisons, sometimes as important tools in pharmacological studies. In all cases the total amounts produced worldwide are rather limited. For example, by volume, possibly the largest production involves the alkaloids quinine and quinidine. For their isolation 5000-10,000 metric tons of Cin-

1.

3

PLANT BIOTECHNOLOGY

TABLE I NUMBER OF UNIQUE STRUCTURES IN MAJORALKALOIDS CLASSES IN NAPRALERT DATABASE^ Class

Number

Indole Isoquinoline Protoalkaloids Quinoline Pyrrolidine/piperidine Diterpene Quinolizidine Pyrrolizidine Steroidal Tropane Pyridine Indolizidine Sesquiterpene Homospermidine/spermidine

4125 4045 1147 718 714 642 57 I 562 440 300 24 1 170 132 129 15,947

October, 1988. Totals include alkaloids isolated from plant, animal, and marine sources. N. R. Farnsworth, personal communication.

chona bark is processed yearly, yielding 300-500 metric tons of quinine and quinidine (I). The alkaloid ajmalicine, used as an antihypertensive, has a yearly market volume of about 3600 kg (2), for which probably 200-300 tons of roots of Catharanthus roseus is needed. Compared to laboratory-scale isolations, these seem impressive amounts, but compared to agricultural crops they are only very small volumes. Several compounds of interest are isolated from plants which need several years to develop. For example, Cinchona trees need 10-12 years before they can be harvested, and Coptisjaponica rhizomes, a source of barberine, require 5-6 years of growth before harvesting. In many cases little plant breeding has been done to improve yields of the alkaloids, and in some cases one simply relies on collection of plant material from the wild (e.g., tubocurarine is isolated from curare, which is collected by Indians from the liana Chondrodendron tornentosurn). Alkaloids are consequently valuable chemicals. Table I1 gives prices per gram of some commonly used alkaloids. As no single source was available for bulk prices of these chemicals, we used the price list of a large supplier of fine chemicals. These prices are per gram, higher than the bulk prices, but they give at least some indication of the values of these alkaloids. In this review we shall give a survey of the state of the art of using plant

4

ROBERT VERPOORTE ET A L .

TABLE I1 PRICES,SOURCES, AND PRODUCT YIELDSBY MEANSOF ALTERNATIVE BIOTECHOLOGICAL METHODS OF SOME COMMONLY USED ALKALOIDS

Alkaloid Ajmalicine Vinblastine Vincristine Ajmaline Reserpine Rescinnamine Vincamine Strychnine Brucine Yohimbine

Coronaridine Quinine Quinidine Ellipticine 9-Hy droxyellipticine

Camptothecine Emetine Physostigmine Pilocarpine Caffeine Theobromine Atropine

Scopolamine

Cocaine Berberine Sanguinarine Berbamine Tu bocurarine

Plant source Catharanthus roseus Catharanthus roseus Catharanthus roseus Rauvolfia species Rauvolfia species Rauvolfia species Vinca species Strychnos species Strychnos species Rauvolfia species, Corynanthe species Tabernaemontana species Cinchona species Cinchona species Ochrosia elliptica Ochrosia elliptica Camptotheca acuminata Cephaelis ipecacuanha Physostigma venenosum Pilocarpus microphyllus Coffea, Camellia Theobromu Atropa belladonna, Datura species, Hyoscyamus species Duboisia species, Hyoscyamus species Erythroxylon coca Coptis juponica, Berberis Eschscholtzia, Papaver Menispermaceae Chondrodendron tomentosum

Price (DMlgram) 56.00 15,800.00 37,800.00 15.50 12.50 27.50 29.50 1.45 1S O 6.50

Production by cell culturesa Source

Yield

C. roseus SC C. roseus ShC C. roseus ShC RauvolJa SC Rauvolfia SC No data available Vinca minor SC No reports on PCTC No reports on PCTC No data available

0.2 g/liter Traces Traces 0.04 g/liter 0.002 g/liter

Tabernaemontana species SC Cinchona ShC Cinchona ShC 0. elliptica SC

Traces

C. acuminata SC

0.00025% DW

39.50

Cephaelis RC

0.3-0.5% DW

79.00

No reports on PCTC

25.50

No reports on PCTC

980.00 0.75 1.40 3940.00 4680.00 720.00

3.3 g/liter

0.01-0.1% DW 0.01-0.1% DW 0.005% DW

Coffea SC Coffea SC Atropa HR Datura HR

0.48 g/liter

26.00

Duboisia HR Hyoscyamus HR

0.08 glliter 0.4% DW

17.00 16.50

No reports on PCTC C. juponica SC Thalictrum SC Eschscholtzia SC Papaver SC Stephania RC No reports on PCTC

7 g/liter 0.87 g/liter 0.16 g/liter 0.25 g/liter 0.55% DW

0.12 0.95 5.20

72.00 265.00 126.00

0.1-0.2 g/liter 0.1-0.2 g/liter

(continued)

5

1. PLANT BIOTECHNOLOGY TABLE I1 (Continued)

Alkaloid Papaverine Noscapine Narceine Morphine Codeine Nicotine Colchicine Hamngtonine Aconitine Conessine Solasodine Shikonin'

Plant source Papaver somniferum Papaver somniferum Papaver somniferum Papaver somniferum Papaver somniferum Nicotiana species Colchicum autumnale Cephalotaxus harringtonia Aconitum species Holarrhena antidysenterica Solanum species Lithospermum erythrorhizon

Price (DM/gram) 0.43 13.50 16.00 510.00 25.50 1.55 75.00 NAb 1120.00 NA 170.00

NA

Production by cell cultures" Source

Yield

P . somniferum SC P . somniferum SC P . somniferum SC P . somniferum SC P . somniferum SC Nicotiana SC C . autumnale CC

Traces Traces Traces Traces Traces 0.36 g/liter 1.5%DW

C . harringtonia CC

Traces

No reports on PCTC Holarrhena CC

0.001%FW

Solanum SC

O.Ol-i% DW

L . erythrorhizon SC

4 g/liter

Results as reported in the literature. PCTC, Plant cell and tissue culture; CC, callus culture; SC, cell suspension cultures; RC, root cultures; HR, hairy root cultures; ShC, shoot cultures; DW, dry weight; FW, fresh weight. NA, Not available. Shikonin is not an alkaloid, but because it is the first product from a plant cell biotechnological production, it is included in the table to enable comparison with the alkaloids.

cell cultures for the large-scale production of alkaloids. Strategies followed to obtain high production (Section 11) and aspects of technology involved in the large-scale culture of plant cells and the economy of such processes (Section 111) are discussed briefly. Different classes of alkaloids are then discussed separately, with emphasis on production, be it by de nouo biosynthesis or bioconversion of added precursors by plant cells. Patents concerning the production of various alkaloids are also listed. We confine ourselves only to alkaloids derived from higher plants which are presently produced on an industrial scale by extraction of plant materials. Some classes of alkaloids for which production in cell cultures has been studied extensively are thus omitted, for example quinolizidine (lupine alkaloids), pyrrolizidine (Senecio and Symphytum alkaloids), and acridone alkaloids (Ruta alkaloids). For these classes of alkaloids we refer to recent authorative reviews (3-5). For some widely used alkaloids, such as pilocarpine, physostigmine, cocaine, strychnine, and tubocurarine, no studies have been published yet on the plant cell tissue and organ culture (see Table 11). It is obvious that with the rapid developments in biotechnology, alternative biotechnological production methods of plant-derived fine chemicals,

6


like the alkaloids, became of interest. Several possibilities can be considered for applying biotechnology, namely, production of plant compounds by genetically engineered microorganisms; production by means of plant cell cultures; bioconversion of readily available precursors, by using genetically engineered microorganisms, plant cell cultures, or isolated plant enzymes; production by means of genetically engineered plants or plant cell cultures; and production of novel compounds. Besides the possibilities for production of known compounds, biotechnology can also be used to produce new compounds. We shall consider these possibilities in more detail. A. PRODUCTION BY MEANSOF GENETICALLY ENGINEERED MICROORGANISMS Genetic engineering of microorganisms is feasible nowadays. One can thus consider the possibilities of transferring the production of a plant secondary metabolite into a microorganism. To be able to do so one has to know the biosynthetic pathway of the compound concerned; one must identify the enzymes involved and the genes coding for the enzymes. As most plant secondary metabolites result from pathways involving a large number of steps (10-20 is quite normal), at least as many genes are involved. In fact, only a few secondary metabolite pathways are completely known at the level of enzymes, for example, the flavonoid pathway and the biosynthesis of some isoquinoline alkaloids. Consequently only very few genes from secondary metabolism are known (e.g. some of the key genes from the flavonoid pathway). In the case of alkaloids only a few isolated steps from the biosynthetic pathways have been studied to the level of the genes, for example, strictosidine synthase, a key enzyme in indole alkaloid biosynthesis from RauuolJia (see below) (6,7) and tryptophan decarboxylase, another regulated enzyme from indole alkaloid biosynthesis (8,9). Even if all the genes were known, transferring a large number of genes to a microorganism is not feasible, particularly as the enzymes produced have to act in a concerted way. Furthermore, in plants secondary metabolism is often compartmentalized on the subcellular or even cellular level. This will be impossible to realize in microorganisms. BY MEANSOF PLANTCELLCULTURES B. PRODUCTION

As genetic engineering of microorganisms does not seem to be a feasible approach, one should exploit the genetic information of the plant cell itself. Plant cells are totipotent, which means that each cell carries all the genetic

1. PLANT BIOTECHNOLOGY

7

information for all plant functions, including the biosynthesis of secondary metabolites. In theory it is thus possible to have in uitro cultured plant cells produce secondary metabolites. Below, in the review on the state of the art of plant cell biotechnology for the production of various commercially interesting alkaloids, it will become clear that this is only partly true. Table I1 summarizes the results for most of the alkaloids discussed here. Secondary metabolism is a form of differentiation, but cells grown in uitro are rapidly dividing, undifferentiated cells. Only at the end of the growth phase of batch-cultured cells may some form of differentiation occur, connected with the production of secondary metabolites. A plant produces a wide variety of secondary metabolites, all with different, mostly unknown functions. In in uitro cultured cells those compounds which defend the plant against microorganisms, namely, phytoalexins, are often easily formed. For example, Cinchona cell cultures produce large amounts of anthraquinones, but the alkaloids of interest, the quinolines, are produced in trace amounts only. Similarly Papauer cell cultures produce sanguinarine and closely related alkaloids, but no morphinane alkaloids. The various strategies followed to obtain high producing cell lines will be briefly discussed separately (see Section 11).The economics of a plant cell culture production process are discussed below (see Section 111). For cell lines that do not produce, it will be necessary to learn more about the regulation of secondary metabolism in order to eventually be able to use genetic engineering for improving production (see below).

C. BIOCONVERSION OF AVAILABLE PRECURSORS Based on knowledge of a biosynthetic pathway one can select certain steps which could be of interest for bioconversion of (a) readily available precursor(s). This could, for example, be stereospecific reactions, like the reduction of quinidinone in quinine or quinidine and the epoxidation of atropine to scopolamine. For the bioconversion one can consider using plant cells [e.g., the production of L-dopa from tyrosine by immobilized cells of Mucunapruriens (lo)]or isolated enzymes from the plant itself. An interesting example of the latter is the (S)-tetrahydroprotoberberineoxidase (STOX) enzyme, which oxidizes (S)-reticuline but not its stereoisomer (11). This feature can be used in the production of (R)-reticuline from a racemic mixture (see below). Immobilized strictosidine synthase has been successfully used to couple secologanin and tryptamine. The gene for this enzyme has been isolated from Rauuolfia (6) and cloned in Escherichia coli, in which it is expressed, resulting in the biosynthesis of active enzyme (7). The cultured bacteria produced 20 times more enzyme

8


per liter than a plant cell suspension. The genetically engineered microorganism can thus be used for the large-scale production of this intermediate for indole alkaloid biosynthesis, using tryptamine and secologanin as precursors. Strictosidine, with its two secondary nitrogens, two aldehyde groups, a double bond, and an ester group, is an ideal synthon for the (bio)synthesis of a variety of new compounds which could be studied for biological activity (12). The first small steps have been made, but the field of bioconversion still contains numerous possibilities yet to be explored. Cloning of plant genes into microorganisms could be of interest, particularly in the case that cofactors are required. Bioconversions with plant enzymes seem to offer great potential for biotechnological applications.

D. PRODUCTION BY GENETICALLY ENGINEERED PLANTSOR PLANT CELLCULTURES The difficulties of low producing plant cell cultures have already been mentioned. By unraveling the biosynthetic pathways and the regulation thereof on the level of enzymes and genes, it might become possible to identify genes which could be subject for genetic engineering. Various possibilities can be envisioned: combining genes of secondary metabolism with other promoter genes; adding further copies of an already present gene to increase enzyme production; suppressing genes by antisense DNA (e.g., blocking competitive pathways or blocking catabolism); introducing part of a pathway into another plant that is already capable of performing part of the biosynthesis. The latter approach seems particularly interesting. One could consider transferring a pathway from a slowly growing plant into a plant which grows rapidly and is suitable for agriculture (e.g., transferring the final steps of Cinchona alkaloid biosynthesis into Catharanthus roseus). This would mean that the alkaloids could be produced in an annual crop, which is more easily tuned to the demand for the alkaloid. Recently we have been able to introduce the tryptophan decarboxylase gene from Catharanthus roseus into tobacco, resulting in plants producing significant amounts of tryptamine (9), thus again proving that genetic engineering of secondary metabolism in plants and plant cells is feasible nowadays. For all applications of genetic engineering, however, one has to know the mode of regulation of secondary metabolism at the level of a number of enzymes and genes. For the near future this will be a major challenge; at present knowledge is very limited.

1.

PLANT BIOTECHNOLOGY

9

E. PRODUCTION OF NOVELCOMPOUNDS So far only the production of known compounds has been discussed. However, plant biotechnology also offers possibilities for new compounds. A number of plants have been studied phytochemically, sometimes in combination with assays for certain types of biological activity. This has resulted in discovery of numerous compounds with interesting biological activities. Many of the plants studied were collected in remote areas, and the large-scale production of the compounds isolated would be very difficult. Plant cell cultures do offer interesting perspectives, and they could be used to produce on a large scale compounds first found in the plant. Alternatively, one can screen cell cultures for new biologically active compounds. Such an approach has shown to be fruitful (Z3-16). Among others two alkaloids, pericine and apparicine, with activity in the central nervous system (CNS) have been isolated from cell cultures of Picrulima nitida (44). In connection, one might also think about the addition of elicitors to cell cultures; this would lead to the production of antimicrobial compounds (phytoalexins) which could be of interest for futher development as antibiotics. [For a review of new compounds isolated from plant cell cultures, the reader is referred to Ruyter and Stockigt

un.1

Another approach could be to use genetic engineering to introduce a further step in a biosynthetic pathway, leading to (for the plant) new compounds. This approach has, for example, been used to introduce new flower colors (18).It could also be of interest in improving the resistance of plants against microorganisms or predators. However, more insight into the role of alkaloids in plant survival in native ecosystems is needed for this.

11. Strategies to Improve Product Yield: Cellular and Extracellular Aspects

The production of alkaloids in plant cell cultures is a result of an enormously complex set of interactions between cellular and extracellular compartments. The extracellular compartment should at least offer possibilities for survival of the cellular compartment, but often cell growth and cell differentiation are prerequisites. The cellular compartment, however, is continuously changing the extracellular compartment by uptake of nutri-

10

ROBERT VERPOORTE E T AL.

ents and excretion of metabolites. The changed environment of the cells will in turn affect the cellular compartment, and so on. A plant cell culture is thus a dynamic system in which the smallest change can have large, even fatal, consequences. On the other hand, such a system offers many opportunities for manipulation to improve product yields. For alkaloid production the extracellular compartment should supply such conditions, so that the cellular compartment is able to express its secondary metabolism; furthermore one of the compartments should provide secondary product storage facilities. Many studies have dealt with characterization of the extracellular “production-induction’’ conditions. Most of the studies, however, describe only the beginning and the end of the story: for example, omitting component Y from the medium will increase alkaloid formation x times. Little insight into the processes in between or, in general, into the regulation of alkaloid formation is, at present, available. Application of recent developments in enzymology and molecular biology offer great opportunities to fill the gap in our knowledge on the regulation of secondary metabolism. In this section some of the strategies used to improve alkaloid yields in plant cell cultures are discussed.

AND SELECTION A. SCREENING

For development of an alkaloid production system the cellular compartment is the basis of the system. Based on phytochemical or chemotaxonomical data a plant species is selected. From plants of that species, cell and tissue cultures can be initiated from various types of tissue, for example, leaf, anther, or root. From this tissue several types of cultures can be initiated, for example, callus, suspension, shoot, and root. Thus, from the very beginning of the production process one already has to deal with a large number of variables. It is clear that every cell line has its own unique characteristics and that certain strategies for production improvement will only work for that specific cell line. To obtain a high producing cell line, one has to perform a screening or selection procedure. Unlike screening, selection is an active process which deliberately favors only the survival of the wanted variant while the wild-type cells do not survive. This definition was given by Berlin and Sasse (19), who wrote a detailed review of screening and selection procedures. Screening of plant material can be performed at different levels: species, specimens, organ, cell culture, and even single cells or protoplasts. At lower levels of organization selection procedures can be performed, for example, on callus and suspension cultures.

1.

PLANT BIOTECHNOLOGY

11

1. Screening

Various screening procedures have been performed, resulting in cell lines with higher alkaloid contents than that of the parent plant or culture [e.g., for ajmalicine (20) and berberine (21)l. In a comparison of 458 cell lines of C . roseus cv. Roseus, which were all initiated from excised anthers and grown under identical conditions, several different production profiles, with respect to the presence of different types of indole alkaloids, were obtained. Productivity varied from nonproducing (32% of the cell lines) up to 1.5% total alkaloids per cell dry weight (22). This example illustrates the neccesity of screening of various cell lines. Visual screening, facilitated by the color of the alkaloid, yielded highly productive cell lines of berberine (21).The strong fluorescent properties of serpentine allowed the determination of its concentration in individual cells by flow cytometry, and subsequent sorting of the cells with high contents yielded a highly productive cell line (23).The same technique was used for berberine-containing cells (24). 2 . Selection

Selection pressure on a plant cell population can be applied by the addition of selective chemicals to the medium and/or by the creation of selective growth conditions. Selective chemical agents are mainly specific enzyme inhibitors, with which selection pressure is directed to obtain cells with increased (overproduced) enzyme activity. 4-Methyltryptophan was used for the selection of C . roseus cells with high tryptophan decarboxylase (TDC) activity (25).The selected cells contained, besides significantly higher TDC activity, higher levels of tryptamine. However, only one cell line produced higher levels of ajmalicine then the wild-type culture. Using the same selective agent, Berlin et al. (26)obtained a 4-methyltryptophantolerant cell line of Peganum harmala, which produced increased levels of serotonin and, compared to the wild-type culture, similar amounts of P-carboline. Selection with 5-methyltryptophan resulted in C . roseus cells with increased levels of tryptophan; however, it showed no effect on tryptamine and alkaloid levels (27). Nicotinic acid has been used succesfully for the selection of high nicotine-producing hairy root cultures of Nicotiana rustica (28).Attempts to use toxic end products such as vinblastine (29) and quinoline alkaloids (30) as selective agents for obtaining alkaloid-producing cells of Catharanthus and Cinchona, respectively, were not successful. Selection by changed environmental conditions were used for obtaining photomixotrophic and photoautotrophic cells. This yielded leaflike cells

12


with well-developed chloroplasts. Lupine cells of this type were able to produce sparteine and lupanine (31).However, photoautotrophic cells of C . roseus did not produce vindoline (32).

B. CULTURECONDITIONS The environment of the selected plant cells should provide optimum conditions for the cells to express their genetic information concerning secondary metabolite formation, resulting in optimum levels of secondary product. Optimization of environmental conditions is a matter of trial and error because fundamental knowledge on the regulation of alkaloid formation is lacking. Conditions which have been reported to influence the productivity of the culture are, among others, the composition of the culture medium, light, temperature, bioreactor type, and aeration. Several methods have been used to determine systematically the influence of the different parameters on growth and production in a limited number of experiments (33-35). 1. Medium Composition Optimization of medium composition has led to the development of several media (20,36)that induce increased production of indole alkaloids. Some important factors concern the concentration and type of the carbon and nitrogen sources, the phosphate concentration, and the type and concentration of growth regulators. Induction and production media are used in a two-stage process: in the first stage biomass is produced, and the second stage allows alkaloid production. Morris, however, selected conditions for a Catharanthus roseus culture in which high alkaloid accumulation could be combined with high biomass accumulation (37). The initial pH of culture media is generally adjusted to 5.5-6. So far, little is known about the influence of the initial pH of the culture medium on productivity. For Catharanthus roseus cultures contradictory results have been reported: no effect at an initial pH of 5.5, 6.0, or 6.5 (38) but higher productions at pH 5.5 (39) and at pH 7.0 (40), with the results seeming to be cell line specific. In cultures of Hyoscyamus muticus a 7-fold increase in alkaloid production was found on media with an initial pH of 3.5 (41).More alkaloid was released by Nicotiana rustica hairy roots when grown on media with lower initial pH values (i.e., pH 5-5.5); buffering of the media with 50 mM MES decreased growth and total alkaloid production (42). Increased alkaloid production induced by increased osmotic strength of the culture media was detected in Catharanthus roseus suspension cul-

1.

