2.manipulating Production in Bio Reactor - Verpoorte

Phytochem Rev (2007) 6:435457 DOI 10.
1007/s11101-006-9050-0
Manipulating indole alkaloid production by Catharanthus roseus cell cultures in bioreactors: from biochemical processing to metabolic engineering
Jian Zhao Robert Verpoorte
Received: 5 July 2005 / Accepted: 29 November 2006 / Published online: 6 March 2007 Springer Science+Business Media B.V. 2007
Abstract Catharanthus roseus plants produce many pharmaceutically important indole alkaloids, of which the bisindole alkaloids vinblastine and vincristine are antineoplastic medicines and the monoindole alkaloids ajmalicine and serpentine are antihypertension drugs. C. roseus cell cultures have been studied for producing these medicines or precursors catharanthine and vindoline for almost four decades but so far without a commercially successful process due to biological and technological limitations. The research thus focused on the one hand on engineering the bioreactor process on the other engineering the cell factory itself. This review mainly summarizes the progress made on biochemical engineering aspects of C. roseus cell cultures in bioreactors in the past decades and metabolic engineering of indole alkaloid production in recent years. The paper also attempts to highlight new strategies and technologies to improve alkaloid production and bioreactor performance. Perspectives of metabolic
J. Zhao (&) Department of Pediatrics, Childrens Nutrition Research Center, Baylor College of Medicine, 1100 Bates Street, Room 9016, Houston, TX 77030, USA e-mail: jzhao1@bcm.tmc.edu R. Verpoorte Section of Metabolomics, Division of Pharmacognosy, Institute of Biology, Leiden University, 2300 RA Leiden, The Netherlands e-mail: verpoort@chem.leidenuniv.nl
engineering to create new cell lines for large-scale production of indole alkaloids in bioreactors and effective combination of these up- and downstream processing are presented. Keywords Bioreactor process Catharanthus roseus Cell factory Gas regime Indole alkaloid Large-scale cell culture Metabolic engineering Monitoring and autocontroling Abbreviations ABA ABC transporter ASa DCO2 DO2 KLa MeJA SG STR TDC Introduction Plant secondary metabolites encompass a huge number of natural compounds with a wide diver-
Abscisic acid ATP-binding cassette transporter Anthranilate synthase asubunit Dissolved carbon dioxide in liquid medium Dissolved oxygen in liquid medium Oxygen mass transfer coefcient Methyl jasmonate Strictosidine glucosidase Strictosidine synthase Tryptophan decarboxylase
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Phytochem Rev (2007) 6:435457
sity in chemical structure. They provide human beings with unique resources for medicines, food additives, fragrances, and ne chemicals. The daily life, health care, and other well being of humans essentially depend on these plant products. Therefore, production of plant secondary metabolites by cultivation of plants and chemical synthesis are important agronomic and industrial objectives. As a promising alternative to produce plant secondary metabolites, plant cell culture technology has many advantages over traditional eld cultivation and chemical synthesis, particularly for many natural compounds that are either derived from slow-growing plants or difcult to be synthesized with chemical methods. Considering the continuous decrease in arable lands and increased considerations on environmental problems, production of plant secondary metabolites by traditional plant cultivation and chemical synthesis may be largely limited in the future. Large-scale plant cell culture in bioreactors has no such agronomic and environmental concerns since the process is in factory independent of seasons or climates, pathogens or other biofactors that seriously affects eld cultivation. Furthermore, plant cell culture is a renewable resource and environmentally friendly. It is like an industrialized biological factory for production of highquality natural products under strictly controlled conditions. Although most plant cell culture processes are not yet competitive for commercial application due to the high-cost caused by low productivity, to date there are already some successful examples of commercial production of valuable secondary metabolites by plant cell cultures (Alfermann and Petersen 1995; Smith 1995). Shikonin by Lithospermum erythrorhizon cell culture and berberine by Coptis japonica or Thalictrum minus cell cultures are successfully produced by Mitsui Petrochemical Industries (Japan). Paclitaxel is commercially produced by Taxus spp cell cultures in a two-stage process in 2500-l / 75000-l bioreactors by ESCA Genetics (now Samyang Genex, South Korea) and Phyton Catalytic company (USA). Also, ginseng saponin production by Panax ginseng cell or root cultures runs at a 20.000-l scale (Alfermann and Petersen 1995; Smith 1995). These successfully industrialized processes largely depend on either a higher
