Primary and Secondary Metabolism of Plants and Cell Cultures III Primary and Secondary Metabolism of Plants and Cell Cultures III Proceedings of the workshop held in Leiden, The Netherlands, 4-7 April 1993 Edited by J. Schripsema and R. Verpoorte Reprinted from Plant Cell, Tissue and Organ Culture 38: 2/3, 1994 if Springer Science+Business Media, B.V. Library of Congress Cataloging-in-Publication Data Primary and secondary metabolism of plant cell cultures III / edited by J. Schripsema and R. Verpoorte. p. cm. Proceedings from the workshop held in Leiden, Netherlands, April 4th to 7th, 1993. Includes bibliographical references and index. ISBN 978-94-010-4106-5 ISBN 978-94-011-0237-7 (eBook) DOI 10.1007/978-94-011-0237-7 1. Plant cell culture—Congresses. 2. Plants—Metabolism- -Congresses. 3. Metabolism, Secondary—Regulation—Congresses. I. Schripsema, J. II. Verpoorte, R. QK725.P774 1995 581 ' .0724—dc20 94-44744 ISBN 978-94-010-4106-5 Printed on acid-free paper All Rights Reserved © 1995 Springer Science+Business Media Dordrecht Originally published by Kluwer Academic Publishers in 1995 Softcover reprint of the hardcover 1st edition 1995 No part of the material protected by this copyright notice may be reproduced or utilized in any form or by any means, electronic or mechanical, including photocopying, recording or by any information storage and retrieval system, without written permission from the copyright owner. CONTENTS Editorial ix Ajmalicine production by cell cultures of Catharanthus roseus: from shake flask to 85 bioreactor H.J.G. ten Hoopen, W.M. van Gulik, J.E. Schlatmann, P.R.H. Moreno, J.L. Vinke, J.J. Heijnen, R. Verpoorte Production of steroidal alkaloids by hairy roots of Solanum aviculare and the effect of 93 gibberellic acid M.A. Subroto, P.M. Doran Cyclodextrins as a useful tool for bioconversions in plant cell biotechnology 103 W. van Uden, H.J. Woerdenbag, N. Pras Embryogenesis of photoautotrophic cell cultures of Daucus carota L. 115 B. Grieb, U. GroB, E. Pleschka, B. Arnholdt-Schmitt, K.-H. Neumann Semicontinuous cultivation of photoautotrophic cell suspension cultures in a 20 I airlift- 123 reactor U. Fischer, U.J. Santore, W. HOsemann, W. Barz, A.W. Alfermann Studies on the relationship between ploidy level, morphology, the concentration of 135 some phytohormones and the nicotine concentration of haploid and doubled haploid tobacco (Nicotiana tabacum L.) and NICA plants B. Zeppernick, F. Schafer, K. Paasch, B. Arnholdt-Schmitt, K.-H. Neumann Influence of exogenous hormones on the growth and secondary metabolite formation in 143 transformed root cultures M.J.C. Rhodes, A.J. Parr, A. Giulietti, E.L.H. Aird Molecular cloning and expression of key enzymes for biosynthesis of cysteine and 153 related secondary non-protein amino acids K. Saito, N. Miura, M. Yamazaki, K. Tatsuguchi, M. Kurosawa, R. Kanda, M. Noji, I. Murakoshi Thiophene biosynthesis in Tagetes roots: molecular versus metabolic regulation 159 A.F. Croes, J.J.M.R. Jacobs, R.R.J. Arroo, G.J. Wullems Regulatory mechanisms of biosynthesis of betacyanin and anthocyanin in relation to 167 cell division activity in suspension cultures M. Sakuta, H. Hirano, K. Kakegawa, J. Suda, M. Hirose, R.W. Joy IV, M. Sugiyama, A. Komamine The biosynthesis of rosmarinic acid in suspension cultures of Coleus blumei 171 M. Petersen, E. Hausler, J. Meinhard, B. Karwatzki, C. Gertlowski The biosynthetic pathway of the S-alk(en)yl-L-cysteine sulphoxides (flavour precursors) 181 in species of Allium S.J. Edwards, G. Britton, H.A. Collin Elicitor induced secondary metabolism in Ruta graveo/ens L. Role of chorismate 189 utilizing enzymes J. Bohlmann, U. Eilert Constitutive and elicitation induced metabolism of isoflavones and pterocarpans in 199 chickpea (Cicer arietinum) cell suspension cultures