PLANT BIOTECHNOLOGY

13

tures (43,44) and hairy root cultures of Hyoscyamus muticus (45).Osmotic agents that have been used include mannitol(43), NaF (46),and NaCl(47). Increased osmotic stress inhibited cell division and increased the rate of alkaloid production (43). 2. Light Light was found to stimulate serpentine production in Catharanthus roseus cultures (48);an inhibitory effect on alkaloid production was observed in cultures of Cinchona ledgeriana (49),Nicotiana spp. (50,51),and Scopolia parviJEora (52). In a light-grown Agrobacterium tumefacienstransformed culture of C. ledgerianu, 5- to 10-fold lower levels of tryptophan decarboxylase and strictosidine synthase were detected as compared to dark-grown cultures (53). The induction of serpentine production in light-grown Catharanthus roseus cultures probably resulted from a combined effect of light and medium composition: the induction by light was highest in the presence of low concentrations 2,4-dichlorophenoxyacetic acid (2,4-D), phosphate, and mineral nitrogen (48). The influence of light on product formation has been recently discussed (54).In large-scale fermentations the enhancement of product formation by light will be difficult because of technical and economical constraints. 3. Temperature Culture temperature influences both growth rates and productivity. By lowering the culture temperature of C. roseus cells from 27 to 16"C, a strong reduction in growth rate and a strong increase in alkaloid content was observed (55); however, the alkaloid yield per liter medium was not changed substantially. At a culture temperature of 38°C the alkaloid content was strongly reduced. The influence of culture temperature on the growth and productivity of C. roseus cells was also studied by Morris (56,57). For both biomass and alkaloid production the yield curves showed sharp maxima. By changing the temperature the serpentine/ajmalicine ratio could be influenced. Owing to temperature differences in the climate chambers in winter and summer seasons, even seasonal variation might occur (5637). An increased culture temperature may stimulate excretion of products, for example, as has been found for a transformed root culture of Datura stramonium. A 7- to 10-fold higher content of atropine in the medium was obtained at a culture temperature of 30°C compared to a temperature of 25°C (58). 4. Cell Density Cell density affects productivity directly: the more producing cells per liter, the more product per liter. High cell densities require specific pre-

14


cautions with respect to oxygen and nutrient supply (59). By adjusting aeration (oxygen added to the aeration gas) and stirring, Coptis japonica cells were cultured at densities of up to 75 g/dm3 (dry weight) in a culture tank fitted with a hollow paddle-type stirrer (60). Large-scale cultivation of transformed root cultures need specific requirements for growth and harvesting; a 500-liter droplet (or mist-phase) reactor in which the roots are immobilized in a matrix has been developed (61).

5. Gas Composition Aeration of suspension cultures grown in bioreactors needs to be optimized with respect to dissolved oxygen and carbon dioxide levels. High aeration rates may lead to excessive (toxic) concentrations of dissolved oxygen, increased shear forces, and subcritical levels of key volatiles such as gaseous hormones (ethylene) and carbon dioxide (62). Carbon dioxide enrichment of the aeration mixture increased biomass yields (63,64).Van Gulik (65) has recently reviewed this subject in detail. In shake flasks the dissolved oxygen and carbon dioxide levels are strongly determined by the type of closure of the flask. A cotton wool plug is highly permeable. Two layers of aluminum foil rapidly result in oxygenlimited growth of the culture. Silicon foam stoppers combine a good and reproducible gas exchange with low evaporation (66), which allows growth-curve determinations in a single flask by measuring the loss of weight caused by dissimilation (67). 6. Shear Stress

Shear stress caused by impellers in stirred tank fermentors proved to be less detrimental to suspension cultured cells than was expected from first reports (68). Several cell lines were resistent to impeller speeds of 1000 rpm (66,69-72). The various aspects of large-scale plant cell culture have been reviewed recently (73) (see also below). C. ALKALOID STORAGE COMPARTMENTS In plants the site of alkaloid biosynthesis is often separated from the site of storage; for example, tropane alkaloids are produced in the root and stored in the leaves. This means that the alkaloids need to be excreted from the biosynthetically active cells and then transported to and taken up by the storage cells. In undifferentiated tissue like cells in suspension culture this transport mechanism is likely to be seriously affected; this might result in low productivity. Uptake of alkaloids by isolated vacuoles and transport mechanisms over membranes have been studied in detail. Carrier-mediated active transport

1.

PLANT BIOTECHNOLOGY

15

over membranes (and thus a specific uptake) has been postulated for indole alkaloids (74), isoquinoline alkaloids ( 7 3 , quinolizidine alkaloids (76), and pyrrolizidine alkaloids (77). On the other hand, a passive transport mechanism, in which the neutral alkaloids freely diffuse through the membrane and the protonated alkaloids are stored in the acidic vacuole (ion-trap mechanism), has been advocated for indole alkaloids (78-81), quinoline alkaloids (82),and nicotine (83)in cells of C. roseus, Cinchona ledgerianu, and Acer pseudoplantanus, respectively. Ajmalicine diffuses across the tonoplast, driven by the pH gradient between cytosol and vacuole. It is subsequently trapped inside the vacuole in the form of serpentine, which is formed via oxidation of ajmalicine by the basic vacuolar peroxidases (81). Little is known about transport in the opposite direction, that is, excretion. For the ion-trap mechanism the pH difference over the membrane is the driving force for transport. A changed pH gradient caused by, for example, a low extracellular pH, might thus cause excretion. Excretion of alkaloids at low pH has been reported for Nicotiana, Cinchona (42), and Catharanthus cultures (84). Caffeine, an alkaloid with few basic properties, proved to be freely diffusible between cells and medium of a Coffea suspension culture (85). A cell line of Thalictrum minus was reported to excrete large amounts of berberine, a quaternary alkaloid (86); the spontaneous excretion could be inhibited by ATPase inhibitors, indicating an active (energy-requiring) transport mechanism. In some cases nature can be helped by release-promoting techniques such as immobilization, permeabilization, pH cycling ( 8 4 , electroporation (87), and elicitation (see below). Storage capacity in a cell culture is available inside the cells (e.g., vacuoles) or in the extracellular compartment. The storage facilities of the latter can be remarkedly improved by the addition of liquid organic phases, resins, or other sorbents to the medium. Addition of XAD-7 to Catharanthus roseus cultures resulted in increased yields of indole alkaloids (88). Addition of a dimethylsiloxane polymer to Eschscholtzia californica cultures improved yields of benzophenanthridine alkaloids (89), and increased yields of nicotine and anabasine were obtained by adding of XAD-2 and XAD-4 resins to Nicotiana transformed roots (90,91). The internal storage capacity is limited by the number of storage cells. Excreted alkaloids which are dissolved in the medium are exposed to catalytic activities in the medium (e.g., peroxidases) and can thus be degraded; as was demonstrated for quinolizidine alkaloids in lupine cell cultures (92). A role for peroxidases in product degradation has been recently demonstrated for indole alkaloids produced by suspension cultures of Tabernaemontana. Reduction of peroxidase activity by removal of Ca2+ ions from the medium resulted in the formation of the alkaloid

16


apparicine, which is not isolated from cultures with normal Ca2+ concentrations in the medium and thus high peroxidase activity. Apparicine is rapidly degraded after exposure to peroxidases (93). D. ELICITATION Elicitors are compounds which are able to induce the biosynthesis of phytoalexins or, in general, the biosynthesis of stress metabolites. Various elicitor preparations have been used, ranging from homogenates of microorganisms to metal ions. Homogenates of microorganisms may contain actual elicitor molecules, such as oligosaccharides or fatty acid derivatives. Other elicitors, like cellulase, metal ions, and UV light, release endogenous elicitors from the plant cell wall. The microbial and endogeneous elicitors are able to bind (largely unknown) receptors. Following receptor binding a set reactions is induced, in which ethylene, CAMP, and Ca2+play a role and which eventually leads to de nouo biosynthesis of the phytoalexins. This subject has been recently reviewed (94). There are only a limited number of alkaloids whose production can be induced by elicitors; the effects are cell line specific and often transient. Elicitors have been found for the induction of biosynthesis of, for example, indole alkaloids [ajmalicine in C. roseus cultures (95)], benzophenanthridine alkaloids [sanguinarine in Papaver cultures (96)], acridone and furanoquinoline alkaloids [in Ruta gruueolens cultures (97)], and protoberberine alkaloids [berberine in Thalictrum rugosum cultures (9691. When the alkaloids are excreted in the medium after elicitation, the biomass can be recycled and reelicitated. Successful reelicitation procedures have been developed for the production of sanguinarine and ajmalicine in Papaver and Catharanthus cell cultures, respectively (99). E. FEEDING OF PRECURSORS AND BIOCONVERSIONS Production can be increased by addition of precursors to the culture media, in which cases the precursors are not metabolized in the medium and, after uptake, appear in the right compartment of the plant cell. For C. roseus cultures, for example, it was found that increased indole alkaloid production was obtained after feeding with L-tryptophan, tryptamine, secologanin, loganin, loganic acid, or shikimic acid (20). Cell cultures have also been used for biotransformations, for example, the conversion of (-)-codeinone to (-)-codeine in Papauer sornniferum cultures (ZOO). For the tropane alkaloids a large number of precursor feeding and biotransformation studies with cultures of various solanaceous plants have been performed (see below).


17

Bioconversion rates can be optimized by using immobilized cells, cellfree preparations, or immobilized (purified) enzymes. Furuya el al. (101) reported the reduction of codeinone to be more efficient with immobilized P. somniferum cells than with suspended cells. Strictosidine could be produced in large quantities using immobilized strictosidine synthase (102). The potential for this use of plant cell cultures is enormous; however, the number of successful applications is still limited.

F. IMMOBILIZATION Fixation of plant cells in a matrix of, for example, polyurethane foam or entrapment of the cells in calcium alginate beads provides an artificial surrounding for the cells, which protects them from hydrodynamic stress. high cell densities inside the matrix also allow cell to cell contact and communication. Inside the immobilized matrix nutrient and product gradients may occur. Furthermore, immobilized biomass is easily separated from the medium, which is useful in production and biotransformation systems. Immobilization of plant cells has been reviewed (103,104).

Large-scale immobilized alkaloid production systems have been described for C. roseus and Thalictrum rugosum, using glass fiber mats (105). Cinchona pubescens cells were efficiently immobilized in a semirigid matrix of polyurethane foam (106,107). Polyurethane foam was also used for the immobilization of P. somniferum cells; the cells were used for the biotransformation of codeinone to codeine (108). Cells of the same species were surface immobilized on a fabric of loosely woven polyester fibers (72). By elicitation and product adsorption on a hydrophobic resin, sanguinarine yields of 130 mg/g dry weight were obtained. Immobilization of Coffeu cells in calcium alginate resulted in a 13-fold increase of purine alkaloid production (109,110). G. PERMEABILIZATION

Efficient downstream processing of products is facilitated when the product can be recovered from the medium. Besides by elicitation and immobilization, excretion of the product can be obtained by permeabilization of the cells. Various strategies have been used: chemical permeabilization, electrical permeabilization, ultrasonic permeabilization, and iontophoretic permeabilization (111). Chemical permeabilization comprises the use of, among others, organic solvents as DMSO and chloroform and surface active chemicals (as Triton X-100).Results with the various tech-

18


niques used are always achieved at the cost of cell viability, and this might hamper further applications (111). H. DIFFERENTIATION AND CULTURE TYPE The formation in tissue cultures of several types of indole alkaloids was shown to be inseparably connected with morphological differentiation of the cells. For Catharanthus, a time course study has been made of the formation of alkaloids in seedlings, during the first stages of development (112). It was demonstrated that the formation of vindoline, and thus the synthesis of vinblastine, was connected to morphological differentiation. This explains why C . roseus suspension cultures do not produce vinblastine. Recently, a similar experiment was performed with Cinchona seedlings (113). In Cinchona seedlings alkaloid formation is abundantly expressed, but in suspension cultures productivity is very low, if any. Also, the formation of Zboga indole alkaloids in Tabernaernontana tissue cultures proved to be dependent on morphological differentiation (114). On the other hand, cell cultures are capable of producing alkaloids which had not yet been detected in the plant. In a recent review it was reported that a total of 85 novel compounds, including 23 alkaloids, have so far been isolated from 30 different plant cultures (17). Propagation of differentiated tissues as root and shoot cultures offer an alternative when the desired product is not formed in suspension cultured cells. In shoot cultures of C . roseus up to 2.6 pg of 3',4'-anhydrovinblastine per gram fresh weight was detected (115); shoot organ cultures of Cinchona ledgerianu produced 3.5 mg of alkaloids (quinine and quinidine) per gram tissue (116). Root cultures of this species were also able to produce these alkaloids; the productivity was increased 5 times by feeding tryptophan, a precursor of the alkaloids, to the culture (117). Alkaloids were also detected in root cultures of Hyoscyarnus niger (118). For most alkaloid-producing plants hairy root cultures, obtained via infection of the plant material with Agrobacteriurn rhizogenes, have been initiated: Hyoscyarnus, Datura, Atropa, Nicotiana, Catharanthus, Cinchona, and Peganurn. In general the alkaloid contents found in hairy roots are similar to those found in normal plant roots. An interesting aspect of hairy root cultures is that with A. rhizogenes other new genes can also be introduced, for example, genes connected with secondary metabolite production. Because of their rapid growth and the potential of genetic engineering, hairy roots may become superior producers compared to the plant. Hairy roots, or transformed cells, have been shown to be less sensitive to optimization procedures such as medium optimization (26) and elicitation (119).

1.

PLANT BIOTECHNOLOGY

19

I. GENETIC APPROACHES AND GENETIC MODIFICATION Several techniques are now available to change the genetic information in plant cells: transformation with Agrobacterium spp., direct DNA injection, and protoplast fusion. Novel techniques in molecular biology have found rapid application in plant cell biotechnology, as, for example, the use of antisense DNA (9,120,121). Knowledge concerning the genes involved in alkaloid biosynthesis can be exploited in several ways to obtain higher yields. Rate-limiting steps in the biosynthesis may be overcome by increasing the concentration of the enzyme. For this approach knowledge of the rate-limiting steps and characterization of the enzymes are prerequisites. Recently the number of characterized enzymes involved in the biosynthesis of alkaloids has increased exponentially. The pathways leading to berberine (121) and ajmaline (122,123) have been fully characterized. Substantial progress has been made with scopolamine biosynthesis (124-126) and early steps in indole alkaloid biosynthesis (6-9,127-130). After purification of an enzyme one is able to identify the gene encoding for this protein, which opens ways to genetic modification. Genes for various enzymes have been cloned, and several transgenic organisms have been obtained, for example, an E. coli strain producing strictosidine synthase (7) and tobacco plants containing tryptophan decarboxylase activity (9). In some cases microbial genes were also used for manipulation, for example, lysine decarboxylase (131), ornithine decarboxylase (132), and cytochrome P-450 (133). Further genetic strategies may include inhibition of competitive pathways which might lead to increased availability of precursors for alkaloid biosynthesis, or the opposite, the creation of a new branch on an existing pathway. Both strategies can be envisioned for both cell cultures and plants. An interesting option is introducing certain steps of a secondary pathway from one plant to another, with the aim to have the recipient plant produce a certain desired compound. This could, for example, lead to plants which could be harvested every year, instead of after 5-10 years. Introduction of the genes responsible for quinoline alkaloid biosynthesis from Cinchona into Catharanthus could be such an example.

J. COMBINATION OF TREATMENTS: TOWARD HIGHPRODUCTIVITY In the previous sections various treatments have been discussed which might lead to increased product yields. Many of the strategies have only limited value: they only can be used for specific cell lines, the effect is transient, or it causes cell death. At the moment these strategies are,

20


however, the only tools available to manipulate productivity. Most of the epigenetic treatments were reported for the first time more than a decade ago. At present much attention is paid to combined strategies, for example, elicitation of alginate-immobilized Catharanthus roseus cells led to a 45-fold increase of ajmalicine production (90 mg/liter), which was adsorbed from the medium by the resin XAD-7 (134). Combined treatments were also used for P. somniferum cultures (72), where immobilization, elicitation, and product adsorption have lead to high productivity of sanguinarine . For further development of an (economical interesting) production system one has to obtain a stable, with respect to production capacity and growth, cell line, which responds reproducibly to the selected treatments. So far few cell lines have been reported to be stable, but hairy root cultures in particular have a major advantage here (135). Many of the high producing cell lines obtained by various screening and selection procedures proved to be unstable: after a few months of subculture productivity returned to the same level as the wild-type culture. If stability cannot be obtained, a reliable backup system has to be developed, for example, by means of cryopreservation or other storage techniques. For a stable cell line growth and production can be optimized by changes in medium composition. When the product yield is still low even with medium modifications, improvement of product yields by a genetic strategy is the most rational, despite the fact that this may be a long and difficult task. An increase in concentration of a bottleneck enzyme will not necessarily lead to higher product levels as one has to increase the total substrate flow through the whole pathway. Elicitation or induction media might be of help for this. Furthermore storage facilities (and transport mechanisms) should be present in abundance and in such a form that the products are protected against catabolic processes. The latter aspect especially needs more attention as the major research effort is directed to aspects of biosynthesis. Alternative processes can consist of the production of precursors, on which the final steps are performed either by isolated (immobilized) enzymes or by chemical synthesis, or, alternatively, by bioconversion of readily available synthetic precursors with plant cells or isolated enzymes (see Section 111).

111. Large-Scale Production

The number of alkaloids which have been produced on an industrial scale through new biotechnological strategies is still rather small. There are three interrelated categories of problems hampering the development

1.

PLANT BIOTECHNOLOGY

21

of industrial plant biotechnology : biological problems, economical problems, and technological problems. Below we briefly discuss some aspects of these problems. Biological Problems Although there has been a huge effort in investigating plant cell cultures, our fundamental knowledge still is restricted. Essential problems with respect to industrial production of alkaloids are the following: biochemical pathways and their regulation are only partly known; the effects of environmental conditions on growth and production need more investigation; and productivity of selected or genetically modified plant material might be unstable. In section I1 these problemshave already been discussed as well as the strategies used to solve them. Economical Problems By conducting a feasibility study the bottlenecks in the economics of production processes employing plant cells in suspension culture can be assessed. Data from the few studies published on this subject have shown the need for further research. In general the productivity of plant cell cultures per amount of reactor volume per unit time has to be increased considerably in order to make most processes economically feasible. In this section these aspects are discussed in more detail. Technological Problems A general scheme for the development of an industrial process for alkaloid production is depicted in Fig. 1. On the basis of both fundamental research and feasibility studies the decision can be made whether an industrial production process is achievable. For the design of the process (production volume, process type, bioreactor size and type) detailed knowledge of both the kinetics of growth and product formation and physical properties (rheology, shear sensitivity) is essential. The design should be developed in interaction with the downstream processing possibilities. After a process design has been made, the behavior of the process on a large scale can be predicted by scale-up studies. On the basis of these studies a final process design can be made. Problems in the following fields have to be anticipated. Reliable data on the kinetics of growth and product formation as well as data on the physical properties of plant cell cultures are rarely available, especially on a large scale. Determination of the data are difficult and time consuming.

A. ECONOMIC FEASIBILITY A number of cost-price estimations for products of industrial plant cell biotechnology have been published. The main aim of such studies is, of

22


Fundaments

FIG. 1. General design strategy of a biotechnological process.

course, analysis of the economic feasibility of an industrial process, although they may also reveal specific bottlenecks hampering commercialization. Goldstein et al. (141) published a study concerning the production of compounds by plant cell biotechnology . They developed a comprehensive process design and calculated the cost-price of products on the basis of this design. In their study they used growth and production parameters reflecting the state of the art in 1979, as well as more optimistic figures. They assumed production volumes varying from 10 to 1000 tondyear. In a Dutch report (2) the results from the Goldstein study are compared with data form the natural product market (Fig. 2). It is obvious from Fig. 2 that only a few products can be considered as candidates for industrial production; moreover, the first product of plant cell biotechnology, shikonin, is either hardly profitable or the data used deviate from the real situation. Fowler and Stepan-Sarkissian (142) estimated total costs of about U.S. $35,00O/kgfor the production of 375 kg/year of serpentine. Drapeau et a f . (143) reported a cost estimate for commercial-scale production of ajmalicine-rich Catharanthus roseus biomass. At the current state of technology the cost would be approximately U.S. $3215/kg ajmalicine. They suggest the coproduction of catharanthine could relieve the cost of ajmalicine production.