productivity of secondary metabolites in cell cultures like shikonin or extremely high market values like paclitaxel, whereas most other plant secondary metabolites have no such merits. Nevertheless, these encouraging successes are driving research of plant cell cultures towards further breakthroughs, by overcoming biological limitations such as low and unstable production of interesting metabolites, and biotechnological limitations including poor bioreactor performance or uncontrolled processes. In other words, more research efforts will be put in engineering of the process and in the engineering of the cell factory. Catharanthus roseus cell culture is one of such an extremely interesting but unsuccessful examples that has been studied for more then three decades (van der Heijden et al. 2004). The valuable secondary metabolites in C. roseus are terpenoid indole alkaloids, including the anticancer medicines vinblastine and vincristine, as well as the antihypertensive medicines ajmalicine and serpentine. However, the highly valuable drugs vinblastine and vincristine fail to accumulate in in vitro cell cultures due to the absence of the biosynthesis of one precursor vindoline. Ajmalicine and serpentine can accumulate in the cell cultures to high levels, yet their productivities are still too low to compete with eld cultivation. The rapid development of semi-synthesis of vinblastine or vincristine by coupling vindoline and catharanthine provides another opportunity for C. roseus cell cultures as a promising source of catharanthine. In C. roseus plants, vindoline is abundant but catharanthine is limited, but C. roseus cell culture could synthesize much higher level of catharanthine than plants (about 0.1% on dry weight basis) (Misawa and Goodbody 1996). It would be possible to produce catharanthine by plant cell cultures and to obtain vindoline from eld cultures, and then use chemical or biochemical semisynthesis of vinblastine by coupling catharanthine and vindoline (Misawa and Goodbody 1996). Signicant progress has been made in the 1980s on coupling catharanthine and vindoline into vinblastine or other bisindole alkaloids in a much easier way and with an increased efciency (Misawa and Goodbody 1996). In addition, some novel bisindole alkaloid derivatives are further developed as new anticancer medicines, e.g., vindesine and vinorelbine (Souquet et al.
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2002). The advances in developing novel drugs extend the potential applications of bisindole alkaloids in pharmacotherapy and thus create an increased demand for indole alkaloids (Ruszkowska et al. 2003; van der Heijden et al. 2004). Production of indole alkaloids by C. roseus cell cultures still is one of the greatest interests and challenges that attract many researchers to explore the technology. Therefore, C. roseus cell cultures are now a well-developed model system for biosynthesis and regulation of secondary metabolites. Furthermore, it remains a potential alternative for production of indole alkaloids with the expectation of breakthroughs in bottlenecks of the biotechnology such as creating high-alkaloid-yield cell lines by genetically engineering the metabolic ux and improving large-scale performance of bioreactor processing. Most aspects of C. roseus cell cultures affecting production of indole alkaloids have been extensively investigated with respects to optimizing medium components, culture conditions, and bioreactor processing. Van der Heijden and Verpoorte (1989) and Moreno et al. (1995) reviewed more details about the progresses made in the period before their reviews. Obviously, most progress about bioreactor processing was achieved in the 1980s1990s, which reects the large research effort focused on C. roseus cell cultures and indole alkaloid production in this period. But after nding that C. roseus cell cultures were unable to produce bisindole alkaloids and the failure in upscaling the cell culture process for commercial application, research turned away from biochemical engineering of the process and focused more on studies of the regulation of the biosynthetic pathways, i.e., looking for strategies to engineer the cell factory itself. The biosynthetic routes, the enzymes involved and their encoding genes, transcription factors and regulatory signaling compounds for production of indole alkaloids became the focus of the research (for review, see Memelink et al. 2001; van der Heijden et al. 2004; Verpoorte et al. 2002; Zhao et al. 2005a). At present engineering of metabolic uxes is regarded as the key to achieve a commercially viable indole alkaloid production (Verpoorte et al. 2002). Once metabolic engineering of indole alkaloid production is