W. Barz, U. Mackenbrock Regulation of isoflavonoid metabolism in alfalfa 213 N.L. Paiva, A. Oommen, M.J. Harrison, R.A. Dixon Regulation of phenylalanine ammonia-lyase genes in carrot suspension cultured cells 221 Y. Ozeki, J. Takeda Accumulation of anthraquinones in Morinda citrifo/ia cell suspensions. A model system 227 for the study of the interaction between secondary and primary metabolism M.J.M. Hagendoorn, L.H.W. van der Plas, G.J. Segers Calystegines as a new group of tropane alkaloids in Solanaceae 235 B. Drager, C. Funck, A. Hohler, G. Mrachatz, A. Nahrstedt, A. Portsteffen, A. Schaal, R. Schmidt Esterification reactions in the biosynthesis of tropane alkaloids in transformed root 241 cultures R.J. Robins, P. Bachmann, A.C.J. Peerless, S. Rabot Characterization of Coptis japonica cells with different alkaloid productivities 249 F. Sato, N. Takeshihta, H. Fujiwara, Y. Katagiri, L. Huan, Y. Yamada Traits of transgenic Atropa belladonna doubly transformed with different Agrobacterium 257 rhizogenes strains M. Jaziri, K. Yoshimatsu, J. Homes, K. Shimomura Effect of nitrogen and sucrose on the primary and secondary metabolism of trans- 263 formed root cultures of Hyoscyamus muticus K.-M. Oksman-Caldentey, N. Sev6n, L. Vanhala, R. Hiltunen Catharanthine and ajmalicine synthesis in Catharanthus roseus hairy root cultures. 273 Medium optimization and elicitation F. Vazquez-Flota, O. Moreno-Valenzuela, M.L. Miranda-Ham, J. Coello-Coello, V.M. Loyola Vargas A novel 2-oxoglutarate-dependent dioxygenase involved in vindoline biosynthesis: 281 characterization, purification and kinetic properties E. De Carolis, V. De Luca Are tissue cultures of Peganum harmala a useful model system for studying how to 289 manipulate the formation of secondary metabolites? J. Berlin, C. ROgenhagen, I.N. Kuzovkina, L.F. Fecker, F. Sasse Breakdown of indole alkaloids in suspension cultures of Tabernaemontana divaricata 299 and Catharanthus roseus J. Schripsema, D. Dagnino, R.1. Dos Santos, R. Verpoorte The cell culture medium - a functional extracellular compartment of suspension- 307 cultured cells M.Wink Secondary metabolites in hairy root cultures of Leontopodium alpinum Casso 321 (Edelweiss) I. Hook Glycosylation in cardenolide biosynthesis 327 C. Theurer, H.-J. Treumann, T. Faust, U. May, W. Kreis Enzymes in cardenolide-accumulating shoot cultures of Digitalis purpurea L. 337 H.U. Seitz, D.E. Gartner Enzymes involved in the metabolism of 3-hydroxy-3-methylglutaryl-coenzyme A in 345 Catharanthus roseus R. van der Heijden, V. de Boer-Hlupa, R. Verpoorte, J.A. Duine Regulation of 3-hydroxy-3-methylglutaryl-coenzyme A reductase by wounding and 351 methyl jasmonate: implications for the production of anti-cancer alkaloids I.E. Maldonado-Mendoza, R.J. Burnett, M. Lopez-Meyer, C.L. Nessler Index 357 Editorial This volume contains the proceedings from the work shop Primary and Secondary Metabolism of Plants and Plant Cell Cultures III, which took place in Leiden, April 4th-7th, 1993. Since the first two meetings on the topic of primary and secondary metabolism of plant cell cultures, in 1984 and 1988, there has been a clear shift in focus of the ongoing research. In the proceedings from the first meeting, particularly, the cell culture itself and the pro duction of secondary metabolites were the dominant themes. In the second proceedings biosynthetic path ways and the activity of enzymes were major topics. In the proceedings of this third meeting these aspects are linked with genes, such that molecular biology becomes more prominent. These proceedings reflect the state of the art in the field, with contributions on subjects such as fermentation, enzymology of secondary metabolism, catabolism of secondary metabolites, elicitation of pathways and the genetic modification of metabolic pathways. The book includes contributions on the most recent achievements in the research concerning, among oth er topics, tropane and indole alkaloids, phenolics, (iso )flavonoids, terpenes and cardenolides. It gives an excellent review of the progress made in the past years and a perspective on future developments in the field. Leiden. 