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Large-scale plant cell culture
Susan C Roberts and Michael L Shuler∗

Progress towards the commercial-scale use of plant cell Bioreactor considerations
cultures over the past three years has been significant. Several innovative reactor designs have been suggested
Elicitation, particularly with methyl jasmonate, has been that may be useful in the scale-up of shear-sensitive
effective at increasing the product yields of a wide variety plant cell systems: a bubble-free loop fluidized bed
of secondary metabolites, particularly when it is applied bioreactor [2], an external loop air-lift bioreactor [3•], and
synergistically with enhancement strategies such as a centrifugal impeller bioreactor [4•]. Additionally, organ
immobilization and in situ extraction. Rapid advances in cultures for the production of chemicals and the effects
understanding the regulation of the biosynthetic pathways on reactor design have been considered by Doran [5].
of secondary metabolites are allowing the application Quantitative analysis of the effects of shear stress on plant
of enhancement strategies to move from empirical to cells has been discussed by Zhong et al. [6].
semirational. Much of this progress is exemplified by work
on paclitaxel (Taxol), where yields have improved more than Taxus has been cultivated successfully for paclitaxel
100-fold in the past two years. production in pneumatically mixed [7•], stirred tank [7•],
and Wilson-type bioreactors [8]. The similarity of kinetics
of paclitaxel production in all these reactors compared
Addresses
School of Chemical Engineering, 120 Olin Hall, Cornell University, with shake flasks suggests that paclitaxel production
Ithaca, NY 14853-5201, USA is amenable to a variety of reactor configurations in
∗e-mail: mls@cheme.cornell.edu bench-scale sizes and that for scale-up, shake flask data
Current Opinion in Biotechnology 1997, 8:154–159 may be relevant. A continuous production system for
paclitaxel production with a mesh-net cell separator was
Electronic identifier: 0958-1669-008-00154 developed by Seki et al. [9] that increased productivity by
 Current Biology Ltd ISSN 0958-1669 a factor of ten compared with batch operation.
Abbreviations
AS anthranilate synthase Unlike in Taxus cultures, when growth and ajmalicine
ppm parts per million production in Catharanthus roseus cultures were compared
TDC tryptophan decarboxylase
in shake flasks and bioreactors [10], growth was similar,
but ajmalicine production was inhibited in the bioreactor.
Ajmalicine production was restored with the recirculation
of the ventilation gas. These results illustrate the potential
Introduction importance of the gaseous phase in the scale-up of shake
Plant cell culture technology shows promise for the flask data to predict bioreactor performance.
large-scale production of valuable plant products; however,
the commercial use of plant cell cultures is not routine be-
cause of difficulties in achieving acceptable, reproducible Optimization of culture environment
product levels in reasonable periods of time. Much work In the case of ten Hoopen et al. [10], a change in gas phase
has been done in the three years since a prior review [1] to composition was an unintentional consequence of change
overcome this limitation and to determine conditions for in reactor design. In some cases, the gas phase composition
commercial-scale reactors. may be changed intentionally to achieve a desired change
in cell metabolism. Gas phase composition has been
An increase in volumetric productivity is crucial for shown to influence the timing and rate of paclitaxel
potential commercialization. Plant cell culture is a flexible production, with low oxygen concentrations promoting
system that is easily manipulated to increase product early production and high carbon dioxide concentrations
yields. This review focuses on strategies used to enhance inhibiting paclitaxel production [11•]. The most effective
secondary metabolite production that may contribute gas mixture was 10% v/v oxygen, 0.5% v/v carbon dioxide
to the scale-up and commercialization of plant cell and 5 ppm (parts per million) ethylene.