successful based on the understanding of biosynthetic genes and regulatory mechanisms, all the previously developed knowledge on bioreactor processing will be useful to engineer a nal industrial process, which may compete successfully with eld cultivation. This review summarizes the progress made in engineering the bioreactor process of C. roseus cultures for production of indole alkaloids, and highlights recent advances in engineering alkaloid production in the cell factory itself. New technologies in bioreactor processing of other plant cell cultures that might also be useful for transgenic C. roseus cell cultures will be discussed. All aspects from the cell factory, plant cell culture, bioreactor, to downstream bioreactor processing and product recovery will be discussed. Optimization of growth conditions The medium components, growth regulators, pH value, as well as culture conditions including temperature, light, aeration, and agitation are important factors affecting biomass accumulation and indole alkaloid production. The medium is the basic environmental and nutrimental condition for plant cell cultures. Medium optimization and manipulation of culture conditions thus is the most fundamental approach in plant cell culture technology. Such optimization in combination with selection of high-yielding cell lines may lead to a 2030-fold increase of alkaloid production (Verpoorte et al. 1997, 2002). C. roseus suspension cells or hairy root cultures in bioreactors behave generally almost similar as in shake-asks in terms of nitrogen and phosphorus consumption and growth (Bhadra and Shanks 1997). In a two-stage turbine stirred bioreactor process, nitrate depletion in the medium is synchronically correlated with the start of indole alkaloid accumulation (Schlatmann et al. 1995b). Schlatmann et al. (1995a, b) showed the importance of an optimized glucose concentration for ajmalicine production in a 3-l turbine stirred bioreactor (turbine impeller speed at 250 rpm). Growth regulators Growth regulators have signicant effects on indole alkaloid production. Auxins and cytokinins
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are basic requirements for proliferation and growth of in vitro plant cell cultures. However, 2, 4-dichlorophenoxyacetic acid (2, 4-D) signicantly inhibits indole alkaloid biosynthesis whereas cytokinins such as benzyladenine promotes cell differentiation and stimulate indole alkaloid production. Auxins not only suppress expression of biosynthetic genes, but affect precursor supply, e.g., by inhibiting upstream biosynthestic pathways or metabolite trafcking (Whitmer et al 1998a, b; El-Sayed and Verpoorte 2000). Abscisic acid (ABA) is not a necessary growth regulator for plant cell cultures, but it is an important phytohormone that mediates various abiotic stress responses in plants. It was already shown that both ABA, salts or osmotic stress induce an increase in ajmalicine and catharanthine production (for reviews, Van der Heijden and Verpoorte 1989; Moreno et al. 1995). Recently, indole alkaloids and biosynthetic enzymes in salicylic acid-, ethylene-, ABA-, methyl jasmonate (MeJA)-, and gibberellic acid-treated seedling cultures were proled by El-Sayed and Verpoorte (2004). MeJA generally induces production of all indole alkaloids. SA induces serpentine and tabersonine production at lower concentrations and induces vindoline accumulation at higher concentrations. ABA and ethylene promoted metabolic uxes towards ajmalicine, serpentine, tabersonine, and vindoline biosynthesis whereas gibberellic acid has little effect on alkaloid production. Consistently, a recent report showed cytokinin and ethylene or combination of these two hormones to promote expression of the secologanin biosynthetic branch of indole alkaloid production (Papon et al. 2005).
pH and alkaloid storage Effect of pH value on plant cell growth and secondary metabolite production is mainly through inuencing membrane properties and transport activity. The capability of a cell line to produce secondary metabolites depends not only on biosynthesis activity, but also on transport and storage systems. It was shown that ajmalicine and serpentine are stored in the vacuole (Blom et al. 1991). The transport and storage processes for indole alkaloids are affected by the pH. Renaudin and his colleagues and Blom and colleagues have developed a hypothesis named as the ion-trapping-model, which proposed that ajmalicine and serpentine are taken up into the vacuole in unprotonated forms and trapped by protonation (Renaudin 1989; Blom et al. 1991). These studies show that the pH gradient is very important for storage and release of indole alkaloids in C. roseus cell cultures. The biochemical mechanism about transport of indole alkaloids in C. roseus cell cultures recently have been explored at protein and gene levels, indicating that besides iontrapping and diffusion through membranes also active and selective transport by multiple types of ATP-binding cassette (ABC) transporters are involved in alkaloid accumulation in vacuoles (Roytrakul 2004, see in this issue).