1.

PLANT BIOTECHNOLOGY

23

The studies mentioned above are based on the classic biotechnological approach of producing a secondary metabolite, namely, a two-stage process. During the first stage an amount of biomass is produced under conditions favoring growth, and during the second stage the desired secondary metabolite is formed under conditions favoring product formation. In most cases the product is accumulated by the cells and therefore has to be extracted from the biomass. An alternative approach is the use of more advanced technology such as, for example, reuse of biomass. Reuse of biomass can be accomplished either with free cell systems or with immobilized cells. For this approach it is essential that the product be excreted into the culture medium. Van Gulik et al. (I&), in a feasibility study on the production of ajmalicine, compared the classic two-stage process with a method where the produced biomass is used for longer periods of time. The parameters used in this study are given in Table IV. The values for both growth and production parameters represent reasonable figures from the literature available at the time the calculation was performed. The lower biomass concentration in the case of production with release by permeabilization was supported by the fact that a satisfactory recovery of ajmalicine is only feasible when the volume of the biomass does not exceed 30% of the reactor volume. A higher biomass volume results in a larger amount of ajmalicine remaining inside the cells, which results in a lower recovery. For calculation of the fermentation equipment needed, the authors started with the yearly ajmalicine production required and then calculated backward to the total reactor volume needed for the production stage, followed by calculation of the reactor volume needed to produce the inoculum for the ajmalicine production reactors. The investment costs needed for the process were roughly estimated by multiplying the equipment costs with the so-called Lang factor, a factor commonly used for cost calculations in fermentation processes (145).The annual costs were estimated by depreciation of capital equipment over a 10-year period. The medium costs were calculated with glucose as the carbon source. The bulk of the energy needed for the process will be consumed by the compressors and agitators in the reactors (energy costs needed for sterilization were included in the medium costs). A summary of the results of these calculations are given in Table IV. Costs of downstream processing and labor are not included in the calculation. The results show clearly that the stage of product release is a weak point in the current process. This is caused by the necessity to work with a lower final biomass concentration to achieve satisfactory product release. The advantage of reusing the biomass is marginal because the price-determining factor is not biomass production

24


GOLD

0

ILCHICINE OlGOXlN

0

;IKONIN

10'

RESERPINE

0

PILOCARPINE

0

10

PYRETHRINS ATROPINE

0

OPIUN

0

10

I

I

I

1

10

100

,DIOSGENIN

1000

Market volume ( t o n / y e a r )

FIG. 2. Relationship between estimated market price and market volume of a number of different plant products. The upper line represents the relation between the cost-price of a product produced by means of plant cell biotechnology and yearly production, at the current state of the art. The lower line is calculated from more optimistic figures and therefore represents a future situation. Products situated between the lines might be interesting goals for plant cell biotechnology in the near future.

1.

25

PLANT BIOTECHNOLOGY

TABLE I11 PARAMETERS USEDFOR ESTIMATION OF COSTSOF AJMALICINEPRODUCTION BY CELL CULTURES OF Catharanthus roseus Design basis Yearly production of ajmalicine Product loss during recovery and purification Effective yearly operation period Growth parameters Specific growth rate Initial biomass dry weight concentration Inoculation ratio Dry weight yield from glucose Maintenance energy requirement Maximum oxygen uptake rate Single use of biomass Final biomass dry weight concentration Final biomass fresh weight concentration Final ajmalicine content of the biomass after a 21-day production period Repeated use of biomass Specific productivity in case of spontaneous product release Final ajmalicine content of the biomass in case of forced release

3000 kg 20% 300 days 0.029 hr-’ 2.5 kg/m3 1:7 0.61 kg/kg 0.0066 kg/kg . hr 0.0154 kmol/m3 . hr 40 kg/m3 320 kg/m3 0.009 kg/ kg 2.36 x W5kg/kg . hr 0.009 kg/kg

but product formation. For the same reason, increasing the productivity increases the economy of the process substantially.

B. TECHNOLOGICAL ASPECTS 1. Growth and Production Kinetics

To design a production process to obtain alkaloids from plant cell cultures with a promising productivity on a laboratory scale, detailed knowledge of the kinetics of growth and product formation of the cell culture is essential. In order to obtain a quantitative description of the behavior of the cell culture as a function of the external conditions, this knowledge is embedded in a mathematical model. On the basis of the model a process design can be made after which the economic feasibility of the process can be assessed. After implementation of the process the model is subsequently used in process control and optimization. The number of publications dealing with quantitative research on the kinetics of growth and product formation of cultured plant cells has been very limited. For the scarce kinetic research that has been reported batch cultures were used, which were mostly grown

26


TABLE IV BREAKDOWN OF COSTSOF DIFFERENT PROCESSES FOR LARGE-SCALE PRODUCTION OF AJMALICINE BY CELLCULTURES OF Catharanthus roseus

Product extraction Gross reactor volume for product formation (m3) Largest reactor volume for biomass formation (m3) Volume of storage tanks

6 x 145

37

Product extraction (lox increased productivity) 6 X 15

Natural product release 6

X

250

Product release by permeabilization with DMSO 6

335

10

82

3.8

X

3 x 1.2

7 x 150

7 X 150

1 . 1 x lo6

5.4 x 106

5.4 x lo6

0.75 x lo6

0.1 x lo6

3.3

x lo6

0.75 x lo6

0.08 x lo6

1.5

X

4.5 x lo6 1500

3 x 10

(m3) Depreciation costs (U .S .$/year) Medium costs (U.S.$/ year) Energy costs (U.S.$/ year) Total costs (U.S.$/year) Costs (U.S.$/kg ajmalicine)

3 x 106

5.1

X

lo6"

lo6

1.5 x lo6

1.3 x lo6

10.2 x lo6

12 x lo6

430

3400

4000

Including DSMO.

in shake flasks. The use of batch cultures has the advantage that the culture system is relatively simple, while the time needed to conduct an experiment is relatively short. An important disadvantage is that in a batch culture almost every parameter changes in time, thus making it very difficult and sometimes even impossible to draw the right conclusions from an experiment. An alternative that does not suffer these disadvantages is the chemostat culture. In a chemostat the cells can be grown at a constant growth rate at constant concentrations of the various nutrients of the growth medium, thus providing the opportunity to change only one variable at a time. It should be stressed that the chemostat culture is not considered to be a system for alkaloid production but rather a tool to obtain reliable kinetic information. In a future production process the use of batch or batch-fed culture is more likely, owing to the greater flexibility of such a process, the lower complexity of the culture system, and, as a consequence, lower equipment costs. Aspects of the various culture types are discussed in more detail below.

1.

27

PLANT BIOTECHNOLOGY

a. Mathematical Modeling. The character of the mathematical description of a physical, chemical, biological, economical, or social process depends on the character of the process, the available knowledge, and the intended use of the model. Models can be subdivided depending on our knowledge about the process (black box, gray box models), the number of units involved (stochastic or deterministic models), the consideration of particles (continuum or corpuscular models), and the eventual subdivision of the units of the total process (structured or unstructured models). For detailed information on modeling one should consult the specialized literature (e.g., see Refs. 146-148). Although several types of models have been explored in the description of biological systems, the unstructured black box continuum models based on a linear equation for substrate consumption are still most frequently used for the description of fermentation processes. In these models the growth rate of the cells generally is assumed to be a function of the external concentration of the growth-limiting substrate C, according to Monod kinetics : dC,/dt =pmaxx C,/(C,

+ K,)

x C,

The conversion rate of the growth-limiting substrate is divided into a growth rate-dependent term and a growth rate-independent term according to the following linear equation: dC,/dt

=

-(l/Y,,

x dC,/dt

+ m,

x C,)

where C , is the biomass concentration (in kg/m3), t the time (sec), pmax the maximum specific growth rate (l/sec), K , the saturation constant on substrate (kg/m3), C , the substrate concentration (kg/m3), YSxthe net biomass yield on substrate (kg/kg), and m, the maintenance coefficient (l/sec). The growth rate-independent, or maintenance, energy needed is assumed to be supplied by combustion of substrate (149),biomass (150),or a combination of both (151). These models can be extended for product formation. In population biology deterministic or stochastic corpuscular models are used, describing the behavior of a population of single cells or organisms. If cell division is considered a random process and the distribution of cycle times is assumed to be described by a normal distribution, population size models of the form dN(t)/dt = p x N(t)

(3)

where N ( t ) denotes the number of cells at time t , are formulated. Reviews of population size models are given by Eisen (152) and Bertuzzi and Gandolfi (153). Kato and Nagai (154) described the growth of their batch cultures of

28


Nicotiana tabacum with a linear substrate consumption model [Eqs. (1) and (2)]. The model parameters obtained were compared with values reported for Penicillium chrysogenum, Escherichia coli, and Saccharomyces cereuisiae. They concluded that the net yield of N . tabacum biomass on substrate was relatively high whereas the maintenance energy requirement was relatively low compared to the reported values. These observations were explained by the fact that tobacco cells contain large amounts of stored carbohydrates (e.g., starch). Because the energy required for synthesis and maintenance of storage carbohydrates is relatively low this might be the cause of the observed high yield and low maintenance coefficients. Pareilleux and Chaubet (155,156) studied batch suspension cultures of apple cells and Medicago satiua cells, reporting comparable high yield and low maintenance coefficients. Bailey and Nicholson (157) employed in principle the same model structure. However, in order to describe also the changing fresh weight/dry weight ratio, product formation, and cell death and lysis, they extended the model. Two extra variables were added to allow the prediction of the extension phase and a more accurate prediction of the culture death phase caused by shear stress. Another two variables were introduced to describe product formation. In a later paper (158) the authors extended the model further to describe the influence of temperature. With this model an optimal temperature control strategy was predicted. Van Gulik (65,159) performed glucose-limited chemostat experiments with the aim of examining whether the growth of plant cells in suspension cultures could be described satisfactorily with the linear equation for substrate consumption combined with Monod kinetics [Eqs. (1) and (2)]. Cell suspension cultures of two different species were used, Catharanthus roseus and Nicotiana tabacum. For both species the specific substrate consumption rate under steady-state conditions showed a reasonable fit to the linear equation. To give an example, the C. roseus data are shown in Fig. 3. It was reported that the model was of limited value under nonsteady-state conditions. With the model only a poor description of the relationships between growth, substrate uptake, and specific respiration in batch culture could be obtained (Fig. 4).This was partly caused by the fact that during growth in batch culture large amounts of storage carbohydrates are often accumulated. In the case of C. roseus the total amount of stored carbohydrates reached a maximum of nearly 40% of dry biomass concentration, shortly before the depletion of glucose from the medium (Fig. 5 ) . In order to describe a varying biomass composition, however, a metabolically structured model is needed. De Gunst et al. (160-162) used the stochastic corpuscular approach to

1.

29

PLANT BIOTECHNOLOGY

0.05 I A

r

- 0.04

8

; I

0

0.03

E

0.02

Ll

0

v

v,

0.01

0-

0.00 0.00

0.01

0.02

FIG. 3. Relationship between specific glucose uptake rate (4s) and dilution rate (0)in glucose-limited chemostat cultures of Catharanthus roseus.

describe the growth of plant cells in suspension culture. In the proposed model the total cell population is assumed to consist of two subpopulations: dividing cells and nondividing, differentiating cells. The number of dividing cells is assumed to depend on the hormone concentration in the medium, whereas the duration of the cell cycle is a function of the substrate concentration. In Fig. 6 model predictions of the growth of Nicotiana tabacum cells, with respect to dividing, nondividing, and total cells, are shown. The authors compared only the predicted total cell number with experimental data. Although this model needs more experimental validation and the character of the controlling factors (substrate and hormone levels) is not proved, it has some interesting features. First, it corresponds with the observation that cell cultures may consist of different cell types. Second, there are several examples showing that production of alkaloids is a capacity of differentiated cells. Therefore, models describing the development of differentiated cells in a cell culture may be of great importance for the design of alkaloid production processes. b. Conclusions. When black box models, in which biomass is considered as one compartment with a constant composition, do not provide a satisfactory description of the process, one has to develop more sophisticated models. In general this will result in a subdivision of the biomass into more than one compartment. However, increasing the complexity of a model results in an increasing amount of parameters to be determined. The applicability of highly complicated structured models might therefore be doubted. In practice a compromise should be obtained; the complexity of

30

ROBERT VERPOORTE E T A L .

-

0.025

1.00

=5

f

0.80

3 0

0.60

~

-

‘1. 0.40

; I

:

0.020

E

0.010

‘1 . 0

0

0 u

B

h

0)

0.015

v

g

e4

0.20

0.005 ‘ 9

*.I9

0.000

0.00 0

120

360

240

0

120

240

360

Time (h)

Time (h)

0.025 I h

L:

-

z I

0.020

o

0.015

E

0.010

‘1. v

hl

I

C

0.005

0-

0.000

0

120

240

360

Time (h)

FIG. 4. Growth of Catharanthus roseus cells in batch culture. (A) Concentrations of glucose (squares) and biomass (circles), (B) oxygen consumption, and (C) carbon dioxide production are given as a function of culture time. Vertical bars represent the standard deviation of the mean. Solid curves represent the model predictions.

the model should be as low as possible while still providing acceptable accuracy. Furthermore, the structure of the model should allow experimental validation. In order to develop structured models for the description of growth and product formation of cultured plant cells, knowledge of the pathways of primary and secondary metabolism is essential. This knowledge is still limited, however, especially with respect to secondary metabolism. Corpuscular models are not common in process development. It is interesting, however, to realize that corpuscular models of the type developed by de Gunst er al. (160-162) do offer the possibility of describing cell differentiation.

1.

31

PLANT BIOTECHNOLOGY

c

1

.-0,

-.-.-

Q)

-0-

40

3 x

Starch Sucrose

I

73

30

-0-

v) v)

0

E

20

cc

10

0 .a

Glucose Fructose

-+-

Total

0

M

0 0

72

144

288

216

360

T i m e (h)

FIG. 5. Changes in the amounts of intracellular carbohydrates in Catharanthus roseus cells

1,

,*..............

-**

i0

do

\

I

60

I

80

I

-

100 Time (hours)

I

120

I

140

I

160

FIG. 6. Experimental data from the growth of a batch culture of Nicotiana tabacum cells (circles). The solid line represents the prediction of the total number of cells obtained by the corpuscular growth model, the dashed line represents the predicted number of dividing cells, and the dotted line represents the predicted number of nondividing cells.

32


2. Process Design and Bioreactor Type a. Process Design. Whereas a plant is a fully organized structure containing specialized cells forming different types of tissue, a cell suspension culture reflects the totally unorganized situation. Between these two extremes there are several other ways to cultivate plant cells. The commonly used methods to grow plant cells in culture are as follows (with an increasing degree of organization):

Cell suspension cultures Immobilized cells Surface immobilization (biofilm) Gel-entrapped cells Callus cultures Organ cultures Roots Shoots Hairy roots Cell suspension cultures offer some advantages over the more highly organized structures. In a cell suspension culture the transport of nutrients, oxygen, precursors, and/or elicitors to the cells is not hampered by the limited diffusion which may occur in relatively large tissue structures. Another important advantage is the fact that plant cell suspension cultures can be treated almost exactly like cultures of microorganisms. This opens the possibility of using, to a large extent, existing knowledge, equipment, and technology. An important disadvantage which is frequently mentioned in the literature is the low degree of cell differentiation in suspension cultures of plant cells. As product formation in plants largely occurs in differentiated tissue, it seems reasonable that the undifferentiated state might not favor the formation of secondary products. In some cases, for example, the production of ajmalicine in cultures of Catharanthus roseus, it is indeed shown that the product, originally formed in the roots of the plant, is better produced in hairy root cultures than in cell suspension cultures. The use of immobilized cells opens the possibility of keeping the produced biomass in the reactor and using it for prolonged periods of time, under the constraint that the product is excreted by the cells. A disadvantage is the possible occurrence of mass transport limitations inside the biomass beads or in the biofilm. Much of the technology to realize a large-scale process with immobilized cells has yet to be developed, and it might well turn out to be economically unfeasible in most cases.

1.

PLANT BIOTECHNOLOGY

33

Culturing organized tissue such as callus, root, or shoot cultures offers the advantage of some degree of cell differentiation, which is still relatively low in the case of callus cultures but high in the case of root or shoot cultures. The disadvantage in this case is also the necessity of developing new technology. Other important constraints determining the design of the process are the relation between growth and product formation (growthassociated or non-growth-associated), the potential of the plant material to be empolyed for de nouo synthesis or for biotransformation, and the occurrence of product release or accumulation. Fermentation processes can be carried out in batch, fed-batch, or continuous mode. These different process types can be roughly characterized as follows. The batch culture is the simplest process. Nutrients and inoculum are brought into the reactor, and after a period of time (which may be estimated through an on- or off-line measurement) the reactor contents are harvested and processed. Some characteristics of batch culture are limited process control, productivity loss as a result of cleaning and sterilization after each fermentation run, and large flexibility in the process. In fedbatch culture the nutrients are supplied to the reactor during the process. Compared to a batch culture, a fed-batch culture has the advantage of an additional control parameter, namely, nutrient feed rate. In a continuous culture a continuous supply of nutrients and a continuous harvest of reactor contents (medium or medium plus biomass) can be achieved. Some characteristics are better process control, more equipment needed, greater equipment costs, contamination of the process has greater consequences, and relatively low flexibility. Continuous culture without cell retention is only employable with growing biomass (nongrowing biomass will be washed out with the effluent flow). For alkaloid production this type of continuous culture is only suitable if alkaloid formation is growth associated, which greatly restricts application. A further constraint is that large cell aggregates or hairy roots are not easily transported, unless the size of the piping is large compared to the aggregate size. A continuous process with cell retention can be applied when product release can be achieved. Immobilization of the cells, either naturally or artificially, might be an advantage. In particular, biotransformations could be carried out in the continuous mode with biomass retention. From a theoretical point of view the continuous process is often considered as the most attractive choice. In practice, however, the number of continuous biotechnological processes is still very limited. Most processes appear to be performed in the batch or batch-fed mode. The main reasons are the greater flexibility of a batch or a fed-batch process, the lower complexity and thus lower risk of failures, and lower equipment costs.

34

ROBERT VERPOORTE ET A L . TABLE V LARGESCALE PLANTCELLCULTURES

Volume

(m3) 0.134 1.5 20 0.15 5 60

Type

Species

Ref.

Tank Tank, mixing by aeration Stirred Tank Stirred Tank Stirred Tank Stirred Tank

Various Nicotiana tabacum Nicotiana tabacum Lithospermum erythrorhizon Catharanthus roseus Various

136 137 138 59 139 140

b. Bioreactors. An important part of the equipment needed in a largescale production process using plant cell cultures is the bioreactor. There are a great ‘variety of bioreactors for laboratory-scale studies reported in literature. At the industrial scale, however, the variety of applied types of bioreactors are mainly limited to stirred tank reactors, airlift systems, bubble columns, and fluid bed columns. In the fermentation industry the stirred tank reactor is standard equipment. Substantial experience has been gained with this type of reactor. Until recently most workers considered stirred tank reactors to be of limited use in plant cell biotechnology. It was observed that plant cells could not withstand the high shear forces caused by the impeller (165). Therefore, the airlift reactor, which is thought to provide a much friendlier environment for the fragile cells, received much more attention. However, foaming and oxygen transfer, especially at high biomass concentrations, can become a serious problem in airlift reactors (166,167). The fact that since as early as 1960 some successful plant cell fermentations in stirred tank reactors were performed (Table V), however, indicated that it might not be considered impossible. Recently Meijer (66,69) and Scragg and co-workers (70,71) studied the shear sensitivity of plant cell cultures. They found that many plant cell cultures appear to be shear tolerant. The differences between shear-sensitive and shear-tolerant cell lines could not be explained, although there was evidence that well-growing “healthy” cell lines became shear tolerant through regular subculturing. To meet specific needs, some special bioreactor designs for plant biotechnological processes have been described in the literature, including the rolling drum reactor (163),mist reactor (61), hairy root bioreactor (61), and polyurethane foam reactor (164). The lack of experience with respect to the scale-up possibilities provided by these reactors might be a problem. However, particularly in the case of hairy root and organ cultures, a special reactor design might be necessary.