culture processes. Developments in the areas of bioreactor
design, optimization of culture environment, elicitation Schlatmann et al. [12] addressed the question of what is
and signal transduction, and biosynthetic pathway analysis the best time to start the production phase in a two-stage
are discussed. Paclitaxel (Taxol), a promising anticancer batch operation using C. roseus and ajmalicine production
agent, is the focus of worldwide efforts in develop- as a model system. The production phase was started
ing commercial-scale systems for its production. Recent with inocula of different ages and it was found that
progress on paclitaxel production is emphasized. the amount of ajmalicine produced in the late stationary

secondary that jasmonic acid and its methyl ester are signal metabolism could be induced successfully. that although natural jasmonic acid synthesis is initiated soon after fungal elicitation of Agrostis tenuis suspension Synergism of enhancement strategies cultures the synthesis rate is transient. Recent Jasmonic acid was also shown to be an integral part of examples of the successful use of this strategy include the signal transduction pathway leading to the induction work by Choi et al. Methyl jasmonate is emerging maximum effect (eightfold induction) of all treatments as a useful tool to increase the production of secondary and continuous operation was favorable over batch. When methyl jasmonate was combined with ethylene in an elicitation scheme. The treatment of combined effects of immobilization in a spirally wound elicited cells with ibuprofen. As soon secondary metabolites. [13] inexpensive effective elicitors of secondary metabolite examined the effects of calcium and alginate separately production in many other systems. Also. Barium alginate immobilization results in a threefold increase in paclitaxel production did not induce an increase in sesquiterpene production. [22•] and Sajc et al. nitrate might serve as a more general indicator of a switch These compounds accumulated rapidly and transiently to secondary metabolite production was not addressed. Paclitaxel production of 110 mg l−1 in two weeks could lamine and hoscyamine in Datura innoxia Mill. involved the addition of elicitor before phosphate deple- . to induce the production of secondary metabolites in monly used immobilization techniques is calcium alginate 36 different plant species. an immobilized reactor with jasmonate induced a fourfold increase in ajmalicine recycled batch operation. inducing a ten. cell cultures was studied as a factor for enhanced growth Both methyl jasmonate and ethylene are involved in and secondary metabolite production [15–21]. as jasmonic acid synthesis can stimulate secondary metabolite production many could not be reproduced with other types of stresses. This inhibition could be reversed on gossypol production from Gossypium arboreum were with the addition of jasmonic acid. [23•]. [27] showed Taxus cell cultures [18. Whether or not transducers in a wide range of plant cell cultures. Archambault et al. control of the elicitation calcium alginate immobilization and silicone oil in situ processes has yet to be optimized. Blechert et al. The of momilactone A biosynthesis [28•]. an inhibitor of jasmonic acid cotton cloth matrix. The role of calcium in immobilization. 10 µM methyl compared in batch culture. One of the most com.20] and points out the need to consider the interactions of the addition of fructose promoted paclitaxel production in multiple signals to the cell. roseus [15]. there was a discovered that the most successful elicitation schemes 10–30-fold increase. Exogenously applied methyl jasmonate was shown secondary metabolite production. Mirjalili and Linden [26•] have shown of sesquiterpenes in Hyoscyamus muticus by fungal extracts that exogenously applied methyl jasmonate at 10 µM of Rhizoctonia solani [14•]. cultures of Papaver somniferum with chitin elicitation and but when these strategies were combined. all of the treatments were combined. Gundlach et al. and an immobilized bioreactor content and an increase in catharanthine concentration in with continuous operation [22•]. Calcium be induced from Taxus media with the addition of alone was found to be the most effective. When metabolites. Gontier et al. Calcium highest concentration of paclitaxel obtained.19]. jasmonic entrapment. synthesis. when plant suspension cultures of Rauvolfia canescens and Eschscholtzia californica were treated with a yeast The immobilization of plant cells often results in increased elicitor. acid and methyl jasmonate have been shown to be induced elicitation may be important. permeabilization with dimethyl sul. which was was shown to be important for the successful elicitation 153 mg l−1 [P1•]. 100 µM methyl jasmonate [25••]. there was a 23-fold increase in gossypol production. Another recent example Elicitation has also been used extensively in the search is the production of anthraquinones by Frangula alnus for regulatory enzymes in the biosynthetic pathways of Mill. This rate is the highest fold increase in alkaloid production. probably because of productivity reported to date. This effect was The combined effects of various enhancement strategies shown to be highly specific. Nitrate concentration was found to be associated with enzymes responsible for the synthesis of a key indicator of the start of the production phase. The manipulation of the amount and source of sugar in the result was a 19-fold increase in paclitaxel production. This work sucrose levels were favorable in some cultures [15. [24] demonstrated as nitrate levels were depleted in the medium. including Taxus. [29] production extraction [23•]. fold greater than any individual approach and this can including light. and compared these effects to those of calcium alginate immobilization on the production of the alkaloids scopo. Elicitation had the cultures of C. heavy metals. Elevated the metabolic regulatory system in plants. reduced the content of endogenous jasmonic foxide (DMSO). however. although it is not the the activation of N-methylputrescine transferase. An external-loop air-lift bioreactor was utilized with secondary metabolites. be particularly valuable in large-scale systems. Large-scale plant cell culture Roberts and Shuler 155 phase inoculated cultures was greater than fourfold higher Elicitation and signal transduction than the amount produced in the early stationary phase Elicitation is used to induce the expression of genes often inoculated cultures. from Taxus cuspidata cultures. In the past few years. and elicitation with Verticillium dahliae acid and momilactone A. Immobilization and silicone oil measured a twofold increase in sanguinarine production in applied separately increased productivity up to fivefold. and cold or heat shock.