Bioreactor process The bioreactor process is the most important and difcult step in the scale-up of plant cell cultures for production of valuable secondary metabolites. The bioreactors used for plant cell cultures are modied from bacteria fermenters. Because of the different properties of plant cell cultures from microorganisms, such as slower growth rate, adhesive and larger cell walls, sensitive to shear force, and tendency to form cell aggregates, bioreactor behavior of plant cell cultures is very different from that of microorganisms. It is often observed that scale-up of plant cell cultures in bioreactors yields much lower biomass and, particularly, secondary metabolite production compared with that in shake asks (Moreno et al. 1995). Shear stress, gas regime (O2 and CO2,
Light and temperature Light positively affect indole alkaloid production in C. roseus cell and tissue culture (Zhao et al. 2001e), but effects of temperature on C. roseus cell culture are not signicant (see review, van der Heijden and Verpoorte 1989; Moreno et al. 1995). A recent investigation in a two-stage bioreactor process showed that the growth of C. roseus cells and ajmalicine production was found to be optimal at 27.5C (ten Hoopen et al. 2002).
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ethylene, as well as other unknown gaseous compounds), liquidgas mass transfer efciency, and other process parameters all contribute to these problems. Since these parameters are dependent on the bioreactor process design and operation, optimizing bioreactor processing is a great challenge for researchers (Leckie et al. 1991b, c). Reducing shear force to a reasonable level but at the same time increasing mixing efciency is one of main goals during bioreactor design. Regardless the types of bioreactor, also different cultivation modes have been explored for plant cultures. Plant cell cultures may be grown at high-density, as immobilized cells, and in a continuous two-phase cultivation system. Bioreactor design and optimization
biolm as a matrix to support cell cultures and circulated production medium to support living of cells was developed, showing less growth rate but being more effective in maximizing indole alkaloid titers than suspension cultures (Kargi et al. 1990; Kargi and Ganapath 1991). A surfaceimmobilized bioreactor for C. roseus cell cultures had also been tested (Archambault et al. 1990; Archambault 1991). Recently Ramakrishnan and Curtis (2004) developed a trickle-bed bioreactor for root cultures. In terms of operating C. roseus cell cultures in bioreactors, several modes such as batch, semi-batch, fed-batch, immobilized culture, and continuous cultures have been used. But the most common one in the above-mentioned bioreactor processes is the batch culture. Agitation and shear stress
Several types of bioreactors have been used for plant cell cultures. In terms of mixing forces, mechanical stirring (like impeller stirring bioreactor), air sparging (such as airlift bioreactor), and combinations (mechanical stirring together with aeration, like stirred-jar reactor) and variations of mechanical stirring and airlift bioreactors have been used as well as a membrane reactor with two permeable membranes to deliver nutrients and to export waste and products from the plant cells into the center tube. Based on the experience with bioreactor operation and the characteristics of plant cultures, various modied bioreactor congurations were developed, such as loop-uidized bed, spin lter, continuous stirred turbine, hollow ber, membrane stirrer for bubble-free aeration, hybrid reactor with a cell-lift impeller and a sintered steel sparger, as well as a centrifugal impeller bioreactor (for review, see Zhong 2001). A comparison of a variety of bioreactors with different agitation and aeration systems, as to their performance on biomass and secondary metabolite production of C. roseus cells showed the airlift bioreactor as the most suitable system (Misawa 1994). A double helical-ribbon impeller bioreactor with working volume of 11-l was designed for high-density C. roseus cell culture (Jolicoeur et al. 1992), a Maxblend fermentor for high-density culture was developed and tested for rice and C. roseus cell cultures (Yokoi et al. 1993). A biolm reactor that contains a horizontal