35

3. Downstream Processing An essential part of a production process for alkaloids is product recovery. The alkaloids have to be separated from the broth and purified. Application of alkaloids requires a high degree of purity. The high added value of these products, however, permits the use of advanced separation techniques. In the literature there are not many papers on downstream processing. Most studies are concerned with various aspects of regulating the productivity of plant cell cultures; if the productivity reaches an economically interesting level, downstream processing will be studied in direct relation with the development of a commercial process. Results of that research will rarely be published. In a discussion on downstream processing of alkaloids produced by plant cell biotechnology , two quite different cases can be distinguished, namely, product stored in the biomass and product excreted by the biomass. The first case is comparable with the classic production of alkaloids from plant material, although specific problems could arise from the character of the cellular biomass. In the second case a variety of advanced separation techniques could be used. A typical example from plant cell biotechnology is the forced release of alkaloids. In the following sections product recovery from biomass as well as product release and product recovery from spent media are discussed. a. Recovery of Alkaloids from Biomass. The problem of product recovery from biomass can be dealt with in two ways: (1) the biomass could be processed comparably to plant material in a classic alkaloid production process, and (2) the biomass could be fractionated, and, after separation of solids and liquid, the liquid phase could be processed comparably to spent medium containing excreted alkaloids. In classic alkaloid recovery the alkaloids are extracted from the biomass. In general, most alkaloids are basic, and this property is commonly used in most purification methods. One may distinguish three types of extrac tant s . Water-insoluble solvents. Most alkaloids are commonly present in the plant as organic salts. In order to solubilize the alkaloids the crude biomass extract is made basic by the addition of, for example, potassium hydroxide, potassium carbonate, or ammonia. At the resulting high pH the alkaloids are mainly present in their neutral form. The neutral alkaloids are then easily extracted from the aqueous phase with a water-insoluble organic solvent, for example, dichloromethane or chloroform. Water-soluble soluents. The alkaloids are extracted from the biomass with an alcohol, for instance, methanol, ethanol, or 2-propanol. Most

36


alkaloids, both salts and free bases, are readily soluble in alcoholic solvents. AcidiJied water. First the alkaloids are extracted from the biomass with acidified water. At a low pH most alkaloids are protonated and readily soluble in an aqueous solution. Subsequently the extract is made basic, and the neutral alkaloids are extracted from the aqueous phase with an organic solvent. Plant material, especially seeds and leaves, often contain large amounts of fatty compounds. These have to be removed before alkaloid extraction and is most frequently done with petroleum ether. Petroleum ether has the advantage that most alkaloids are not removed with this solvent. Another way to remove fatty compounds and other impurities is acidification of the aqueous extract followed by extraction with an organic solvent. The alkaloids remain in the aqueous phase while the fatty compounds are removed by the organic solvent. The choice of acid for acidification is important as several anions will result in the formation of organic solventsoluble ion pairs with the alkaloids (e.g., chloride and acetate). After extraction with organic solvent, the aqueous phase is made basic, and the alkaloids are extracted with an organic solvent. In literature very little is published on the extraction of alkaloids on an industrial scale. The few papers available were published between 1950 and 1970 and concern the isolation of alkaloids from whole plants or plant parts (168,169). The extraction of catharanthine and vinblastine from C. roseus leaves on a pilot plant scale is described by Atta-ur-Rahman et a f . (170). Svoboda developed a method for the extraction of ajmalicine, vinblastine, and vincristine which has been used by Eli Lilly & Co. (169,171173). Supercritical fluid extraction is a method which is used for the extraction of caffeine from coffee beans. This method also seems of interest for further studies of other alkaloids. b. Forced Release of Alkaloids. For several reasons in a plant cell biotechnological production the release of product from the biomass could be of interest for process design. During cell line selection, product release can be considered as a possible selection criterion. A striking example of this approach concerns the production of berberine by means of Thafictrum cell cultures that excreted almost all the alkaloid produced (see below(523)). Also, during medium optimization product release could be an aim. Therefore it is essential to determine the product of interest not only in the biomass but also in the medium. This might seem obvious, but it is not always done. If spontaneous release of the product is not accomplished, however, there still exist ways to force release of the product. Knowledge of the mechanisms involved in product storage are therefore

1.

PLANT BIOTECHNOLOGY

37

essential. However, studies on product routing are scarce, and the results seem contradictory (see discussion in Section 11). Product release brought about by lowering the pH of the culture medium was shown in case of Cinchona ledgeriana (42,82,174), Nicotiana rustica (42), and Catharanthus roseus (175). Other successful approaches to stimulate product release have also been reported. Several studies have been carried out on the stimulation of product excretion by extraction of the culture broth with an inert organic solvent (176-181). Adsorption of alkaloids from the culture broth with adsorbents, mostly XAD resins, also showed promising results. In several cases product release is stimulated (42,90,181),and in some cases product formation is also increased (88,181). However, growth and/or product formation can be influenced negatively. This might be caused by adsorption of essential medium components by the added adsorbent (42,182,183). Product release has also been reported after immobilization of the biomass (181). The mechanism behind this release, however, is not yet understood. The mechanism of product release by membrane permeabilization with organic solvents, on the other hand, is clear. Positive results with this method have been reported by Brodelius and Nilsson (184). Recently, however, Brodelius (111) concluded that regrowth of the biomass after different permeabilization procedures has not yet been possible. Reuse of biomass has been an important objective in this kind of research. It is not always clear which mechanism is responsible for product release stimulated by a certain chemical compound. Meijer (69) showed product release through the addition of polyethylene glycol. It is difficult to distinguish between the various mechanisms, such as osmotic stress, permeabilization, or extraction. c. Recovery of Alkaloids from the Medium. For the recovery of alkaloids from the medium one can consider two possibilities: (1) liquidliquid extraction, that is, the classic alkaloid extraction procedure using an immiscible organic solvent to extract the alkaloid from the alkalinized medium, and (2) solid-phase extraction, in which the alkaloid is concentrated on a solid phase, for example, ion-exchange resins, C18 reversedphase chromatography materials, or polymer-type resins like XAD. The latter method is of particular interest for continuous removal of the alkaloid from the medium (see above), but it also, in processing the medium, has the advantage of avoiding the need for large-scale liquid-liquid extractions. To our knowledge no data have been published on such large-scale medium extractions. Further studies, also in connection with the processing of alkaloids from plant material, on solid-phase extractions seem of

38


interest, as such methods could probably reduce the use of toxic organic solvents. 4. Scaling Up of Plant Cell Cultures The majority of studies concerning the production of alkaloids by plant cells in suspension cultures are performed with shake flasks. Actual production processes will proceed at a much larger scale, and consequently there is a scaling-up problem. There are no straightforward guidelines to solve this problem because there is an interaction of the various mechanisms involved, for example, stirring speed will have a positive effect on oxygen transfer but might have a negative shear effect on plant cells. Consequently compromises have to be made. In the next section theoretical approaches to this problem are reviewed and applied. a. Theory. Several techniques have been developed to attack the scale-up problems as reviewed by Oosterhuis (185) and Kossen and Oosterhuis (147). The techniques for scale-up methodology are summarized below.

1. Trial and error 2. Rules of thumb 3. Scale-down approachhegime analysis 4. Dimensional analysidregime analysis 5. Semifundamental methods 6. Fundamental methods Trial and error is exactly the approach to be avoided, because it implies expensive trials and errors at a large scale. Dimensional analysis, semifundamental methods, and fundamental methods are based on considerable and wide-ranging quantitative knowledge of the mechanisms involved in the process. In plant biotechnology this knowledge is partly lacking. Therefore, a combination of rules of thumb and the scale-down approach with regime analysis is the methodology preferred. This approach, reviewed by Sweere (186), is shown schematically in Fig. 7. First, regime analysis must &veal which mechanisms are rate limiting. Whether there is one ruling mechanism (pure regime), or more (mixed regime), and whether there may be a change in ruling mechanism going from the small to the large scale must be determined. Second, experiments on a laboratory scale, aimed at process optimization, should be designed under the same ruling mechanism. Third, in the small-scale experimental setup the effects of the ruling mechanism can be studied. Finally, if the experiments reveal measures for process optimization, they should be translated to the production scale.

39

1. PLANT BIOTECHNOLOGY Production scale \

\

Regime

Application \

\

A

---------_-V

Simulation

\

\

’

\

Optimization Modeling

\

Laboratory scale

FIG. 7. Scale-down procedure for a biotechnological process.

The scale-down approach can be applied both in the case of an existing process and for a new process. In the latter case a process design is made for the new process from available process data, experimental evidence, and rules of thumb. Regime analysis is applied to this process design. Based on the results a further small-scale research strategy is developed, and eventually changes are made in the process design. In the following paragraphs this approach will be applied to production of an alkaloid from plant cells cultured in a bioreactor. Regime analysis can be performed by comparison of characteristic parameters of the mechanisms involved in the process. Here the characteristic time concept will be used. The characteristic time is a measure for the rate of a mechanism. A fast mechanism has a short characteristic time. Other terms used are relaxation time, process time, or time constant. A time constant is formally only defined for first-order linear processes. Not all mechanisms involved in a plant cell production process are first order, therefore the term characteristic time is used. The characteristic time is defined as the ratio of a capacity and a flow; for example, the characteristic time for oxygen transfer to the liquid phase in a aerated bioreactor to,Lbecomes to,^ = ( C o , d m - Co,L)lkLa ( C O , G-~ CO,L) ~ = 11kLa

(5)

where CO,Gis the oxygen concentration in the gas phase (in mol/m3),CO,L the oxygen concentration in the liquid phase (mol/m3),m the Henry coefficient, kLa the volumetric oxygen transfer coefficient (llsec),

40


(C0,Glrn - Co,L) the oxygen capacity of a (C0,Glrn - C O , ~the ) oxygen flow to a solution.

solution,

and kLa

When inferring the mechanisms involved in a process one should realize that mechanisms with characteristic time magnitudes larger or smaller than the process time can be ignored. In a microbial fermentation, for example, metabolic reactions in the cell are too fast (characteristic times less than lop4sec) and microbial selection is too slow (more than lo6 sec) to influence the process.

b. Process Design. In the following a hypothetical process for the production of an alkaloid is studied. The main process parameters are briefly summarized in Table VI. The process comprises two stages: (1) in the growth phase biomass is grown in a fed-batch manner in a fermentor cascade, and (2) in the production phase (stationary phase) production medium with a high glucose concentration is fed into the fermentor to induce product formation. After 21 days a product concentration of 2.5% of dry weight is reached. Data are partly based on growth kinetics research on Catharanthus roseus (65), values adopted from literature, and reasonable assumptions. From the data in Table VI the total volume of the production phase can be calculated to be 150 m3. To diminish the risk of loss of production owing to contamination or equipment failure and to increase process flexibility, it TABLE VI PROCESS PARAMETERS Parameter Design basis Production Product loss during downstream processing Operation period Growth parameters Specific growth rate Doubling time Initial biomass dry weight concentration Inoculation ratio Biomass yield coefficient on glucose Maintenance coefficient on substrate Biomass yield coefficient on oxygen Maintenance coefficient on oxygen Maximum oxygen uptake rate Maximum substrate uptake rate Final biomass dry weight Production parameters Final product concentration Production period

Measure kg/year %

day slyear

Value 500 20 300

c,,/ce, C,,lC,,lhr C,,/mol mol/C,,lhr mol/m3/hr mol/m3/hr kg/m3

0.018 38.5 2.5 12.5 0.65 0.0074 2.1 0.0073 10.6 23.4 20

kgk days

0.025 21

hr-' hr kg/m3 %

41

1. PLANT BIOTECHNOLOGY TABLE VII DIMENSIONS OF INDUSTRIAL-SCALE FERMENTOR AND GEOMETRICALLY DOWN-SCALED LABORATORY FERMENTOR Scale

Gross volume (m3) Net volume (m3) Height (m) Diameter (m) Liquid height (ungassed) (m) Number of impellers Impeller diameter (m) Impeller blade width (m) Baffle diameter (m)

Industrial

Laboratory

25 20 5.44 2.42 4.35 3

0.005 0.004 0.316 0.141 0.253 3 0.045 0.009 0.013

0.77

0.16 0.22

is adequate to perform the production phase in, for example, six bioreactors of 25 m3 each. Therefore the growth phase has to be performed in six parallel three-stage fermentor cascades of 0.063, 1.25, and 25 m3. In Table VII the dimensions of such a 25-m3 bioreactor and a geometrically downscaled 5-liter fermenter are given. Assuming further the general process data given in Table VIII, the operating conditions can be calculated using a methodology for analyzing agitator performance and mass transfer in multiturbine production fermentors developed by Bader (187). The application of this approach provides a method for determining axial dissolved oxygen profiles under conditions of known mass transfer rates as a function of agitation-aeration characteristics. A stagewise approach is used which divides the fermentor into a series of mixing cells. The results of the calculations are presented in Table IX. In the design of the large-scale fermentor it was assumed that the volumetric oxygen transfer coefficient kLa must be sufficiently high to provide nonlimiting oxygen transfer under worst case conditions (at the end of the exponential growth phase).

TABLE VIII GENERALPROCESS DATA Parameter

Measure

Broth density Kinematic medium viscosity Process temperature Back pressure Gas flow/reactor volume/time

kg/m3 m-*/sec "C N/m2 m3/m3/sec

Value 1030 I x 25 5 x 104 0.005

42


TABLE I X OPERATING CONDITIONS FOR INDUSTRIAL-AND LABORATORY-SCALE FERMENTORS FOR CONSTANTIMPELLER TIP SPEED( u ; ) AND POWERINPUT (PlV) v(m3) Parameter Gas flow (m3/sec) Superficial gas velocity (m/sec) H0Id-up Oxygen transfer coefficient (sec-I) Impeller speed (sec-I) Impeller tip speed (m/sec) Power input (W/m3)

25 0.1 0.02 0.043 0.0072 1.28 3.1 400

0.005 (Ui = C )

0.005 (P/V-C)

3.3 x 10-5 0.0014 0.039 0.03 21.9 3.1 7000

3.3 x 10-5 0.0014 0.016 0.004 8.6 1.2 400

c. Regime Analysis. For equations to calculate the various time constants in the hypothetical plant biotechnological process described above one should consult the literature (185). In Table X the results of the time constant calculations are presented. The growth and production time are the longest characteristic times in the process, thereby determining the total process time. By comparing the characteristic times of the 25-m3 fermentor, it can be concluded that there may be some problems in supplying oxygen to the plant cells. Oxygen transfer and oxygen consumption

TABLE X TIMECONSTANTS OF A 25-m3 FERMENTOR AND A DOWN-SCALE 5-LITER FERMENTOR FORCONSTANT TIP SPEED(ui) AND CONSTANT VOLUMETRIC POWERINPUT (PlV)

COMPARISON OF

Time constant

25

Mixing Oxygen transfer Oxygen consumption (growth) Oxygen consumption (production) Heat production Heat transfer Substrate consumption (growth) Biomass production Product formation

32 139 139 303 4800 4800 1500" 2 x 105 18 X lo'

a

Fed-batch process in which C , is maintained at 10 mollm'.

0.005 C)

(Ui =

2 33 139 303 1000 1000 1500"

2 x 105 18 x 105

0.005

(PlV-C) 8 250 139 303 4800 4800 1500" 2 x 105 18 x 105

1.

PLANT BIOTECHNOLOGY

43

times are equal on a large scale because design has been based on the critical oxygen transfer coefficient. However, mixing time is short compared to oxygen transfer time, so oxygen depletion is unlikely to occur in badly mixed regions of the fermentor. Heat transfer and heat production time are equal. The calculation is based on the assumption that all heat produced is exchanged. No temperature gradients are expected because the time of heat production is long compared to the mixing time. In the 5-liter fermentor for constant volumetric power input, oxygen limitation will occur because the oxygen transfer time is longer than the oxygen consumption time. When ui is constant the oxygen transfer time is much shorter than the oxygen consumption time, so growth will not be hampered by oxygen limitation. When the process is operated in a fedbatch manner, problems can occur when the substrate supply is limiting. From the characteristic time of substrate consumption, it can be concluded that in this case substrate limitation will occur after 25 min. It can be concluded that experiments to simulate the large-scale process cannot be performed on a 5-liter scale for constant volumetric power input. Under these conditions oxygen transfer will be the ruling mechanism for growth instead of maximum specific growth rate. For ui constant oxygen transfer is more than sufficient, so small-scale experiments should be preferably performed under this condition. d. Shear Effects. Characteristic times for shear effects on the cell population cannot be calculated because of various reasons: various effects, each dependent on the flow regimes in the fermentor, are acting simultaneously on the cells; and it is not possible to define a rate for the incompletely understood shear effects. An analysis of shear effects should therefore be based on comparison of the variety of mechanisms in relation to flow characteristics. This approach has been described by Cherry and Papoutsakis (188) for a system of animal cells on microcarriers. This methodology was also used by Meijer (69) to analyze a plant cell culture with the aim of selecting the optimal scale-down criterion for laboratoryscale shear studies.

e. Present Situation. Several large-scale plant cell cultures have been reported, both in airlift bioreactors and in stirred vessels (Table 111). One might conclude that the theoretical analysis given above is superfluous, given that successful large-scale plant cell fermentations have already been carried out. However, successful does not necessarily mean optimal. In a large-scale fermentor it is not at all easy to study factors that may eventually be limiting, although these limitations (e.g., in oxygen transfer or mixing) may have a deep impact on productivity and therefore the economics of a process. Further studies on the various aspects of large-

44


scale culturing of plant cells are thus of great importance to the eventual improvement of the economic feasibility of plant cell biotechnological production of plant secondary metabolites.

IV. Nicotine Tobacco is one of the major model systems in plant cell and tissue culture studies. Particularly in developing techniques for genetic engineering has it played a major role. Consequently numerous papers have described various aspects of tobacco plant biotechnology . For example, almost 200 entries for plant cell culture media for tobacco are given in the tabulation by George et al. (189). Some of the most commonly used media for plant cell cultures were, in fact, developed for tobacco callus cultures (190,191). Indeed, root cultures of several Nicotiana species were described as early as 1938 by White (192). Collins and Legg (193) reviewed the use of cell and tissue culture methods for the improvement of tobacco. Although the price of nicotine is very low, extensive studies have been made on the production of this alkaloid in cell cultures. Some patents have been filed (Table XI). Studies on the production of nicotine (1)and related alkaloids (2-4) in cell cultures are summarized in Table XII. A. SECONDARY METABOLITES IN CELLCULTURES From Table XI1 it is obvious that the alkaloid levels found in the cell and tissue cultures of Nicotiana species vary widely. The cultures do produce a variety of other secondary metabolites as well: sterols and triterpenes (245,246), ubiquinone (243, cinnamoyl putrescines (248-251), and the phytoalexin capsidiol and related sesquiterpenes (252-254). TABLE XI PATENTSCONCERNING PRODUCTION OF NICOTINE IN PLANTCELL AND TISSUECULTURES

I. Shio and S. Ota. Ajinomoto Co., Ltd. Jpn. Kokai, JP 480912187, 28 Nov 1973 Showa. JP 72-18357, 22 Feb 1972. Alkaloid production by tissue culture. Chem. Abstr. 80, 11839613. H. Smith and D. W. Pearson. Gallaher Ltd., Eur. Pat. Appl. EP 7244, 23 Jan 1980. GB 78-30381, 19 Jul 1978. Nicotine by culturing a Nicotiana strain. Chem. Abstv. 92, 194784b. A. S. Weaving, A. J. N. Bolt, D. J. Barlett, and S. W. Purkins. Imperial Group PLC. GB 2203022, 10-12-1988. Smoking material containing substrate of tobacco cells or plant material and cellulosic material with mixtures of lactose and glucose.

1.

45

PLANT BIOTECHNOLOGY

(1)Nicotine, R = CH3 (2) Nornicotine, R = H

(3) Anabasine

(4) Anatabine

TABLE XI1 OCCURRENCE OF NICOTINE IN PLANTCELLAND TISSUECULTURES Alkaloid Nicotine Nicotine Nicotine, nornicotine, anabasine Nicotine, anabasine Nicotine Nicotine Nicotine, anatabine Nicotine Nicotine, anatabine, anabasine Nicotine Nicotine Nicotine Nicotine Nicotine, nornicotine, anatabine, anabasine Nicotine Nicotine Nicotine Nicotine, nornicotine Nicotine Nicotine Nicotine

Plant species

Type of culture

Yield

Ref.