addition enzymes were suppressed in some cases and enhanced in of precursors. roseus by converting tryptophan into tryptamine. Reduced sulfate concentration in the production of tropane alkaloids in genetically engineered medium decreased thiophene accumulation fourfold. There was no increase in reduced experimentation time. control mechanisms can be studied by varying the mineral Hashimoto and Yamada [33] have provided an excellent concentration. and the transformation and overexpression others. rate-limiting steps. pathway enzyme levels were not of signal molecules in natural culture filtrates. Cell cultures of carrot were elicited for anthocyanin to better understand the regulatory role of AS.156 Biochemical engineering tion. measuring enzyme levels use of nonspecific elicitors because levels of desired in cell lines of different biosynthetic capabilities. Currently. alkaloid accumulation. to be dependent on both the tissue density and the free elicitor concentration in the medium. but alkaloid accumulation in both cell lines suspension cultures were elicited with cell extracts and was raised to similar levels (fivefold increase for the culture filtrates of four different fungi and arachidonic high-accumulating cell line and 100-fold increase for the acid [31•]. root cultures. used to better understand the paclitaxel biosynthetic ranocoumarins by Ruta graveolens L. point of the shikimate pathway for control of the inhibition of metabolic steps. [35•] studied two cell lines of Tabernaemon- play a more general role as a signal molecule in the tana divaricata with different biosynthetic capabilities for elicitation process. and those eliciting both paclitaxel and other taxanes equally. indicating different biosynthetic sites of The enzyme tryptophan decarboxylase (TDC) catalyzes a action. This trend of investigating the control of secondary metabolite production is important in moving If a secondary product contains an element in its molecule plant cell technology from a soley empirical approach to that is derived from an environmental mineral nutrient. This production [30] with culture filtrates and cell extracts study provides a good model for an approach to identify of bacteria and yeast as well as various abiotic ions. Taxus increased. those eliciting only other limited by the availability of precursors. of pathway genes. [32]. Characterization of the paclitaxel biosynthetic pathway is Bohlmann and Eilert [34] have investigated the branch a critical factor in attempts to increase yields. [36•] increased TDC levels by transformation and overexpression. but is rate controlling for a precursor compound. promoting a twofold increase in anthocyanin production. tor in alkaloid biosynthesis. which A method for predicting elicitor dosages for plant cell led to an increase in tryptamine content but no increase in culture reactor systems was developed by Singh et al. By adding results may be complicated by the heterogeneous mix the precursor loganin. taxanes. [37•]. These results indicate secondary metabolites by identifying the rate-limiting that there is a limitation in the terpenoid pathway that steps in the biosynthetic pathways. Thiophene contains sulphur and its biosyn- overview on the molecular aspects of alkaloid biosynthesis thesis in Tagetes can be controlled by sulfate concentration that includes a discussion of their pioneering work on the in the medium [38]. and precursor feeding were production of acridone epoxides. pathway [39••]. not be a direct precursor of paclitaxel. Elicitation. [25••] resulted in preferential enzyme is present at low levels and may be a limiting fac- increases in paclitaxel over other taxanes. although the interpretation of these the production of terpenoid indole alkaloids. By mathematically Additional insights into the metabolic control of terpenoid characterizing elicitation. indicating that it may Dagnino et al. This study of Yukimune et al. The addition of various inhibitors and a com- anthranilate synthase (AS) does play a key role in parison of kinetic profiles suggest that baccatin III need regulating the production of acridone epoxides. Three categories of elicitors were identified: low-accumulating cell line). roseus cell cultures are found in the added to cell cultures can be easily determined with work of Moreno et al. both arachidonic acid in the above key step in the biosynthesis of terpenoid indole alkaloids study [31•] and the addition of methyl jasmonate in the in C. They determined. Several approaches may cause a shortage of secoiridoid precursors for alkaloid can be effective: elicitation followed by monitoring the biosynthesis. indicating that biosynthesis is those eliciting only paclitaxel. indicating that the TDC-catalyzed A physical interpretation of elicitation is presented as an reaction is not rate limiting for the production of these elicitor–receptor binding event. Interestingly. It also demonstrates the limitations of the activities of pathway enzymes. that chorismate mutase is not a pyrophosphate source were both found to be primarily key enzyme in the induction of furanocoumarins but plastidic. Paclitaxel production and isopentenyl after fungal elicitation. Some enzymes early Biosynthetic pathway analysis and control in the indole alkaloid pathway were induced upon A lot of recent research on plant cell cultures has elicitation and others were inhibited whereas enzymes focused on determining the control of production of further along were not elicited. furoquinoline and fu. metabolite production towards a semirational approach. the amount of elicitor to be production in C. Goddijn et al. Elicitor dosage was found alkaloids. Conceptual models the purification of AS and cDNA cloning are underway were formulated to describe carbon flow and simple . Calcium was the most effective. ajmalicine accumulation upon elicitation with a cell-free filtrate of Pythium aphanidermatum.