1994 J. Schripsema and R. Verpoorte Plant Cell, TIssue and Organ Culture 38: 85-91, 1994. © 1994 Kluwer Academic Publishers. Ajmalicine production by cell· cultures of Catharanthus roseus: from shake flask to bioreactor Hens lG. ten Hoopen1, Walter M. van Gulikl, Jurriaan E. Schlatmann1, Paulo R.H. Moreno2, J.L. Vinke1, J.1. Heijnen1 & Robert Verpoorte2 Biotechnology Delft Leiden, Project Group Plant Cell Biotechnology, Department of Biochemical Engineering, I Delft University of Technology, lulianalaan 67, NL-2628 BC Delft, The Netherlands; 2Division of Pharmacog nosy, LeideniAmsterdam Center for Drug Research, Leiden University, P.O. Box 9502, NL-2300 RA Leiden, The Netherlands Key words: Ajmalicine, bioreactor, Catharanthus roseus, growth model, scale-up Abstract The productivity of a cell culture for the production of a secondary metabolite is defined by three factors: specific growth rate, specific product formation rate, and biomass concentration during production. The effect of scaling-up from shake flask to bioreactor on growth and production and the effect of increasing the biomass concentration were investigated for the production of ajmalicine by Catharanthus roseus cell suspensions. Growth of biomass was not affected by the type of culture vessel. Growth, carbohydrate storage, glucose and oxygen consumption, and the carbon dioxide production could be predicted rather well by a structured model with the internal phosphate and the external glucose concentration as the controlling factors. The production of ajmalicine on production medium in a shake flask was not reproduced in a bioreactor. The production could be restored by creating a gas regime in the bioreactor comparable to that in a shake flask. Increasing the biomass concentration both in a shake flask and in a stirred fermenter decreased the ajmalicine production rate. This effect could be removed partly by controlling the oxygen concentration in the more dense culture at 85% air saturation. Introduction iments are necessary to optimize a set of conditions (for example, medium optimization through statistical Commercial application of the production of secondary experimental design, Tuominen et al. 1989). metabolites by plant cells in suspension culture is main To perform a process on an industrial scale at least ly hampered by the too low productivity of the cultures. two scale-up steps are necessary (Fig. I). First, the Three factors determine the productivity ofthe cell cul developed system has to be reproduced in a laboratory ture: the specific growth rate, the specific product for scale bioreactor. Secondly, the process has to be scaled mation rate, and the biomass concentration during the up in one or more steps to the process-size bioreactor. production phase. To increase productivity, these three The first step is the most difficult one, because a shake factors have to be optimized. Particularly, the product flask and a bioreactor are completely different systems formation rate of plant cells shows a broad variation. in geometry, mixing and gas regime. Transferring a Several techniques are available to increase the prod process to a larger bioreactor of the same type generates uct formation rate: screening and selection of cell lines, also problems, but these problems can be solved with optimizing culture conditions (medium composition, the general scale-up approaches developed in fermen light, temperature, gas composition, genetic modifica tation