N. tabacum N. tabacum N . alata, N. glauca, N. paniculata, N . rustica, N . silvestris N . glauca

Roots Roots Roots

2.9% DW 0.2% DW

194,I 95

Roots

0.76% DW

I98

N . tabacum N . rustica N . tabacum

Callus Roots Callus

0.01-0.1% DW -

I99 200 201,202

N . tabacum N . tabacum

Callus Callus

0.26% DW 0.013% DW

203 204

N . tabacum

0 0.01% DW 0.5-1.1% DW 0.29% DW 1-3.4% DW

205

N . tabacum N . tabacum N . rustica N . tabacum

Callus Suspension Callus Callus Callus Callus

N . tabacum N . tabacum N . tabacum N . tabacum

Callus Callus Callus Callus

0.3-3.38% DW 0.04% FW 0.75% DW 0.004% DW

50,211-214 215 216-218 219

N . tabacum N . tabacum N . tabacum

Callus Suspension Callus Roots

0.17-0.75% DW 0.16% DW 3.2% DW

220 22 I 222

196 197

206,207 208 209 210

(continued)

46


TABLE XI1 (Continued) Alkaloid Nicotine Nicotine nornicotine, anatabine Nicotine anatabine, anabasine, myosmine, anatalline, nicotelline Nicotine Nicotine, nornicotine, anabasine Nicotine

Nicotine Nicotine Nicotine, nornicotine, anatabine Nicotine Nicotine, nornicotine, anatabine, anabasine Nicotine, nornicotine, anatabine, anabasine Nicotine, nornicotine, anatabine, anabasine Nicotine, nornicotine anatabine, anabasine Nicotine, nornicotine anatabine, anabasine

Plant species

Type of culture

Yield

Ref.


Suspension Callus

2.2% DW, 0.14 g/liter 0.16-0.7% DW

223 224-227

N . tabacum

Suspension

0.2% DW

228

N . tabacum, N . rustica N . tabacum

Suspension Callus

2.9% DW, 0.36 g/liter 1.6-3.75% DW

229 230-233

N . tabacum, N . rustica, N . debneyi, and somatic and sexual hybrids of these species N . tabacum N . tabacum N . tabacum

Suspension

0.007-0.041% DW, 0.007-0.018% DW, 0.005% DW

234

Callus Hairy roots Hairy roots

235-237 0.05% DW 90 1.5 mg/liter/day 0.03% (FW), 0.01 g/liter 238

N . rustica N . rustica

Hairy roots Hairy roots

0.065% FW 0.085% FW

239 28

N . tabacum

Hairy roots

0.12% FW

240

N . rustica

Hairy roots

0.04% FW

240

N . hesperis

Hairy roots

0.09% FW

240

N . africana

Hairy roots

0.12% FW

240

(continued)

47

1. PLANT BIOTECHNOLOGY TABLE XI1 (Continued) Alkaloid Nicotine, nornicotine anatabine, anabasine Nicotine, nornicotine anatabine, anabasine Nicotine, nornicotine anatabine, anabasine Nicotine, nornicotine anatabine, anabasine Nicotine, nornicotine anatabine, anabasine Nicotine Nicotine, nornicotine, anabasine Nicotine

Plant species

Type of culture

Yield

Ref.

N . umbricata

Hairy roots

0.1% FW

240

N . velutina

Hairy roots

0.14% FW

240

N . cavicola

Hairy roots

0.06% FW

240

N . rustica

Hairy roots

0.08% FW

241

N . hesperis

Hairy roots

0.1% FW

242


Suspension Hairy roots

0.003% DW 0.11% FW

243 244

N . rustica

Hairy roots

0.08% FW

132

B. SCREENING, SELECTION, AND STABILITY After establishing a cell culture, alkaloid production may decrease during successive subculturing (208,2091, but stable, high producing cell lines can be obtained (299,208-220,222). Fluctuations in alkaloid production during successive subculturing may also occur (224).Shiio and Ohta (208) reported that during initial subcultures a considerable difference in alkaloid content exists for calli derived from different parts of the same tobacco plant, root calli being the best producers. For N. rustica such differences could not be observed (209), but a large variation in alkaloid content of single-cell clones derived from a callus cell line was noted. Based on this a selection program was conducted in order to obtain high producing cell lines. By a repeated single-cell cloning procedure high producing (1-3.4% alkaloid on a DW basis) N. tabacum cell lines were obtained (220). Ohta and co-workers (211,224) successfully selected a

48


stable high nicotine-producing (2.29% DW) tobacco callus line by screening at the level of calli Kinnersley and Dougall (215,220) found a strong correlation between alkaloid levels in the plant and those in the derived callus cultures. However, Roper et al. (229)could not find such a correlation. Great variation in the nicotine content of cultured cells of three Nicotiana species and their somatic and sexual hybrids have been found (234). Some of the cell lines showed stable production for a year, whereas others showed a change in nicotine production after a year of subculturing (100 subcultures). It was concluded that making sexual or somatic hybrids is an interesting way to obtain new cell lines (or plants) with improved characteristics such as nicotine production. Differentiation into root-type tissue results in increased alkaloid production (194-197,199,220,222,225,230,231), which is in agreement with the fact that the roots are the major site of nicotine biosynthesis in the plant (I 94,196). Plants regenerated from non-alkaloid-producing cultures did regain the capacity of alkaloid biosynthesis (207). Robins et al. (28) proposed the use of a biosynthetic precursor, nicotinic acid, as a selective agent for obtaining high nicotine-producing hairy root cultures of N. rustics. Nicotine did not work as a selective agent in the media, whereas nicotinamide proved to be toxic. C. EFFECTSOF GROWTHCONDITIONS A number of factors have been studied for their influence on nicotine production. Of these the negative effect of auxins, and in particular 2,4-D, on alkaloid production is worth mentioning (202-204,211,220, 225,226,229,255). In root cultures the addition of indoleacetic acid (IAA) also reduces alkaloid production (196). Light was reported to inhibit nicotine formation (50,255). In a green cell suspension, however, increased nicotine levels were found on illumination (229).Ikemeyer and Barz (243) reported that a photoautotrophic cell line of N. tabacurn did not produce nicotine, whereas a heterotrophic cell line did accumulate this alkaloid. Elicitation with a preparation of the fungus Phytophthora megasperma did not affect the nicotine levels of these cell lines. Addition of organic acids to the medium resulted in increased alkaloid formation in callus cultures (up to 3.25%) (230). For a review of the various cultural factors which influence secondary metabolism, the reader is referred to Mantel1 and Smith (255).

D. LARGE-SCALE SUSPENSION CULTURES From Table XI1 it is clear that most studies concerned callus cultures of Nicotiana species. For industrial applications however, suspension cul-


49

tures are of more interest. The first report on a suspension culture in a bioreactor was by Kato et al. (256). Tobacco cells were cultured in a 30-liter jar fermentor in a semicontinuous mode. A biomass density of 20 g/liter was reached in batch cultures; the mean optimal growth rate during the exponential phase was a doubling time of 24 hr (specific growth rate, p , 0.69 day-'). In a semicontinuous culture the yields (dry weight) were 12-13 g/liter/day. The biomass was stirred by a disk turbine-type of impeller. Subsequently, a continuous culture in a 1500-literfermentor was reported (137). Mixing of the culture was achieved by aeration, and no agitator was present in the fermentor. The specific growth rate was less than that in a 15-liter fermentor (0.62 compared to 0.69 day-'). At a dilution rate, D , of 0.42 day-' and a sucrose concentration of 26 g/liter in the influent medium, maximum cell productivity was obtained (3.82 g/ liter/day). Although mixing of the culture broth in the 1500-literfermentor presented some problems, the authors considered the system to be suited for the production of tobacco raw material. Further optimization of the medium for batch cultures resulted in a further increase of the maximum specific growth rates from 0.80 to 0.96 day-' (257). In a two-stage continuous culture system using 60-liter fermentors, stirred by flat blade turbine impellers, Kato and co-workers (138,258)further improved the production of tobacco raw material; an average production of 6.9 g DW cell material/ liter/day was reported. In a batch culture on a 20,000-liter scale the cells showed a highest growth rate of 15 hr doubling time ( p = 1.09 day-') (138). Hashimoto et al. (221) reported the continuous culture of tobacco cells on a 20,000-liter scale, in a 66-day experiment. During steady state the biomass density was 16.5 g/liter (DW), the dilution rate was 0.35 day-', and a productivity of 5.82 g/liter/day was achieved. In this type of experiments biomass as a substitute for tobacco plant material was the major goal. No data on the nicotine production were given. A fed-batch fermentation on a 10-liter scale for the production of cinnamoyl putrescines by tobacco cells was reported by Schiel et al. (251). Flat blade turbines were used for stirring. Sahai and Shuler (259) described a multistage continuous culture of tobacco cells in chemostats of the Wilson type. Steady states under different conditions were maintained; concerning secondary metabolites, only the production of phenolics was measured. Mantel1 et al. (223) cultured tobacco cells in 5-liter Wilson-type batch fermentors. Mixing was by the aeration through air inlet tubes extended to the base of the vessel. By using media having a high sucrose (5%) and low phosphate concentration, nicotine levels of 2.2% (DW) could be reached, corresponding to 0.14 g/liter. Optimization of tobacco cell suspension cultures, with the aim of largescale production of nicotine, was studied by Roper et al. (229). A fast growing cell line with a high nicotine content was selected. Various combi-

50


nations of growth and production media were tested. Best results were obtained by a Murashige-Skoog (MS) medium containing 0.2 ppm naphtylacetic acid (NAA), 0.02 ppm kinetin, and casein hydrolysate (1 g/liter). The medium contained 4% sucrose; glucose was found to inhibit nicotine production. This medium was suited for both growth and production, and after 1 week of preculture on this medium the cells were subcultured on the same but fresh medium. After 21 days from the start of the preculture the nicotine content of the cells was 2.9% (DW), the cell yield was 1015 g/liter. The maximum yield obtained for this system was 0.36 glliter. With a mixotrophic green cell culture biomass yields of 14-27 g/liter were obtained, the nicotine production being 0.92 g/liter over a 2-week period. All these experiments were performed in shake flasks. When the cell lines were tested in fermentors different results were obtained. They did not grow well in a 20-liter airlift fermentor; in a 20-liter stirred fermentor good growth (doubling time 45 hr) was observed for one of the cell lines, but the nicotine production (0.006 glliter) was far below the 0.36 g/liter level observed for this cell line in shake flasks. Berlin and co-workers (250,251) reported on the production of cinnamoyl putrescines by tobacco cells in 70-liter fermentors, operated in the fed-batch mode. They also found considerable difference between results obtained in shake flasks versus fermentors. The large-scale culture of tobacco cells, claimed to contain 5-30% nicotine, for the preparation of a tobacco-smoking substitute have been patented (see Table XI). Hallsby and Shuler (260) reported the growth of tobacco cells immobilized in a membrane-type reactor using different flow patterns.

E. ROOTCULTURES As nicotine biosynthesis is connected with the roots, an obvious solution for obtaining high producing, stable systems is the transformation of tobacco cells with Agrobacterium rhizogenes, yielding the so-called hairy root cultures. Hamill et al. (238)first reported such cultures of N. rustica. These cultures did produce alkaloids, of which a substantial proportion was excreted into the medium. The alkaloid level was slightly higher than in 6- to 8-week-old plants. Via protoplasts single-cell clones were obtained from N. rustica hairy roots. Hairy root cultures regenerated from the clones showed variation in morphology, alkaloid formation, and T-DNA structure. Some clones showed increased alkaloid production (239).Also the accumulation ratio, that is, the amount of alkaloid released into the medium compared to the amount in the cells, varied largely. Parr and Hamill (240) compared alkaloid production of the transformed hairy root

1.

PLANT BIOTECHNOLOGY

51

cultures of a number of Nicotiana species with that of the plant roots. The biosynthetic capacity of the hairy root cultures was similar to that of the intact plant. Differences in accumulation ratios observed for the various hairy root cultures did not correlate with the ability of the plants to transport alkaloids from the roots to aerial parts. The N. rustica hairy roots were successfully grown in a packed-bed fermentor, yielding biomass densities (DW) of 10 g/liter (90). The growth rate was comparable with a cell suspension culture. The fermentor was operated as a batch fermentor for 11 days, after which is was run as a continuous culture. Nicotine was isolated from the medium during the continuous operation. The alkaloid production rate was estimated to be 1.54 mg/liter/day during this phase. To improve the release of alkaloids by the hairy roots, a continuous removal of the alkaloids from the medium with XAD-4 as an adsorbent was tested. Hairy roots cultured in flasks did not produce more alkaloids in the presence of sachets with XAD-4. However, the amount of alkaloid released into the medium increased; in other words, the accumulation ratio was affected by the adsorbent. The effect of addition of various precursors of nicotine such as nicotinic acid (28,195,198,211,222,228),ornithine (198,211,222,228), and putrescine (211,222,228) on alkaloid production has been studied. The results of these studies are of particular interest in connection with a better understanding of the regulation of the biosynthesis of nicotine. The recent and extensive studies in this field (227,230-233,235-237,241,242,244,261-263) [for a review, see Wagner (264)],particularly those concerning the enzymes involved in the biosynthetic pathway of nicotine, hold the promise that in the near future the genes regulating this pathway will be isolated. This will open the way for genetic modification as a tool to improve (or block) nicotine production in plants or plant cells. The feasibility of such an approach was recently proved by Hamill et al. (132).By overexpression of a yeast ornithine decarboxylase (ODC) in transgenic roots of N. rustica, the production of nicotine in the root culture could be enhanced about 2-fold. The yeast ODC gene was combined with the CaMV35S promoter and introduced in the plant using a binary vectorlAgrobacterium rhizogenes system.

F. CONCLUSION Despite the extensive studies of tobacco cell and tissue cultures, so far no stable and high producing, large-scale production system for nicotine has been achieved. The technology of growing tobacco cells on a large scale is available, but suitable cell lines remain to be developed. Although the hairy roots are fast growing, the large-scale production of such cultures

52


needs further study. The alkaloid production in these cultures probably needs to be enhanced before such a production becomes economically feasible. In the near future, genetic engineering might offer new perspectives in this area.

V. Tropane Alkaloids The tropane alkaloids represent from pharmaceutical point of view one of the most important groups of alkaloids, on the one hand because of the alkaloids atropine (5)* and scopolamine (6), both widely used in pharmacotherapy, and on the other hand because of cocaine, most known for is its abuse as a stimulant. The former two alkaloids are extracted from a variety of Solanaceae, and the latter alkaloid is isolated from the leaves ofErythroxylon coca. For the plant cell and tissue culture of the latter plant we have not been able to find any literature. For the Solanaceae, however, many studies have been published. Several genera of this family have been studied extensively, for example, Anisodus, Atropa, Datura, Duboisia, Hyoscyamus, and Scopolia. Of these Datura has widely been used as a model system for the development of various techniques in plant cell and tissue culture and for basic studies of cultured plant cells, without reference to alkaloid production. Scopolamine is a more costly derivative of atropine: it has an epoxide function (6). Therefore, many studies concern the bioconversion of atropine (I-hyoscyamine) to scopolamine. The isolation of the enzymes involved and the subsequent cloning of the genes open new avenues for the production of scopolamine, either by genetic engineering of plants or by introducing the gene(s) responsible into microorganisms which could then effectively perform the desired bioconversion. The industrial interest in the tropane alkaloids is well reflected in the large number of patents concerning the plant cell biotechnology of tropane alkaloids (Table XIII). The first reports of the occurrence of tropane

* We shall here use the name atropine, which stands for the racemic mixture of 1- and d-hyoscyamine, to avoid confusion, as in none of the publications concerning tropane alkaloids is the optical rotation of the alkaloid isolated mentioned, and even the co-occurrence of atropine and hyoscyamine in cell cultures has been reported (265-270)!

1.

53

PLANT BIOTECHNOLOGY

'*a

ofiH

H

0-c-c

II I

0 CHzOH (5) Atropine

(6) Scopolamine

alkaloids in cultured cells or tissues are quite old. James (271) reported the presence of alkaloids in meristems of some solanaceous plants. The first report on a suspension culture of Datura tumor tissues was in a patent of 1956 (see Table XIII). Telle and Gautheret (272) and Stienstra (273) reported the production of tropane alkaloids in cultured roots of Hyoscyamus niger and Datura stramonium, respectively. Reinouts van Haga (274,275) studied the biosynthesis of tropane alkaloids in root cultures of Atropa belladonna. Root callus cultures of Atropa belladonna were reported to contain atropine (276). In Table XIV through XVIII the occurrence of alkaloids in various types of cell and tissue cultures of Atropa, Datura, Duboisia, Hyoscyamus, and various other species in the family Solanaceae is summarized. From these data it is clear that the production of tropane alkaloids in cell suspension cultures is rather low. Only in root cultures has production similar to, or even higher than, the original plant been obtained. For this reason an extensive discussion on efforts to improve production in cell suspension cultures is not useful; instead, we briefly deal with the application of plant biotechnology for the improvement of the tropane alkaloid-producing plants. Finally, we discuss the bioconversion of added precursors.

A. PLANTBIOTECHNOLOGY

The cell and tissue culture of the major tropane alkaloid-producing species does not apparently offer any special problems. The regeneration of plantlets from callus and tissue cultures seems to be routine (286,307,309,323,325,332,350-352). Plants have also been regenerated from protoplasts of Atropa belladonna (353),Duboisia rnyoporoides (354), and Hyoscyamus muticus (355,356). Cryopreservation has been reported for Anisodus and Datura species (349,357).

54

ROBERT VERPOORTE ET AL. TABLE XI11 PATENTSCONCERNING TROPANE ALKALOIDS IN PLANTCELLAND TISSUECULTURE

1956 J. B. Routien and L. G. Nickell. U.S. Patent 2,747,334, 29-05-1946. Cultivation of plant tissue. 1974 American Cyanamid Co. D1-108-769. 26.07.72-D1-164695 (05-10-74). Cell culture production of plant metabolites by inducing redifferentiation of undifferentiated plant cell cultures ( e g , Datura). I980 Eisei KK. JP 024116, 29-02-1980. Isobutyroyl-tropine and valeryl-tropine preparation by culturing callus derived from Duboisia plant and extracting cultured cells. A. R. Saint-Firmin. U.S. Patent 4241536 30-12-1980. GB 76-46866 10-11-1976. Embryogenesis in uitro, induction of qualitative and quantitative changes in metabolites produced by plants and products (e.g., Hyoscyamus muticus). Chem. Abstr. 94, 171236b. I986 Y. Mano, N. Nabeshima, and H. Okawa. Sumitomo Chemical Co., Ltd. Jpn. Kokai Tokkyo Koho. JP 61254195 A2 11-11-1986. JP 85-97326,7-05-1985. Tropane alkaloid production by tissue culture (hairy roots, Scopolia and Datura). Chem. Abstr. 107, 174434n. 1987 Seitaikinou Riyou. J62248429 A 29-10-1987, 86JP-089975;21-04-1986. Tissue cultivation of Duboisia plants by infecting callus, shoot, or adventitious root cultures with Agrobacterium rhizogenes. H. Ideno and C. Habara. Seito Kino Riyo Kagakuhin Shinseizo Gijutsu Kenkyo Kumiai. Jpn. Kokai Tokkyo Koho. JP 62006675 A2, 13-1-1987. JP 85-143882,2-7-1985.Tropane alkaloid production by Duboisia tissue cultures. Chem. Abstr. 107,5796b. H. Yamagata. Seito Kino Riyo Kagakuhin Shinseizo Gijutsu Kenkyo Kumiai. Jpn. Kokai Tokkyo Koho. JP 62006674 A2 13-1-1987. JP 85-143881, 2-7-1985. Tropane alkaloid manufacture by Duboisia tissue culture. Chem. Abstr. 107, 17443513. Y. Yamamoto and R. Mizuguchi. Nippon Paint Co., Ltd. Jpn Kokai Tokkyo Koho. JP 62186790 A2, 15-9-1987. JP 86-30501, 13-2-1986. Manufacture of 15N-containing compounds by plant cell culture (Datura taluta) Chem. Abstr. 108,73743~. H. Kamata and H. Saga. Lion Corp. Jpn Kokai Tokkyo Koho. JP 62205792 A2, 10-91987. JP 86-47046,4-3-1986. Manufacture of alkaloids by genetically engineered Solanaceae plant cell culture (hairy roots, Atropa belladonna). Chem. Abstr. 108, 73775q. 1988 H. Kamata and H. Saga. Lion Corp. Jpn Kokai Tokkyo Koho. JP 63039595 A2,20-21988. JP 86-181532, 1-8-1986. Medicinal alkaloid production by plant tissue culture (hairy roots, Datura). Chem. Abstr. 108,220369r. H. Kamata and H. Saga. Lion Corp. Jpn Kokai Tokkyo Koho. JP 63039596 A2,20-21988. JP 86-181533, 1-8-1986. Manufacture of alkaloids by cultivating genetically engineered Solanaceae plants (hairy roots, Datura innoxia). Chem. Abstr. 109,53249~. H. Kamata and H. Saga. Lion Corp. 88JP-064650, 17-03-1988. EP-283051,21-09-1988. Alkaloid($ preparation from Solanaceous plants by transformation with Agrobacterium rhizogenes, culturing hairy roots produced, and continuous recovery from alkaloids (Hyoscyamus, Scopolia). (continued)