Progress in The use of plant cell culture for the production of the next three years will be critical. 5:165–174.26•] and productivity. Three years ago. Plant Cell Tissue one. Kut OM. Biotechnol Prog 1995. USA) announced in 1995 that they A very important insight into paclitaxel synthesis comes were licensing the Phyton Inc (Ithaca. (taxadiene-5-hydroxylase). Particularly predicting rate-limiting steps. particularly when details of important will probably be the need to better control gas pathways and compartmentation are unknown. moieties. This is the first report of an alternative pathway of a pharmaceutical. to facilitate application and removal of elicitors. Should the plant cell culture process being used in plants and may be important for other plant for paclitaxel become a successful reality. Sajc L. large-scale plant cell culture technology.18. to 2-taxa-4(5). doors for the serious consideration of plant cell culture for the commercial-scale production of other pharmaceuticals. Now. Taxadiene synthase catalyzes the initial conversion of geranylgeranyl The increased appeal of natural products for medicinal pyrophosphate. Taxol strategy that appears to be broadly useful and can result is a registered trademark of Bristol-Meyers Squibb. the cDNA encoding taxadiene synthase has paclitaxel was considered significant and two years ago been isolated [43] and the authors speculate that it the majority of reports described low paclitaxel levels should soon be possible to engineer cells that overexpress [19. The use of methyl jasmonate is one (grant BES-9632961). the universal precursor of diterpenoid purposes coupled with the low product yields and supply compounds. the presence of even low levels of Recently. Should the process for paclitaxel falter. • of special interest •• of outstanding interest Further. Vukovic D. Dubuis B. and the American Association of University Women. Bugarski B: Hydrodynamics and the point when conditions are optimal for the production • mass transfer in a four-phase external loop air lift bioreactor. 11:420–428. pathways for secondary metabolism will provide the basis to move from a brute-force empirical approach for the 2. The next step concerns of plant harvestation has renewed interest in is the hydroxylation of taxa-4(5).46]. the United States Department of Agriculture (grant 58-1907-2-044). in some cases. chemicals and pharmaceuticals has made great strides building on advances in plant sciences. USA) plant cell from radiolabeled studies with Taxus chinensis in which it culture process for paclitaxel production. Lee CW. Taticek RA.45. Curr Opin Biotechnol 1994. This methodology can be useful in synergistic product-enhancement strategies. in rapid improvements in productivity in a short period of time. Prenosil JE: Pilot-scale culture of Coffea optimization of production conditions to a semirational arabica in a novel loop fluidised bed reactor.11(12)-diene. These initial two steps were found to be very slow compared to subsequent metabolic transformations. The interrelationship of methyl jasmonate signaling with other signal compounds is beginning to emerge and will supplement the widely demonstrated strategy of using References and recommended reading Papers of particular interest. NY. 43:171–183. to facilitate the use of in situ The enzymes catalyzing the initial steps of paclitaxel product-removal strategies. Model results Although further improvements in bioreactor design may and experimentation suggest that paclitaxel production is be anticipated. contrary to expectations. the increased use of genetic tools and an emerging picture of the structure and regulation of 1. one can then predict a great reduction in the time Organ Cult 1995. Bristol-Meyers Squibb (Princeton. it will open products. phase composition. resulting in enhanced paclitaxel are common from academic laboratories [8. . Obradovic B.21. NJ. Large-scale plant cell culture Roberts and Shuler 157 mathematical simulations were performed. of commercially acceptable levels of product. multiple productivity enhancement techniques to achieve have been highlighted as: synergistic increases in production. The past three was shown.11(12)-dien-5a-ol by a cytochrome P450 enzyme agent paclitaxel has been the focus of recent research. Better molecular Acknowledgements understanding of elicitation and signal transduction in The authors acknowledge funding from the National Science Foundation plants is emerging. biosynthesis have been identified [40–42]. required to move from the establishment of a culture to 3.11(12)-diene to taxa. published within the annual period of review. it will significantly Conclusions reduce interest in large-scale plant cell culture. these studies will be important primarily limited by the ability of the cells to convert phenylalanine as vehicles that allow the more effective application of to phenylisoserine. The anticancer 4(20). It is speculated that additional which has shifted from establishing paclitaxel-producing oxygenation steps may be catalyzed by similar P450 cultures to enhancing productivity in these cultures. and. that the taxane ring years have witnessed tremendous progress towards the system is not synthesized through the mevalonate pathway commercialization of plant cell culture for the production [44•]. much higher levels (153 mg l−1 [P1•] and 110 mg l−1 [25••]) have been reported from industrial groups. reports of paclitaxel levels of 10–22 mg l−1 these two enzymes. Shuler ML: Large-scale insect and plant cell culture.