industry. The type of bioreactor is an essential tion) (Verpoorte et al. 1991). Some of these approach factor in these studies. In this paper a standard stirred es have to be carried out at a small scale in shake and aerated bioreactor is used. This type is common flasks, petri dishes or culture tubes, either because the in fermentation industry and therefore the best option technique demands very small amounts of biological to introduce a new process for the production of a material, or because large numbers of parallel exper- plant product by plant cells in suspension culture. Fur- 86 Plant Molecular Sciences, Leiden University. The cul li ture was initiated from seeds and grown in suspension culture since 1983. Cell lines were subcultured every I 14 days (BIX) or every 7 days (BXV) by adding 35 -W- ml of suspension to 165 ml of fresh growth medium. ~ The cultures were grown in lO00-ml Erlenmeyer flasks * 'mening- ----. with silicon stoppers (Shin Etsu, Tokyo, Japan) on a * selection rotary shaker at 100 rpm in the dark. * medium optimization * ventilation Media * pathway studies (gaseous compounds) * genetic studies * Growth medium, described by Linsmaier & Skoog stirring (1965) supplemented with 2.0 mg 1-1 naphthalene (shear) acetic acid, 0.2 mg 1-1 kinetin, and 30 g 1-1 glucose. The medium was adjusted to pH 5.8 before sterilization (20 min, 121°C). Fig. 1. Scaling-up in plant cell biotechnology. Production medium: Growth medium, depleted of nitrate, ammonium, phosphate and honnones, and sup plemented with 80 g 1-1 glucose. thennore, scale-up procedures for aerated and stirred bioreactors are well developed. Shake flask experiments In this study the first step of the scale-up procedure, from shake flask to bioreactor, will be investigated. The Shake flask experiments were perfonned in 250 ml production of ajmalicine by cell cultures of Catharan Erlenmeyer flasks with silicon stoppers on a rotary thus rose us was selected as a model system, because a shaker (100 rpm) at 25°C in the dark. The total cul great deal of experience with this system was available ture volume was 60 ml; the inoculum size was 10 ml, from previous studies. Experiments by Meijer (1990) except for the comparison of low and high biomass revealed that the cell line was very shear-tolerant. It concentration. In that case 50 ml of production medi grew well on LS-medium with either sucrose or glu um was inoculated with respectively 5 g and 20 g of cose as carbon source. The productivity of the cell line fresh weight. used in these studies has been increased to a great deal by Schlatmann et al. (1992, 1993); an optimal produc Bioreactor experiments tion medium for this cell line was selected. In litera ture, a positive effect on biomass growth of condition Bioreactor experiments were carried out in a commer ing factors in the medium is mentioned several times cially available 3-1 turbine stirred tank reactor with a (Stuart & Street 1971; Wijnsma et al. 1988). There working volume of 1.8-2.1 1 (Applikon Dependable are conflicting data on the effect of carbon dioxide in Instruments, Schiedam, The Netherlands). The culture the gaseous phase on the growth (Ducos & Pareilleux was aerated through a sintered steel sparger. The flow 1986; Ducos et al. 1988; Hegarty et al. 1986; Maurel was kept at 113 vvrn with a mass flow control system & Pareilleux 1985). Taking into consideration these (Brooks, Veenendaal, The Netherlands). Exhaust gas observations, the scale-up effects on the three aspects was led through a glass condenser cooled by a cryo of productivity: biomass growth, product fonnation stat (Lauda Messgeriite, Lauda, Gennany) at 4°C, in and biomass concentration were investigated. order to minimize evaporation. Two six-bladed turbine = impellers (D 45 mm) were used for mixing; rota