55

TABLE XI11 (Continued) S. Takayama. Kyowa Hakko Kogyo Co., Ltd. Jpn. Kokai Tokkyo Koho. JP 63105689 A2, 10-5-1988. J P 86-252759, 23-10-1986. Scopolamine and its manufacture with plant tissue culture. Chem. Abstr. 109, 168983r. H. Ideno, Seito Kino Riyo Kagakuhin Shinseizo Gijutsu Kenkyo Kumiai. Jpn. Kokai Tokkyo Koho. J P 63087991 A2, 19-4-1988. JP 86-230156, 30-9-1986. Scopolamine and hyoscyamine manufacture enhancement by tissue culture of tropane alkaloid-producing Solanaceae (Duboisia myoporoides). Chem. Abstr. 109,228787s. K. Shimomura, A. Yagi, and N. Okumura. National Institute of Hygeinic Sciences. Jpn. Kokai Tokkyo Koho. JP 63059897 A2, 15-3-1988. JP 86-203184, 29-8-1986. Therapeutic tropane alkaloid manufacture by plant tissue culture (Duboisia). Chem. Abstr. 110, 22332b. H. Ideno and H. Yamagata. Seito Kino Riyo Kagakuhin Shinseizo Gijutsu Kenkyo Kumiai. Jpn. Kokai Tokkyo Koho. JP 63084497 A2, 15-4-1988.JP 86-230155, 30-9-1986. Therapeutic tropane alkaloid manufacture by plant tissue culture (Duboisia myoporoides). Chem. Abstr. 110,22334d. T. Emoto, H. Ideno, and T. Yoshioka. Seito Kino Riyo Kagakuhin Shinseizo Gijutsu Kenkyo Kumiai. Jpn. Kokai Tokkyo Koho. JP 63116691 A2,20-5-1988. JP 86-26-0768, 4-1 1-1986. A plant tissue culture method using a medium containing adsorbents (Duboisia). Chem. Abstr. 110, 37782~. H. Ideno, T. Emoto, and F. Ito. Seito Kino Riyo Kagakuhin Shinseizo Gijutsu Kenkyo Kumiai. Jpn. Kokai Tokkyo Koho. JP 63167790 A2, 11-7-1988, JP 86-309270, 27-121986. Tropane alkaloids and their manufacture with plant tissue culture (Duboisia myoporoides root cultures). Chem. Abstr. 110,210958j. Y. Mano, Y. Yamada, and H. Okawa. Sumitomo Chemical Co., Ltd. Jpn. Kokai Tokkyo Koho. JP 63226280 A2, 20-9-1988. JP 87-63092, 17-3-1987. Tropane alkaloids and their manufacture with plant tissue culture of Duboisia (hairy roots, Duboisia leichhardtii). Chem. Abstr. 110, 17182213. H. Ideno, T. Emoto, and F. Ito. Seito Kino Riyo Kagakuhin Shinseizo Gijutsu Kenkyo Kumiai. Jpn. Kokai Tokkyo Koho. JP 63167790 A2, 11-7-1988. JP 86-309270, 27-121986. Tropane alkaloids and their manufacture with plant tissue culture (Duboisia myoporoides root cultures). Chem. Abstr. 110,210958j. H. Kamata and S. Marumo. Nippon Kayaku Co., Ltd. Jpn. Kokai Tokkyo Koho. JP 63216491 A2, 8-9-1988. JP 87-49949, 6-3-1987. Manufacture of atropine and scopolamine from indoleacetic acid derivatives by plant tissue culture (Duboisia, Hyoscyamus, Scopolia, and Datura species). Chem. Abstr. 110,210979s. H. Ideno and H. Yamagata. Seito Kino Riyo Kagakuhin Shinseizo Gijutsu Kenkyo Kumiai. Jpn. Kokai Tokkyo Koho. JP 63167793 A2, 11-7-1988. JP 86-309271, 27-121986. Tropane alkaloid manufacture with plant tissue culture (Duboisia myoporoides root cultures). Chem. Abstr. 111,22173~. Seitaikinou Riyou. 563129982 A 02-06-1988. 86JP-276787; 21-1 1-1986. Plant tissue culturing method, by culturing plant tissue in liquid medium containing surfactant and discharging out secondary metabolic products of plants from cells. Seitaikinou Riyou. 563226278 A 20-09-1988. 87JP-001118; 08-01-1987. Plant tissue culturing method - using culture medium containing one or more sulfoxide(s), polyhydric alcohol(s), organic acids, and steroids. Seitaikinou Riyou. 563226281 A 20-09-1988. 86JP-308544; 26-12-1986. Tissue culturing to release secondary metabolic products of plant from cells comprises using liquid medium containing sodium or potassium ions, or calcium or magnesium ions. (continued)

56


TABLE XI11 (Continued) 1989 T. Takahashi, Y. Kita, and K. Koide. Oji Paper Co. Ltd. Jpn Kokai Tokkyo Koho. JP 01 168294 A2, 3-7-1989. JP 87-323744, 23-12-1987. Manufacture of tropane alkaloids by plant tissue culture of Solanaceae (Hyoscyamus niger). Chem. Abstr. 112,34439~. S. Takayama and H. Tanaka. P.C.C. Technology, Inc. Jpn Kokai Tokkyo Koho. JP 01243991 A2, 28-9-1989. JP 88-69745, 25-3-1988. Plant metabolites manufacture with differentiated plant tissue (Atropa belladonna root cultures). Chem. Abstr. 112, 137584~. S . Kitani and H. Ideno. Seito Kino Riyo Kagakuhin Shinseizo Gijutsu Kenkyo Kumiai. Jpn. Kokai Tokkyo Koho. JP 01124383 A2, 17-5-1989. JP 87-280893,9-11-1987. Tropane alkaloid enhanced manufacture with arylmethylbutanediacids in plant tissue culture (Duboisia myoporoides). Chem. Abstr. 112, 1567042. M. Sakai and H. Ideno. Seito Kino Riyo Kagakuhin Shinseizo Gijutsu Kenkyo Kumiai. Jpn. Kokai Tokkyo Koho. JP 01273597 A2, 1-11-1989. JP 88-102735,27-4-1988. Manufacture of tropane alkaloids by plant tissue culture with adenosyl compounds or cyclic adenosine phosphates (Duboisia myoporoides). Chem. Abstr. 112, 15670q.

1. Atropa

Eapen et al. (286) studied the morphogenesis of haploid (obtained from anthers) and diploid tissue cultures of Atropa belladonna. The haploid tissue was found to regenerate more readily; the diploid tissue also lost its regenerative potential more rapidly on prolonged subculturing. The regenerated plants showed alkaloid production similar to plants grown from seeds. Alkaloid (atropine) production increased with increased differentiation. The loss of regenerative potential was in agreement with observations made by Rashid and Street on haploid suspension cultures (358).The suspension cultures showed a decrease in embryogenic potential on succesive subculturing, paralleling a decline in the proportion of haploid cells. Jung and Tepfer (269) regenerated plants from Atropa belladonna roots which were transformed with Agrobacteriurn rhizogenes. The transformed plants had similar alkaloid levels in the root as the nontransformed plants, but the leaves had a 3 times lower alkaloid content. These experiments thus show that genetic engineering is feasible for this species. 2 . Datura

Hiraoka and Tabata (307) were able to grow plants from cellular aggregates formed in cell suspension cultures of Datura innoxia. Most of the plants obtained were diploid (82%). Surprisingly only a few were aneuploid or polyploid, given that in the suspension culture only 32% of the cells were diploid. In the development of plants from the non-alkaloidproducing suspension cells, an increase in alkaloid production was ob-

1.

57

PLANT BIOTECHNOLOGY

TABLE XIV OCCURRENCE OF TROPANE ALKALOIDS IN Atropa PLANT CELLA N D TISSUECULTURES A1kaloid Atropine Atropine Atropine scopolamine, tropine, cuscohygrine Atropine Atropine Atropine, Choline Atropine, scopolamine, tropine Atropine Atropine Atropine Atropine, scopolamine, cuscohygrine, plus 14 tropane derivatives Atropine, scopolamine hydroxyhyoscyamime Atropine, scopolamine Atropine Atropine, scopolamine Atropine, scopolamine, cuscohygrine Atropine Atropine, scopolamine Atropine, scopolamine Atropine, scopolamine

Plant species

Type of culture

Yield

Ref.

A . belladonna A . belladonna A . belladonna

Roots Callus Roots

0.5% DW 0.05% DW 0.096% DW

274,275 2 76 277

A . belladonna

Suspension, aggregates Callus, suspension Roots Callus Callus Callus

-

278

0.45% DW 0.53% DW

279-283 284 285

0.0012% DW

286

A . belladonna A . belladonna A . belladonna A . belladonna

Callus Roots Shoots Roots Suspension

0.75% DW 0.5% DW 0.81% DW 0.0005% DW

287 288 289 290

A . belladonna

Roots

0.34% DW

291,292

A . belladonna

Hairy Roots

0.4% DW

293

A . belladonna A . belladonna

Immobilized roots Hairy roots

0.2% DW -

294 268

A . belladonna

Hairy roots

1.3% DW

269

A . belladonna A . belladonna, A . caucasica A . belladonna

Shoots Hairy roots

295 296

Callus

0.01% DW 0.2% DW, 0.12% DW 0.09%DW

A . belladonna

Roots

0.25% DW

298

A . belladonna A . belladonna A . belladonna A . belladonna

-

297

TABLE XV OCCURRENCE OF TROPANE ALKALOIDS I N DATURA PLANTCELLA N D TISSUECULTURES Alkaloid

Plant species

Alkaloids Atropine, scopolamine Atropine, scopolamine Scopolamine

D. stramonium D. tatula D. metel D. stramonium

Atropine, scopolamine Choline, pseudotropine, cuscohygrine Atropine, scopolamine Choline, scopolamine Atropine, choline Scopolamine

D. quercifolia D. innoxia D. metel D. stramonium D. stramonium D. tatula D . metel D. innoxia

Atropine Aposcopolamine Choline, atropine, scopolamine Atropine

D. D. D. D.

Atropine Atropine, scopolamine Atropine, tropine, scopolamine

D. innoxia D. innoxia D. innoxia, D. stramonium D. clorantha

metel meteloides metel metel

Type of culture Roots Roots Roots Callus suspension Callus Callus Callus Suspension Suspension Callus Callus Callus Shoots Callus Suspension Callus Callus suspension Callus Suspension Callus

Yield 0.06% DW -

0.03% DW, 0.003 g/ liter 0.02% DW 0.06% DW 0.006% DW -

0.18% DW 0.04% DW 0.01% DW 0.015% DW

Ref. 2 73 299,300 301 302

303 304 305 306 265 307

0.08% DW 0.01 1% DW

308 309 266 310

0.0016 g/liter 0.15,0.1, 0.15% DW

31 I 312 313

Atropine, scoplamine, h ydrox yh yoscyamine Atropine Atropine, scopolamine

VI

Scopolamine Atropine, scopolamine Atropine, scopolamine, apoatropine Atropine Atropine, scopolamine Scopolamine Atropine, scopolamine, plus 17 tropane derivatives

D. stramonium, D. innoxia, D. leichhardtii, D. fastuosa D. stramonium D. chlorantha D. ferox D. fastuosa D. innoxia D. metel D. meteloides D. quercifolia D. rosei D. sanguinea D. stramonium D. stramonium var. inermis D. stramonium var. stramonium D. strumonium var. tatula D. stramonium D. candida D. stramonium D. wrightii Datum F, hybrid D. strumonium, D. innoxia, D. lanosa, D. pruinosa D. kymatocarpa D. candidu

Roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Hairy roots Callus Hairy roots

0.42, 0.47, 0.19, 0.27% DW 0.6% DW, 0.1 g/liter 0.25% DW 0.23% DW 0.2% DW 0.22% DW 0.31% DW 0.08% DW 0.26% DW 0.17% DW 0.13% DW 0.31% DW 0.22% DW 0.19% DW 0.4% DW 0.56% DW 0.68% DW 0.18 giliter -

291,292 314 296

315 316 317 317 317 318 319

60


TABLE XVI OCCURRENCE OF TROPANE ALKALOIDS IN Duboisia PLANTCELLAND TISSUE CULTURES Alkaloid Atropine, scopolamine Atropine, scopolamine, hydroxyhyoscyamine, valtropine Nicotine, tropine esters Nicotine, anabasine Atropine, scopolamine, nicotine Atropine, scopolamine, nicotine, nornicotine, anabasine Atropine, scopolamine, nicotine Atropine, scopolamine Atropine, scopolamine, hydroxyhyoscyamine Atropine, scopolamine Atropine, scopolamine Atropine, scopolamine Atropine, scopolamine, nicotine

Plant species

Type of culture

Yield -

Ref. 320 32 1

D . myoporoides Duboisia hybrid

Callus Shoots

D. leichhardtii D. myoporoides D. leichhardtii

Callus Callus Roots

0.8% DW

322 323 324

D . myoporoides

Roots

0.02% DW

325

D . myoporoides, D . leichhardtii, D . hop woodii Duboisia hybrid D . leichhardtii

Roots

0.4, 1.69, 1% DW

326

Roots Roots

0.65% DW

32 7 291,292

D. myoporoides Duboisia hybrid D . leichhardtii D.leichhardtii

Hairy roots Hairy roots Hairy roots Roots

1% DW 0.13% DW 2.1% DW 0.1% DW

328 296 329 298

served with an increase in differentiation, and production of scopolamine (the major alkaloid in the plant) was associated with the formation of roots. Some of the plants obtained showed abnormal alkaloid metabolism; for example, in some plants hydrolysis of scopolamine was observed during drying of harvested leaves, and in another plant alkaloid production occurred only after flowering. Kibler and Neumann (312) reported that diploid plants of D . innoxia had higher alkaloid contents than plants obtained from haploid tissue. Androgenic diploid plants obtained from anther cultures of a Datum innoxia plant showed considerable variation in alkaloid content. High scopolamine-producingplants could be obtained in this way (352).Plantlets were regenerated from a Datum candida hybrid hairy root culture (326). For a more extensive review on the in uitro propagation of Datum, the reader is refered to Petri and Bajaj (357). 3 . Duboisia

Conditions for the tissue culture and plant regeneration of Duboisia myoporoides have been described (323). The alkaloid content was fol-


61

lowed during the development of the plants (351). A clear difference was found between the alkaloid content of a seedling, which has most of the alkaloids in the leaves, and a regenerated plantlet, where most of the alkaloids are found in the roots, the leaves being devoid of alkaloids. During development of the regenerated plant various patterns of alkaloids were observed in the leaves. In the fully developed plant, however, the alkaloid distribution was similar to that in the mother plant. In a further study it was found that in the regenerated plants very low levels of atropine esterase (a tropane alkaloid-hydrolyzing enzyme) activity in the roots coincided with the absence of alkaloids in the leaves (359). Griffin (360) reviewed various aspects of Duboisia species, including plant cell culture work. He concluded that it ought to be possible to develop high producing strains of Duboisia by means of plant tissue culture methods in combination with a sensitive quantification method for scopolamine. Kitamura (361)reviewed the in uitro regeneration of Duboisia species.

4. Hyoscyamus Corduan (309) reported methods of obtaining haploid and homozygous diploid plants form anthers of Hyoscyamus niger. Wernicke and Kohlenbach (350) described a method to generate plants from cultures obtained from microspores. Regeneration of Hyoscyamus muticus from callus was reported by Grewal et al. (332).Wernicke et al. (355)was able to obtain plants from leaf protoplasts isolated from a haploid Hyoscyamus muticus. From in uitro cultures of anthers of Hyoscyamus niger and H . albus, plants could be obtained which produced scopolamine as the major alkaloid, like the mother plants; however, qualitative and quantitative differences existed in the total spectra of alkaloids present (267).Considerable somaclonal variation was found in cell cultures derived from protoplasts of Hyoscyamus muticus (339,341,343).The plants also show considerable variation in alkaloid content. High alkaloid-producing plants can be developed via selection, and plants with a scopolamine content of 4% have been obtained (342,362). Introduction of genes in Hyoscyamus muticus with Agrobacterium tumefaciens (363) and electroporation (364) has proved feasible. An extensive review of the in uitro propagation, plant breeding, and cultivation of Hyoscyamus species is given by Strauss (365).

5. Conclusion Summarizing, most methods for in uitro propagation and plant breeding in modern plant biotechnology have successfully been applied to the major tropane alkaloid-producing Solanaceae species. Even transformation with Agrobacterium has been applied extensively. So far, new genes, coding for certain desired traits, have not been introduced into these plants, but

TABLE XVII ALKALOIDS I N Hyoscyamus PLANT CELL AND TISSUE CULTURES OCCURRENCE OF TROPANE Alkaloid

8

Plant Species

Alkaloids Atropine, scopolamine, cuscohygrine Atropine Atropine, scopolamine

H. niger H. niger H . muticus H. niger

Atropine Atropine Scopolamine Atropine, scopolamine

H . niger H. muficus H . niger H. niger H . albus H . gyorffi H . pusillus H . muticus H. bohemicus

Type of Culture Roots Suspension Callus Suspension Roots Callus Hairy roots Hairy roots Callus Roots Callus Roots Callus Roots Callus Roots Callus Roots Callus Roots

Yield 0.075% DW 0.11% DW 0.09% DW, 0.01 g h t e r 0.3% DW

0.61% DW 0.55% DW 150"C, UV, CD, IR, MS, 'Hand I3C NMR (545,546)

2. 5 . Physarochrome A

ALKALOIDS FROM MUSHROOMS

32 1

Physarum polycephalum (Myxomycetes)

Isolation, structure, amorphous, [aID +7.2” (MeOH), UV, IR, MS, ‘H and I3C NMR (546,547)

6. Piperine

Ulocladium species (H yphomycetes)

Isolation, identification, UV, IR, MS, ‘H NMR (548)

7. Phomopsin A

Phomopsis leptostromiformis (Coelomycetes)

Isolation, structure, mp 205°C (dec.), UV, IR, ‘H and 13C NMR (549,550)

8. Rhizonin A

Rhizopus microsporus (Mucorales, Zygomycetes)

Isolation, structure, conformational analysis in solution and crystal, IH and I3C NMR, X-ray

COCH,

I H O P : %H2N”

(551,552)

322

R62A ANTKOWIAK AND WIESEAW Z. ANTKOWIAK ~

~

9. AK-toxin I R = CH3 AK-toxin I1 R = H

~~

Alternaria alternata (Hyphomycetes)

Isolation, structure determined by chemical, spectral, and X-ray crystallography studies (553,554);total synthesis AK-toxin I1 (555)

Acremonium coenoohialum (Hyphomycetes)

Isolation, semisynthesis, 'H and "C NMR studies of derivatives (556); total synthesis (557)

Fusarium equiseti (Hyphomycetes)

Isolation, structure, UV, CD, IR, MS, 'H and I3C NMR, phenylboronic ester derivative in structure assignment (558); total synthesis (559)

Ascochyta chrysanthemi (Coelomycetes)

Isolation, mp 193"C, [aID -37.2" (PyH), UV, IR, 'H and 13CNMR, X-ray structure (560)

Cylindrocladium ilicicola (Hyphomycetes)

Biosynthetic study with I3Clabeled acetates, "Nlabeled phenylalanine, and I4C-labeledphenylalanine

,,a

R r

10. Loline (festucine)

&CH3

11. Equisetin

HO,

*-Me

12. Chrysanthone O M -e

0

13. Ilicicolin

OH

(561)

H

2. ALKALOIDS FROM MUSHROOMS

323

14. Gliovictin

Asteromyces cruciatus

Isolation, identification [a],, -62" (CHC13), 13C NMR

15. Bohemamine

Aztinosporangium species (Actinomycetes) (Fungi Imperfecti)

Isolation, structure, mp 199200°C (dec.), UV, IR, MS, ' H and I3C NMR, X-ray (563)

