Primary and Secondary Metabolism of Plants and Cell Cultures III: Proceedings of the workshop held in Leiden, The Netherlands, 4–7 April 1993

265 Pages·1995·16.33 MB·English

by  Hens J. G. ten Hoopen

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