tion speed was 250 rpm. The bioreactor was equipped Materials and methods with 3 baffles to improve mixing. The temperature was maintained at 25°C with a thennostated stainless steel Cell material pipe in the culture fluid. In experiments on growth medium the pH was maintained at 5.0 by the addition Cell suspension culture of Catharanthus rose us (L.) of either 0.25 N NaOH or 0.25 N HCI using AD! 1030 G. Don MP183D was obtained from the Institute of Bio-controllers (Applikon) equipped with sterilisable 87 .. pH-electrodes (Ingold) and peristaltic pumps for alkali _ _. -0- Shake- Fermentor and acid. Dissolved oxygen concentrations were mea flasks 0.5 vvm sured with a sterilisable oxygen electrode (Ingold). The 18 formation of foam was prevented by adding, at regular time intervals, a silicon-based antifoaming agent (1 % -.r..-.-:-l . .--0'. j " w/w, BDH, Poole, England), or adding 18 mg 1-1 to .b..J ) " '-' 12 the medium in the case of continuous culture experi ..c: I ' ments. The bioreactor was wrapped in black plastic to bJ) <ll keep out light. The bioreactor was inoculated with one ~ 6 part suspension culture and five parts of medium. In ;>, H the incoming as well as the exhaust air the oxygen (Ser 0 ~'II~~"'D' vomex 1101 paramagnetic O analyzer, Crowborough, 0 2 UK) and the carbon dioxide concentration (Rosemount 0 100 200 300 400 analytical 870, La Habra, USA) were measured on line. Investigations on the effect of high biomass con Time (h) centrations were performed in 2 identical 15-1 turbine Fig. 2. Comparison ofthe growth of C. roseus cell cultures in shake stirred bioreactors with a working volume of 11.5 flasks and in bioreactors at 0.5 vvm aeration rate (Van Gulik et aI. 1 (Applikon, Schiedam, The Netherlands). One six J993b). = bladed turbine impeller (D 75 mm) was used for 18 mixing; rotation speed was 200 rpm. The aeration rate was 0.3 vvm. The bioreactor was equipped with 3 baf fles to improve mixing. In the experiments in which 12 two treatments were compared, two bioreactors were inoculated simultaneously with the same inoculum. Analytical procedures 6 The determinations of biomass dry weight, packed cell volume, storage carbohydrates, glucose, phosphate, o ajmalicine, tryptamine were performed as described o 100 200 300 400 previously (Van Gulik et al. 1992; Schlatmann et al. 1993). Time (h) Results and discussion .. 0.03 ~. CO , Used medium .2.0 % CO 2 withdrawn Biomass growth L. 0.03 % CO , Used medium 020 % CO 2 re-added All gas~ing rales 0.5 vvm Figure 2 compares the growth in a shake flask and a bioreactor. There is no significant difference during lag Fig. 3. Effects of C02 enrichment of the aeration gas on the growth of C. roseus cell suspensions with and without conditioning factors or growth phase. Apparently, neither the difference in in the medium in stirred ferrnenters (Van Gulik et aI. J993b). shear, nor the difference in gas regime between shake flask and stirred fermenter influences the growth of this cell line significantly. If the gas regime does not ditioning factors in the medium. The growth curves affect growth, an effect of variations in carbon diox in Fig. 3 show that there is no significant difference in ide concentration in the gaseous phase on the biomass growth between cultures at 0.03% CO2 or at 2.0% CO2 growth is not very likely. This aspect was investigated concentration. On the other hand, the positive effect of in detail by comparing growth in stirred bioreactors at conditioning factors in the inoculum is clearly proven different CO2 concentrations. To exclude an eventual for both CO2 concentrations. interference with the effect of conditioning factors the For scale-up to larger fermenters, the availabili experiments were carried out with and without con- ty of a mathematical growth model is very useful. A