16. Olivoretin A R = CH3 Olivoretin D R = H

Streptoverticillium olivoreticuli (Actinomycetes)

Isolation, structure, mp 251253"C, [ a ] -314.9" ~ (CHC13)for A, mp 228229"C, [ a ]-141.5" ~ (MeOH) for D; UV, IR, MS, CD, ' H and "C NMR, X-ray study of olivoretin D (564)

17. Meleagrin R=R'=H

Penicillium rneleagrinum

Isolation, structure, mp 250°C (dec.), UV, IR, MS, CD, 'H NMR, X-ray analysis of 9-0-pbromobenzoyl derivative

OyNH

OR

(565,566)

L I

Me0 R

18. Peramine

Acremonium lolli (Hyphomycetes)

Isolation, HBr salt: mp 242243"C, UV, MS, ' H and 13C NMR, biological activity (567)

324

R 6 Z A ANTKOWIAK A N D WIESJZAW 2. ANTKOWIAK

Acknowledgments

The authors express their deep gratitude especially to Professors W. Boczon, A. Brossi, W. Gessner, K. H@iland, S. Rapior, W. Steglich, and T. Stijve for kind compliance with literature requests, to Mr. M. Court and Mrs. E. Krygier-Court for watching over our English, to Professor A. Bujakiewicz for introducing us to the world taxonomy of fungi, and to Mrs. K. Sternal, Miss L. Sadek, and Mr. J. Bartoszewicz for technical assistance. We also thank the Institute of Organic Chemistry, Polish Academy of Sciences, for financial support (Grant No. CPBP-01.13.2.19). REFERENCES 1. D. L. Hawksworth, B. C. Sutton, and G. C. Ainsworth, “Ainsworth and Bisby’s

Dictionary of the Fungi,’’ 7th Ed., Commonwealth Mycological Institute, Kew, Surrey, England, 1983. 2. A. Koivikko and J. Savolainen, Allergy 43, 1 (1988). 3. V. Marteka, “Mushrooms Wild and Edible.” Norton, New York and London, 1980. 4. R. Phillips, “Mushrooms and Other Fungi of Great Britain and Europe.” Pan Books Ltd., London, 1981. 5. G. Pacioni, “Simon and Schuster Guide to Mushrooms” (G. Lincoff, ed.). Simon and Schuster, New York, 1981. 6. J. H. Birkinshaw and C. E. Stickings, Fortschr. Chem. Organ. Naturstofle 20,1(1962). 7. S. Kohlmunzer and J. Grzybek, Wiad. Bot. 16, 35 (1972). 8. C. H. Eugster, Naturwissenschaften 55,305 (1968). 9. C. H. Eugster, Fortschr. Chem. Organ. Naturstofle 27,261 (1969). 10. T. Wieland, Science 159,946 (1968). 11. M. C. Pirrung and C. V. DeAmicis, Tetrahedron Lett. 29, 159 (1988). 12. C. H. Eugster and P. G. Waser, Experientia 10,298 (1954). 13. F. Jellinek, Acta Crystallogr. 10,277 (1957). 14. C. H. Eugster and E. Schleusener, Helu. Chim.Acta 52,708 (1969). 15. E. Schleusener and C. H. Eugster, Helu. Chim.Acta 53, 130 (1970). 16. R. J. Stadelmann, E. Mdller, and C. H. Eugster, Helu. Chim. Acta 59,2432 (1976). 17. T. Stijve, Coolia 25,94 (1982). 18. P.-C. Wang and M. M. Joullie, in “The Alkaloids” (A. Brossi, ed.), Vol. 23, Chap. 6, p. 327. Academic Press, Orlando, Florida, 1984. 19. H. Bollinger and C. H. Eugster, Helu. Chim. Acta 54,2704 (1971). 20. P.-C.Wang and M. M. Joullie, J. Org. Chem. 45,5359 (1980). 21. C. H. Eugster, Helu. Chim.Acta 39, 1023 (1956). 22. S. E. Unger, A. Vincze, R. G. Cooks, R. Chrisman, and L. D. Rothman, Anal. Chem. 53,976 (1981). 23. S. Wilkinson, Q. Rev., Chem. SOC. 15, 153 (1961). 24. M. Chmielewski and P. Guzik, Heterocycles 22, 7 (1984). 25. M. Chmielewski, P. Guzik, B. Hintze, and W. M. Daniewski, J. Org. Chem. 50,5360 (1985). 26. J. Jurczak, M. Tkacz, and U. Majchrzak-Kuczyfiska, Synth. Commun., 920 (1983). 27. M. Chmielewski, P. Guzik, B. Hintze, and W. M. Daniewski, Tetrahedron 41, 5929 (1985). 28. R. Amouroux, B. Gerin, and M. Chastrette, Tetrahedron Lett. 23, 4341 (1982).

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525. R. Sauter, E. J. Thomas, and J. P. Watts, J. Chem. Soc., Perkin Trans. 1 , 519 (1989). 526. P. F. Dowd, R. J. Cole, and R. F. Vesonder, U.S. Patent Appl. U.S. 201,143, November 15, 1988; Chem. Abstr. 111,73094d (1989). 527. R. J. Cole, D. M. Wilson, J. L. Harper, R. H. Cox, T. W. Cochran, H. G. Cutler, and D. K. Bell, J. Agric. Food Chem. 30, 301 (1982). 528. M. Nukina, Agric. Biol. Chem. 51,2625 (1987). 529. M. Nukina, Tennen Yuki Kagobutsu Toronkai Koen Yoshishu 29, 705 (1987); Chem. Abstr. 108, 91432e (1988). 530. K. Neupert-Laves and M. Dobler, Helu. Chim. Acta 65, 1426 (1982). 531. W. Keller-Schierlein and E. Kupfer, Helu. Chim. Acta 62, 1501 (1979). 532. B. M. Trost, M. Ohmori, S. A. Boyd, H. Okawara, and S. J. Brickner, J. A m . Chem. SOC.111,8281 (1989). 533. E. Kupfer and W. Keller-Schierlein, Helu. Chim. Acta 62, 1525 (1979). 534. A. C. Stierle, J. H. Cardellina 11, and G. A. Strobel, J . N a t . Prod. 52,42 (1989). 535. S. Iwasaki, H. Muro, S. Nozoe, S. Okuda, and Z. Sato, Tetrahedron Lett., 13 (1972). 536. N. Umetsu, J. Kaji, and K. Tamari, Agric. Biol. Chem. 36,859 (1972). 537. M. H. Lebrun, L. Nicolas, M. Boutar, F. Gaudemer, S. Ranomenjanahary, and A. Gaudemer, Phytochemistry 27,77 (1988). 538. P. S. Steyn and C. J. Rabie, Phytochemistry 15, 1977 (1976). 539. M. J. Nolte, P. S. Steyn, and P. L. Wessels, J . Chem. SOC.,Perkin I, 1057 (1980). 540. H. Kayakiri, S. Takase, H. Setoi, I. Uchida, H. Terano, and M. Hashimoto, Tetrahedron Lett. 29, 1725 (1988). 541. M. J. Schneider, F. S. Ungemach, H. P. Broquist, and T. M. Harris, Tetrahedron 39,29 (1983). 542. F. P. Guengerich, S. J. DiMari, and H. P. Broquist, J. A m . Chem. Soc. 95,2055 (1973). 543. M. H. Ali, L. Hough, and A. C. Richardson, J. Chem. Soc., Chem. Commun., 447 (1984). 544. P. R. Dorling, S. M. Colegate, and C. R. Huxtable, Toxicon 3, (Suppl.) 93 (1983). 545. V. I. Casser, B. Steffan, and W. Steglich, Angew. Chem. 99,597 (1987). 546. W. Steglich, Pure Appl. Chem. 61,281 (1989). 547. B. Steffan, M. Praemassing, and W. Steglich, Tetrahedron Lett. 28,3667 (1987). 548. J. S. Dahiya, D. L. Woods, and J. P. Tewari, Phytochemistry 27,2366 (1988). 549. C. C. J. Culvenor, J. A. Edgar, M. F. Mackay, C. P. Gorst-Allman, W. F. 0. Marasas, P. S. Steyn, R. Vleggaar, and P. L. Wessels, Tetrahedron 45,2351 (1989). 550. C. C. J. Culvenor, P. A. Cockrum, J. A. Edgar, J. L. Frahn, C. P. Gorst-Allman, A. J. Jones, W. F. 0. Marasas, K. E. Murray, L. W. Smith, P. S. Steyn, R. Vleggaar, and P. L. Wessels, J. Chem. SOC.,Chem. Commun., 1259 (1983). 551. M. Potgieter, P. S. Steyn, F. R. van Heerden, P. H. van Rooyen, and P. L. Wessels, Tetrahedron 45,2337 (1989). 552. P. S. Steyn, A. A. Tuinman, F. R. van Heerden, P. H. van Rooyen, P. L. Wessels, and C. J. Rabie, J. Chem. Soc., Chem. Commun., 47 (1983). 553. T. Nakashima, T. Ueno, and H. Fukami, Tetrahedron Lett. 23,4469 (1982). 554. T. Nakashima, T. Ueno, H. Fukami, T. Taga, H. Masuda, K. Osaki, H. Otani, K. Kohmoto, and S. Nishimura, Agric. Biol. Chem. 49,807 (1985). 555. K. Ando, T. Yamada, Y. Takaishi, and M. Shibuya, Heterocycles 29, 1023 (1989). 556. R. J. Petroski, S. G. Yates, D. Weisleder, and R. G. Powell, J. Nut. Prod. 52,810 (1989). 557. J. J. Tufariello, H. Meckler, and K. Winzenberg, J. Org. Chem. 51,3556 (1986). 558. N. J. Phillips, J. T. Goodwin, A. Fraiman, R. J. Cole, and D. G. Lynn, J . A m . Chem. Soc. 111,8223 (1989). 559. E. Turos, J. E. Audia, and S. J. Danishefsky, J. A m . Chem. Soc. 111, 8231 (1989).

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CUMULATIVE INDEX OF TITLES Aconitum alkaloids, 4,275 (1954), 34,95 (1988) diterpenoid, 7,473 (1960) Ci9 diterpenes, 12,2 (1970) Czoditerpenes, 12, 136 (1970) Acridine alkaloids, 2, 353 (1952) Acridone alkaloids, experimental antitumor activity of acronycine, 21, 1 (1983) Actinomycetes, isoquinolinequinones, 21,55 (1983) N-Acyliminium ions as intermediates in alkaloid synthesis, 32,271 (1988) Ajamaline-Sarpagine alkaloids, 8, 789 (1965), 11,41 (1968) Alkaloid structures forensic chemistry of, 32, 1 (1988) spectral methods, study, 24,287 (1985) unknown structure minor alkaloids, 5,301 (1955), 7,509 (1960) unclassified alkaloids, 10,545 (1%7), 12,455 (1970), 13,397 (1971), 14,507 (1973), 15,263 (1975), 16,511 (1977) Alkaloids histochemistry of, 39, 165 (1990) Alkaloids in Cannabis satiua L., 34,77 (1988) the plant, 1, 15 (1950), 6, l(1960) Alkaloids from Ants and insects, 31, 193 (1987) Aspergillus, 29, 185 (1986) Puuridiantha species, 30,223 (1987) Tabernaemontana, 27, 1 (1986) Alstonia alkaloids, 8, 159 (1965), 12,207 (1970), 14, 157 (1973) Amaryllidaceae alkaloids, 2,331 (1952), 6,289 (1960), 11,307 (1968), 15,83 (1975), 30, 251 (1987) Amphibian alkaloids, 21, 139 (1983) Analgesics, 5, 1 (1955) Anesthetics, local, 5,211 (1955) Anthranilic acid, related to quinoline alkaloids, 17, 105 (1979), 32,341 (1988) Antimalarials, 5, 141 (1955) Antitumor alkaloids, 25, 1 (1985), 37, 1, 205 (1990) Taxus alkaloids, 25,6 (1985) Sesbania alkaloids, 25, 18 (1985) Pyrrolizidine alkaloids, 25,21 (1985) Acronycine, 25,38 (1985) Emetine, 25,48 (1985) Cephalotaxus alkaloids, 25,57 (1985) Colchicine, 25,69 (1985) Camptothecine, 25,73 (1985)

341

342

CUMULATIVE INDEX OF TITLES

Ellipticine, 25, 89 (1985) Maytansinoids, 25, 142 (1985) Phenanthroindolizidines,25, 156 (1985) Bisisoquinolines, 25, 163 (1985) Benzophenanthridines, 25, 178 (1985) Protoberberines, 25, 188 (1985) Amaryllidacea alkaloids, 25, 198 (1985) Apocynaceae alkaloids, steroids, 9, 305 (1967) Aporphine alkaloids, 4, 119 (1954), 9, 1 (1967), 24, 153 (1985) Aristolochia alkaloids, 31,29 (1987) Aristotelia alkaloids, 24, 113 (1985) Aspidosperma alkaloids, 8,336 (1965), 11,205 (1968), 17, 199 (1979) Azafluoranthene alkaloids, 23,301 (1984) Bases simple, 8, 1 (1965) simple indole, 10, 491 (1967) Benzodiazepine alkaloids, 39,63 (1990) Benzophenanthridine alkaloids, 26, 185 (1985) Benzylisoquinoline alkaloids, 4, 29 (1954), 10,402 (1967) Betalains, 39, 1 (1990) Bisbenzylisoquinoline alkaloids, 4, 199 (1954), 7, 439 (1960), 9, 133 (1967), 13, 303 (1971), 30, 1 (1987) occurrence, 16,249 (1977) structure, 16,249 (1977) pharmacology, 16,249 (1977) synthesis, 16, 319 (1977) Bisindole alkaloids, 20, 1 (1981), 37, 1 (1990) isolation, structure elucidation, and biosynthesis of, 37, 1 (1990) medicinal chemistry of, 37, 145 (1990) pharmacology of, 37,205 (1990) therapeutic use of, 37,229 (1990) Bums alkaloids, steroids, 9,305 (1967), 14, 1 (1973) Cactus alkaloids, 4,23 (1954) Calabar bean alkaloids, 2,438 (1952), 8,27 (1965), W, 213 (1971), 36,225 (1989) Calabash curare alkaloids, 8,515 (1%5), 11, 189 (1968) Calycanthaceae alkaloids, 8,581 (1965) Camptothecin, 21, 101 (1983) Cancentrine alkaloids, 14,407 (1973) Canthin-6-one alkaloids, 36, 135 (1989) Capsicum species, pungent principle of, 23,227 (1984) Carbazole alkaloids, 13, 273 (1971), 26, 1 (1985) Carboline alkaloids, 8,47 (1965), 26, 1 (1985) P-Carboline congeners and ipecac alkaloids, 22, 1 (1983) Cardioactive alkaloids, 5,79 (1955) Celestraceae alkaloids, 16,215 (1977) Cephalotaxus alkaloids, 23, 157 (1984) Chemotaxonomy of papaveraceae and fumariaceae, 29, 1 (1986) Chinese medicinal plants, alkaloids, 32,241 (1988)


343

Chromone alkaloids, 31,67 (1987) Cinchona alkaloids, 14, 181 (1973), 34,331 (1988) chemistry, 3, 1 (1953) Colchicine, 2, 261 (1952), 6, 247 (1960), 11,407 (1968), 23, 1 (1984) Configuration and conformation, elucidation by X-ray diffraction, 22,5 1 (1983) Corynantheine, yohimbine, and related alkaloids, 27, 131 (1986) Cularine alkaloids, 4,249 (1954), 10,463 (1967), 29,287 (1986) Curare-like effects, 5,259 (1955) Cyclic tautomers of tryptamines and tryptophans, chemistry and reactions, 34, 1 (1988) Cyclopeptide alkaloids, 15, 165 (1975) Daphniphyllum alkaloids, 15,41 (1975), 29,265 (1986) Delphinium alkaloid, 4,275 (1954) diterpenoid, 7,473 (1960) Clo-diterpenes, 12, 2 (1970) Czo-diterpenes, 12, 136 (1970) Dibenzopyrrocoline alkaloids, 31, 101 (1987) Diplorrhyncus alkaloids, 8, 336 (1965) C 19-Diterpenealkaloids Aconitum, 12,2 (1970) Delphinium, 12, 2 (1970) Garrya, 1 2 , 2 (1970) structure, 17, l(1970) synthesis, 17, l(1979) Czo-Diterpenealkaloids Aconitum, 12, 136 (1970) chemistry, 18,99 (1981) Delphinium, 12, 136 (1970) Garrya, 12, 136 (1970) Distribution of alkaloids in traditional Chinese medicinal plants, 32,241 (1988) Diterpenoid alkaloids Aconitum, 7,473 (1960), 12,2 (1970) Delphinium, 7,473 (1960), 12, 2 (1970) Garrya, 7,473 (1960), 12,2 (1960) general introduction, 12, xv (1970) C19-diterpenes, 12, 2 (1970) Cz0-diterpenes, 12, 136 (1970) Eburnamine-Vincamine alkaloids, 8,250 (1965), 11, 125 (1968), 20,297 (1981) Elaeocarpus alkaloids, 14,325 (1973) Ellipticine alkaloids and related compounds synthesis and antitumor activity of, 39,239 (1990) Elucidation, by X-ray diffraction structural formula, 22,51 (1983) configuration, 22,51 (1983) conformation, 22,51 (1983) Enamide cyclizations, application in alkaloid synthesis, 22, 189 (1983) Enzymatic transformation of alkaloids, microbial and in uitro, 18, 323 (1981) Ephedra bases, 3,339 (1953), 35,77 (1989) Ergot alkaloids, 8,726 (1965), 15, 1 (1975), 38, 1 (1990)

344


Erythrina alkaloids, 2,499(1952), 7,201(1960),9,483 (1967),18,1 (1981) Erythrophleum alkaloids, 4,265 (1954), 10,287 (1967) Eupomatia alkaloids, 24, 1 (1985)

Forensic chemistry, alkaloids, 12, 514 (1970) by chromatographic methods, 32,1 (1988) Galbulimima alkaloids, 9,529 (1%7), 13,227 (1971) Gardneria alkaloids, 36,1 (1989) Garrya alkaloids diterpenoid, 7,473 (1960) C19 V-diterpenes, l2,2(1970) Czo-diterpenes, 12, 136 (1970) Geissospermum alkaloids, 8,679(1965), 33,84(1988) Gelsem‘um alkaloids, 8,93 (1965),33,83 (1988) Glycosides, monoterpene alkaloids, 17,545 (1979) Guatteria alkaloids, 35,1 (1989) Haplophyton cimicidum alkaloids, 8,673(1965) Hasubanan alkaloids, 16,393 (1977), 33,307(1988) Holarrhena group, steroid alkaloids, 7,319 (1960) Hunreria alkaloids, 8,250(1965) Iboga alkaloids, 8,203(1965),11,79(1968) Imidazole alkaloids, 3,201 (1953), 22,281 (1983) Indole alkaloids, 2,369 (1952), 7,1 (1960),26, 1 (1985) distribution in plants, 11,1 (1968) simple, including P-carbolines and P-carbazoles, 26, 1 (1985) Indole bases, simple, 10,491 (1967) Indolizidine, simple and quinolizidine alkaloids, 28, 183 (1986) 2.2’-Indolylquinuclidinealkaloids, chemistry, 8,238(1965),11,73(1968) Ipecac alkaloids, 3,363 (1953),7,419 (1960), 13,189 (1971),22, 1 (1983) P-Carboline alkaloids, 22,1 (1983) Isolation of alkaloids, 1,1 (1950) Isoquinoline alkaloids, 7,423 (1960) biosynthesis, 4,1 (1954) I3C-NMR spectra, 18,217(1981) simple isoquinoline alkaloids, 4,7(1954),21,255 (1983) Isoquinolinequinones, from actinomycetes and sponges, 21,55 (1983)

Khat alkaloids, 39,139 (1990) Kopsia alkaloids, 8, 336 (1965) Lead tetraacetate oxidation, 36,69(1989) Local anesthetics, alkaloids, 5,211(1955) Localization of alkaloids in the plant, 1,15 (1950),6,1 (1960) Lupine alkaloids, 3,199 (1953), 7,253(1960), 9,175 (1967),31,116 (1987) Lycopodium alkaloids, 5,265(1955),7,505 (1960), 10,306(1967),14,347(1973),26,241

(1985)

Lythracae alkaloids, 18,263(1981), 35,155 (1989)