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Large-scale plant cell culture Roberts and Shuler 159 30. 93:6431–6436. in- 31. 10:365–371. Dagnino D. Williams 33. The results suggested that there is a limitation in the terpenoid pathway. which may cause a shortage of secoiridoid precursors for alkaloid biosynthesis. Ciddi V. Jacobs JJMR. the literature. Chem Biol 1996. Eilert U: Elicitor induced secondary metabolism in Ruta graveolens L. Verpoorte R: Effects • of elicitation on different metabolic pathways in Catharanthus roseus (L. 18:99–107. taxane production by Taxus chinensis cell cultures. and an increase in other taxanes only. 1995. 35. Croteau R: Cyclization of geranylgeranyl diphosphate to an increase in paclitaxel only. Nicholson SA. Srinivasan V. and precursor feeding. Arteca RN: Taxus cell suspension Enzyme levels were increased by transformation and overexpression. Lewis NG. TDC-catalyzed reaction is not a limiting step in alkaloid biosynthesis. metabolite production from root cultures. 4:315–323. US5407816. 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Reddy GR. Plant Cell Tissue Organ Cult 1994. Bringi V. NG. duction may be limited by the conversion of phenylalanine to phenylisoserine. 41. Schilperoort 45. Moreno PRH. Lewis were observed: an increase in paclitaxel and other taxanes in equal amounts. 39:341–349. Hezari M. taxa-4(5). indicat- enzymes may be limiting in alkaloid production. 271:9201–9204. 42. Bringi V.11(12)-dien-5a-ol: the 45:257–285. Hoge JHC: Overexpression of a tryptophan DiCosmo F: Improved Taxol yield by aromatic carboxylic acid decarboxylase cDNA in Catharanthus roseus crown gall and amino acid feeding to cell cultures of Taxus cuspidata. Phytochemistry 1995. Prog 1996. . Croes AF. 36. 46. These taxa-4(5). suggesting that there are multiple control points in the taxane pathway. Transgenic Res 1995. Rajendran L. was shown that strictosidine synthase and geraniol 10-hydroxylase activities The incorporation of radiolabeled glucose and acetate into taxuyunnanine were much higher in the high-accumulating cell line. Paclitaxel production was • cell cultures for production of Taxol. Hezari M. It examines a wide variety of enhancement strategies. V. Biotechnol Prog 1994. Shuler ML: Metabolic inhibitors.11(12)-diene to taxa-4(20). Venkataraman LV: 39. Suvarnalatha G. • RA. 322:437–444. Curtis WR: Use of binding measurements brevifolia) that catalyzes the first committed step of Taxol to predict elicitor dosage requirements for secondary biosynthesis. 12:457–465.Don cell suspension cultures. Singh G. Arroo RRJ. increased terpenoid indole alkaloid production. Srinivasan V. Zajicek J. Wickremesinhe ERM.11(12)-diene is the first committed step of Taxol data were the first evidence of a differential response of the taxane pathway biosynthesis in Pacific yew. The paclitaxel biosynthetic pathway was characterized using elicitation. Ciddi V. Vogel BS. J Biol Chem 1995. 44:967–971. Annu Rev Plant Physiol Plant Mol Biol 1994.