345

Mammalian alkaloids, 21, 329 (1983) Marine alkaloids, 24,25 (1985) Maytansinoids, 23,71 (1984) Melanins, chemistry of, 36,253 (1989) Melodinus alkaloids, 11,205 (1968) Mesembrine alkaloids, 9,467 (1967) Metabolic transformation of alkaloids, 27, 323 (1986) Microbial and in uitro enzymatic transformation of alkaloids, 18,323 (1981) Mitragyna alkaloids, 8, 59 (1965), 10, 521 (1967), 14, 123 (1973) Monoterpene alkaloids, 16,431 (1977) glycosides, 17, 545 (1979) Morphine alkaloids, 2, 1 (part 1, 1952),2, 161 (part 2, 1952),6,219 (1960), 13, 1 (1971) Mushrooms, alkaloids from, 40, 189 (1991) Mydriatic alkaloids, 5,243 (1955) a-Naphthaphenanthridine alkaloids, 4,253 (1954), 10,485 (1967) Naphthylisoquinoline alkaloids, 29, 141 (1986) Narcotics, 5, 1 (1955) I3C-NMR spectra of isoquinoline alkaloids, 18,217 (1981) Nuphar alkaloids, 9,441 (1967), 16, 181 (1977), 35,215 (1989) Ochrosia alkaloids, 8, 336 (1965), 11,205 (1968) Ourouparia alkaloids, 8, 59 (1965), 10, 521 (1967) Oxaporphine alkaloids, 14,225 (1973) Oxazole alkaloids, 35,259 (1989) Oxindole alkaloids, 14,83 (1973)

Papaveraceae alkaloids, 10,467 (1967), 12,333 (1970), 17,385 (1979) pharmacology, 15,207 (1975) toxicology, 15,207 (1975) Pavine and isopavine alkaloids, 31,317 (1987) Penraceras alkaloids, 8,250 (1965) Peptide alkaloids, 26,299 (1985) Phenanthrene alkaloids, 39,99 (1990) Phenanthroindolizidine alkaloids, 19, 193 (1981) Phenanthroquinolizidine alkaloids, 19, 193 (1981) P-Phenethylamines, 3,313 (1953), 35,77 (1989) Phenethylisoquinoline alkaloids, 14, 265 (1973), 36, 171 (1989) Phthalideisoquinoline alkaloids, 4, 167 (1954), 7,433 (1960), 9, 117 (1967), 24, 253 (1985) Picralima alkaloids, 14, 157 (1973) Picralima nitida alkaloids, 8, 119 (1965), 10,501 (1967) Piperidine alkaloids, 26,89 (1985) Plant Biotechnology, for production of alkaloids, 40, 1 (1991) Plant systematics, 16, 1 (1977) Pleiocarpa alkaloids, 8,336 (1965), 11,205 (1968) Polyamine alkaloids, putrescine, spermidine, spermine, 22,85 (1983) Pressor alkaloids, 5,229 (1955) Proroberberine alkaloids, 4,77 (1954), 9,41 (1967), 28,95 (1986), 33, 141 (1988) Protopine alkaloids, 4, 147 (1954), 34, 181 (1988) Pseudocinchona alkaloids, 8,694 (1965) Purine Alkaloids, 38,225 (1990)

346


Putrescine and related polyamine alkaloids, 22,85 (1983) Pyridine alkaloids, 1, 165 (1950), 6, 123 (1960), 11,459 (1968), 26,89 (1985) Pyrrolidine alkaloids, 1,91 (1950), 6, 31 (1960), 27, 270 (1986) Pyrrolizidine alkaloids, 1, 107 (1950), 6, 35 (1960), 12, 246 (1970), 26, 327 (1985) Quinazolidine alkaloids, see Indolizidine Alkaloids Quinazoline alkaloids, 3, 101 (1953), 7, 247 (1960), 29,99 (1986) Quinazolinocarbolines, 8 , 5 5 (1965), 21,29 (1983) Quinoline alkaloids other than Cinchona, 3,65 (1953), 7,229 (1960) related to anthranilic acid, 17, 105 (1979), 32,341 (1988) Rauwo&a alkaloids, 8,287 (1965) Reissert synthesis of isoquinoline and indole alkaloids, 31, 1 (1987) Reserpine, chemistry, 8,287 (1965) Respiratory stimulants, 5, 109 (1955) Rhoeadine alkaloids, 28, 1 (1986) Salamandra group, steroids, 9,427 (1967) Sceleriuim alkaloids, 19, 1 (1981) Senecio alkaloids, see Pyrrolizidine alkaloids Secoisoquinoline alkaloids, 33,23 1 (1988) Securinega alkaloids, 14,425 (1973) Sinomenine, 2,219 (1952) Solanum alkaloids chemistry, 3,247 (1953) steroids, 7,343 (1960), 10, 1 (1967), 19,81 (1981) Sources of alkaloids, 1, 1 (1950) Spectral methods, alkaloid structures, 24, 287 (1985) Spermine and related polyamine alkaloids, 22, 85 (1983) Spirobenzylisoquinoline alkaloids, 13, 165 (1971), 38, 157 (1990) Sponges, isoquinolinequinones, 21, 55 (1983) Sremona alkaloids, 9,545 (1967) Steroid alkaloids Apocynaceae, 9,305 (1967), 32,79 (1988) Buxus group, 9,305 (1967), 14, 1 (1973), 32,79 (1988) Holarrhena group, 7,319 (1960) Salamandra group, 9,427 (1967) Solanum group, 7,343 (1960), 10, 1 (1967), 19,81 (1981) Verarrum group, 7,363 (1960), 10, 193 (1967), 14, (1973) Stimulants respiratory, 5, 109 (1955) uterine, 5, 163 (1955) Structural formula, elucidation by X-ray diffraction, 22,51 (1983) Strychnos alkaloids, 1,375 (part 1, 1950),2,513 (part 2, 1952), 6, 179 (1%0), 8,515, 592 (1965), 11, 189 (1%8), 34,211 (1988), 36, 1 (1989) Sulfur-containing alkaloids, 26,53 (1985) Taxus alkaloids, 10,597 (1%7), 39, 195 (1990) Toxicology, Papaveraceae alkaloids, 15,207 (1975)


347

Transformation of alkaloids, enzymatic, microbial and in uitro, 18,323 (1981) Tropane alkaloids, 1,271 (1950), 6, 145 (1960), 9,269 (1967), 13,351 (1971), 16,83 (1977), 33, (1988) Tropoloisoquinoline alkaloids, 23,301 (1984) Tropolonic Colchicum alkaloids, 23, 1 (1984) Tylophora alkaloids, 9,517 (1967) Uterine stimulants, 5, 163 (1955) Verarrum alkaloids chemistry, 3,247 (1952) steroids, 7,363 (1960), 10, 193 (1967), 14, 1 (1973) Vinblastine, 37, 133 (1990) Vinblastine-Type Alkaloids, 37,77 (1990) “Vinca” alkaloids, 8, 272 (1965), 11,99 (1968), 37, 1 (1990) Voacanga alkaloids, 8,203 (1965), 11,79 (1968)

X-Ray diffraction, elucidation of structural formula, configuration, and conformation, 22, 51 (1983) Yohimbe alkaloids, 8,694 (1965), 11, 145 (1968), 27, 131 (1986), see also Coryantheine


A Acetylenic amino acids, in mushrooms, 303 17-O-Acetylnorajmaline, 146 Acetylnorrauglucine, 147 Acetylrauglucine, 147 Acetyltropine, 69 Aflatoxin, 234 Aflatrem, 250 Aflavinine, 247,249 Agaricone, 279 Agaridoxin, 300 Agaritine, 276 biosynthesis of, 278 synthesis of, 278 Agroclavine, 236 Ajmalicine, 110, 114, 117 Ak-toxins, 322 Aladeoxoviroidin, 219 Alaviroidin, 219 Alkaloids from, Lactarius necator, 272 Alkaloids, production by genetically engineered microorganisms, 6 production by plant cell cultures, 8 production by plants, 8 production of, 2 production prices, 4 types of, 2 precursor bioconversion, 7 Allomuscarine, 195 synthesis of, 197 Amanin amide, 218 Amanin, 218 Amanita, physiological principles, 194 Amanitin, 217, 218 Amanullin, 218 Amanullinic acid, 218 Amatoxins, 218 Amauronine, 253

Amino acids, in mushrooms, 289 4-Aminopyridine-2,3-dicarboxylicacid, 295,296 Anabasine, 45 Anatabine, 45 Anatmanide, 225 3',4'-Anhydrovinblastine, 139 Antiphallotoxin, 225 Apparicine, 9 Apparicine, 151 Arcyriarubin B, 230 Aromelic acids, 290,291 synthesis of, 293, 294 Aromoline, 76 Ascochalasin, 3 11 Aspergillic acids, 227 Aspochalasin, 319 Atropine, 52 Austamides, 249,251

B Baeocystine, 229 Benzylisoquiniolines, biosynthesis of, 79 Berbamine, 76 Berbamunine, 76 Berberine, from Berberis species, 94 from Coptis species, 95 occurrence in plant tissues, 98 patents on bioproduction, 96 Betalamic acid, 209,215 Betalanine alkaloids, 208 Betanidine, 209 Betaxanthins, 209 4,4'-Binecatorone, 271 Biosynthesis of, ajmalicine, 19J berberine, 19,72,94 indole alkaloids, 19 349

350

INDEX

ipecuhana alkaloids, 72 scopolamine, 19 Bisbenzylisoquinoline alkaloids, production by cell cultures, 75 patents, 75 Bissecodehydrocyclopiazonic acid, 253 Bohemamine, 323 Brevianamides, 249,252 Bufotenine N-oxide, 227 Bufotenine, 226 Bufothionine, 227 C

Caffeine, 154,155 Camptothecine, 153 Carbolin-1-propionic acid, 231 p-Carbolines, 231 p-Carbolines, 231 p-Hydroxyhyoscyamine epoxidase, 71 Cardycepin, 283 Catharanthine, 114,139 Cathenamine, 151 Cephaeline, 73 Cephalotaxine, 75 Cephalotaxus alkaloids, 75 production by cell cultures, 75 Chaconine, 157 Chaetoglobosin, 310 Chaliciporones, 305 Chanolclavine I, 236 Cheilanthifoline, 79 Chelerythrine, 88 Chelilutine, 88 Iq 2 Chelirubine, 88, 12 Chrysanthone, 322 Cinchona alkaloids, 104 plant biotechnology, 105 cell cultures, 105 extraction of, 107 bioconversions, 108 patents, 105 Cinchoninone, 108 Clavine alkaloids, 236 Clithioneine, 291 Clitidine, 281 Clitocine, 285,286 Codeine, 77,81 Codeinone, 84 Colchicine, 74

Conessine, 161 Connatin, 280 Connatin, 304 Coprine, 298 Cortinarins, 268 Cryptopine, 83,86 Culture types, 18 Cuscohygrine, 68 Cyclopiazonic acid, 253 Cyclopiazonic acid imine, 253 Cyclostizolobic acid, 213 Cytochalasanes, 307-310 Cytochalasins, 312-318 D Dehydrobufotenine, 227 Dehydrocommersonine, 159 Deoxophomin, 311 Deoxoviroidin, 219 Deoxoviroisin, 219

lO-Deoxy-4,4’-binecatorone, 271 10,10‘-Dideoxy-4,4’-binecatorone, 271 Dihydrochelerythrine, 88 Dihydrochelirubine, 88 Dihydromarcapine, 88 Dihydrosanguinarine, 87 Dihydrosetoclavine, 244 Domoic acid, 291

E Echinulines, 249,251 Elegagnine, 233,235 Ellipticine, 150 Elymoclavine, 236 Emetine, 73 Engleromycin, 315 Epchrosine, 151 Epiallomuscarine, 195 Epimuscarine, 195 synthesis of, 197 Epivincamine, 150 Equisetin, 322 Ergine, 237 Ergoannam, 246 Ergobasine, 238 Ergocornam, 245 Ergocorninam, 245

35 1

INDEX

Ergocornine, 245 Ergocorninine, 245 Ergocristam, 243,245 Ergocristam, 245,246 Ergocristine, 245 Ergocristinine, 245 Ergocryptam, 245 Ergocryptianm, 245 Ergocryptines, 245,246 Ergoline, 236 Ergometrine, 237 Ergonine, 245 Ergoninine, 245 Ergonovine, 237 Ergonovine, 239 Ergonovinine, 239 Ergopeptide alkaloids, 242 Ergopeptine, 242 Ergopeptide alkaloids, tabulation of, 245 Ergophine, 245 Ergophinine, 245 Ergosine, 245 Ergosinine, 245 Ergostetrine, 238 Ergostine, 245 Ergostinine, 245 Ergot alkaloids, 233 Ergotamine, 242,243,246 Ergotaminine, 245 Ergothioneine, 295,2% Ergotocin, 238 Ergovaline, 245 Ergovalinine, 245 Eritadenine, 287, 288 Escholtzia species, alkaloid production, 93 Eudistomine S, 232

F Fuligorubin A, 320 Fumitremorgins, 249, 251

G Genetic engineering, 142 Genetic modification, 19 Gliovictin, 323 -~-Glutaminyl-3,4-benzoquinone, 301 ~-Glutaminyl-4-hydroxybenzene, 300

Grzymaline, 267 Gyromitrine, 275

H Harman, 232,235 Harringtonine, 75 Hercynine, 195 L-Hercynine, 297 Homodeoxyhamngtonine, 75 3-Hydroxyvoafrine A and B, 152 4-Hydroxymethylphenylhydrazine,277 5-Hydroxytryptophan, 227 6-Hydroxymethylnebularine,287 6P-Hydroyhyoscyamine, 64 &Hydroxyergine, 237 %Hydroxyerginine, 237 Hyoscyamine-P-hydroxylase, 71 1-Hyoscyamine, 64 Hydrazine alkaloids, 275 Hydroxyinfractin, 232, 233 1

Ibotenic acid, 195,203 biosynthesis of, 208 photoreaction of, 205 synthesis of, 207 Ilicicolin, 322 Illudalic acid, 274 Illudin, 274 Illudinine, 272, 274 Illudol, 274 Indicaxanthin, 212 Indigo, 229 Indirubin, 230 Indole alkaloids biotechnology of, 109 patents on bioproduction, 111 high producing cell lines, 110 culture medium, 113 precursor feeding, 122 bioregulators, 123 large-scale culturing, 129 economics, 137 production of dimeric alkaloids, 138 bioconversions, 138 plant biotechnology, 141 Infractin, 233

352

INDEX

Infractopicrin, 231 Isobetanidine, 210 Isofumigaclavine, 236 Isolysergic acid, 237 Isoquinoline alkaloids, 72 Isoreserpiline, 151 Isothebaine, 83

J Jatrorrhizine, 76,95 K

Kainic acid, 291,292

L Lanosulin, 251 Lentinacin, 287 Lentysine, 287 Lepistine, 305 Leucoagaricone, 279 Loline, 322 LSD, 241 Lyophyllin, 230 Lysergic acid amide, 237 Lysergic acid, synthesis of, 239 Lysergol, 236

M Macarpine, 38 Macleaya species, Magnoflorine, 83,86, 101 Marcofortines, 252 Melanin, 215 Meleagrin, 323

N-Methyl-N-formylhydrazine,275 Methyl-10-methoxy-paspalate, 238 6-Methylnebularine, 287 10-Methoxyellipticine, 150 Miraxanthins, 212 Monogagamine, 153 Morphine, biosynthesis of,79 Muscaflavin, 210,215 Muscapurpurin, 214 Muscapurpurinic acid, 215 Muscaridine, 195 Muscarines, 194 synthesis of, 197

Muscarufin, 209 Muscaurins, 210,212 Muscazone, 195 Muscimol, 195, 203

N Narceine, 77 Nebularine, 286 Necatorin, 269 Necatorone, 269 synthesis of, 270 Necatorones, 269 Neobetanidine, 209 Neoechinulines, 249, 251 Nicotine, by root cultures, 50 in large-scale suspension cultures, 48 production in tissue cultures, 44 Nominine, 247,249 Nonmorphinan alkaloids, 83 Norbaeocystine, 229 2-Norberbamunine, 76 P-Nitroaminoalanine, 304 biosynthesis of, 7,72 N(8)-Norphysostigmine, 154 precursor bioconversion, 7 synthesis of, 293,294 Norlluorocurarine, 151 Nornicotine, 45 Norsanguinarine, 88 Noscapine, 83 Noscopine, 77 I

0 Olivoretins, 323 Opium alkaloids, biotechnology of, 78 patents on biotechnology methods, 78 bioconversions of, 84 Optisine, 95 Orellanine, 253 Orellanine, 254 demethylation of, 258 fluorescence of,263 mass fragmentation of, 262 model compounds, 261 photodecomposition of, 266 synthesis of, 256,257,259 thermal decomposition of, 259,260

353

INDEX

toxicity of, 264 X-ray analysis of, 262 Orientalidine, 83 Oxysanguinarine, 87

P Palmatine, 95 Pantherine, 203 Papaver species, alkaloid production, 93 Papaverine, 77 Paraherquamide, 252 Paspalic acid, 238 Paspaline, 250 Paspalinine, 250 Paspalitrem B, 250 Paxilline, 250 Penitrems, 250 Penniclavine, 236 Peptide alkaloids, 216 Peramine, 323 Pericine, 9 Phallacidin, 219 Phallacin, 219 Phallisacin, 219 Phallisin, 218 Phalloidin, 217, 219 Phalloin, 219-221 Phallolysin, 224 Phallotoxins, 218, 220 Phomopsin A, 321 Physarochrome A, 321 Physostigmine, 154 Picralina nitida, alkaloids of, 9 Piperine, 321 Pistillamine, 305 Plant biotechnology, cell density, importance of, 13 cell populations, selection of, 11 cellular transport, 15 culture conditions, 12 elicitation in alkaloid formation, 16 feeding of precursors, 16 gas composition, 14 immobilization of plant cells, 17 light, importance of, 13 medium composition, 12 pH, influence of, 12 premeabilization of cells, 17

screening, 11 storage compartments, 14 temperature, importance of, 13 Plant cell cultures, new compounds from, 9 Pleiocarpamine , 15 1 Proamanullin, 218 Process design of, 32,40 Production of alkaloids bioreactors, 34 cost estimation, 25 design stratergy for production of, 22 economic feasibility, 22 forced release of alkaloids, 36 large-scale production of, 20 market volume of alkaloids, 24 process parameters, 40 recovery from biomass, 35 recovery from medium, 37 Prophalloin, 219,221,223 Protophomin, 310 Protopine, 79 Proxiphomin, 3 11 Psalliotin, 230 Psilocin, 223 Psilocybin, 228 Pulcherriminic acids, 227 Pyrichalasin, 3 17 Pyridine alkaloids, 253

Quinidine, 104 Quinidinone, 108 Quinine, 104 Quinone, 301

Q

R Raucaricine, 145 Rauglucine, 147 RauvolJa alkaloids, 142 plant biotechnology, 144 cell cultures, 144, 147, 148 patents on biotechnology of, 147 enzyme production, 149 (R)-Reticuline, (S)-Reticuline, 79 Rhizonin A, 321 Roquefortine, 25 1 Rugulovasine A, 236

354

INDEX

S

Sanguinarine, 79,85 antimicrobial properties, 85 elicitation, 87 growth conditions, 86 patents for bioproduction, 86 Scopolamine, 52,64 Scoulerine, 79 Secologanin, 7 Serotonin, 225 Serpentine, 110, 114, 117 Silybin, 225 Solamargine, 157, 159 Solanidine, 157 Solanine, 157 Solasodine, 157, 159 Solasonine, 157, 159 Stizolobic acid, 210,212,215 Stizolobinic acid, 215 STOX (S)-tetrahydroberberine oxidase, 7,72 Strictosidine lactam, 150 Strictosidine synthase, 6 , 7 Stylopine, 79,83 T Tabersonine, 151 Tenuazonic acid, 320 Tetrahydroalstonine, 151 Tetrahydroharman-3-carboxylic acid, 23 1 from Thalictrum species, 100 Thalifendine, 101 Thebaine, 77,81 Theobromine, 154, 155

Tomatidine, 161 Trichomolic acid, 204 Tropane alkaloids, 68 large-scale cultures, 68 precursor feeding, 69 plant biotechnology, 71 Tropine, 69 Tryptathionine thioether, 223 Tryptophan decarboxylase, 6 Tryptophan, 227 Tubigensin A and B, 250

V Vermcofortin, 25 1 Vermculogen TR-2,249,251 Vinblastine, 109, 137, 139 Vinca alkaloids, 149 Vincamine, 149 Vincrictine, 109, 137 Vindoline, 139 Viroidin, 219, 222 Viroisin, 219 Virotoxins, toxicity of, 221 Voafrine A and B, 152 Vomilenine, 145 Vulgaxanthins, 212

X Xanthodermin, 279,280 Z

Zygosporins, 3 14-3 16

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