Mechanical agitation and sparging aeration are very important parameters for the culture of plant cell suspensions. They are responsible for mixing the plant cells with the medium and thus to facilitate homogenous nutrient uptake, and also for providing a good O2 and CO2 supply. However, mechanical agitation and sparging aeration cause hydrodynamic forces on the cells. The cells subjected to these shear forces show many physiological and morphological changes, such as aggregate size and shape, cell wall composition, oxygen uptake rate, cell integrity and viability, and eventually biomass accumulation and secondary metabolism. The effect of shear force on C. roseus cell cultures has been investigated in various bioreactors (Meijer et al. 1987; Leckie et al. 1991b, c; Smith et al. 1990; Kargi et al. 1990; ten Hoopen et al. 1994). The major conclusions of these studies were different from each other for many years and this was the paradigm for developing a plant cell culture system. Plant cells are not extremely sensitive for shear forces, depending on the cell lines; some are moderately or even almost not sensitive for shear. (Fig. 1) Because of the close relationship between agitation and oxygen supply, many studies have been conducted on the effect of these parameters on C. roseus cell cultures in bioreactors. DO2 in cell culture can be controlled by both agitation speed and aeration rate, which further will affect
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hydrodynamical conditions and gas regime in the cell culture. C. roseus cell cultures are more sensitive to hydrodynamic forces than bacteria. However, due to the much slower growth of plant cells, there is no need for high stirrer speeds and high aeration levels. Most bioreactors for plant cell cultures are from origin microbial fermenters.
In such fermenters C. roseus cells showed a lower growth rate and viability under higher agitation speed and aeration rate due to a high shear force and DO2 supply. On the other hand, C. roseus cells tend to form even larger aggregates at lower agitation speeds, and the cell morphology and aggregate size also largely depend on the
U ps t r ea m process
study on signaling and regulatory components, related genes and enzymes, biosynthetic and metabolic pathways, protein trafficking and metabolite transport mutagenesis and screening, or metabolic engineering f o r c r e a ti n g h i g h - y i e l d c e l l l i n e s , b a t c h c u l t u r e t e s t , optimization of medium and culture conditions L a r g e - s c a l e c u l t ur e i n b i o r e a c t o r probes b i or e a c t o r d e si g n , air te st an d i m p ro v em en t me di um zoom-in of cell factory of Catharanthus roseus suspension cells f o r b i os y n t h e si s o f i n d o l e a l k a l o id s
release
transporters alkaloids
Bioreactor proc es s m o n itor, process evaluation and i mp ro v e m e n t on-line monitor and auto-control of whole processing release and recovery of alkaloids f r om m e d i u m cell ex traction, purification to final products
ajmalicine catharanthine vindoline ABC tra nsp orter alkaloids

SG
serpentine b i s in d o l e alkaloids
enzymes
GC C STR
strictosidine STR TFs

G 10H
ORCA3
air
Tr p JA signalling signal t ra n s d u c t i o n [Ca2+]cyt spiking Elicitation
Down -s t r e a m process
ajmalicine catharanthine vindoline
geraniol
vindoline precursors t ran spo rter JA biosynthesis
Fig. 1 Schematic illustration of engineering the bioreactor process and cell factory of Catharanthus roseus cell cultures for indole alkaloid production. Upstream engineering is mainly to generate high-alkaloid-yield cell lines for large-scale production of indole alkaloids in bioreactor. A simple bioreactor model shows an airlift bioreactor with various probes for plant cell culture process. Several important steps were shown in frames with or without recycle arrows, which show these steps are developing technologies and processes. A chromatography column is used to show recovery and purication at downstream process. A zoom-in C. roseus cell shows the cellular process of indole alkaloid biosynthesis. Hormonal and environmental stimulations (elicitation) initiate signal transduction leading to jasmonate (JA) biosynthesis in plastids, which signaling further activates activation of transcription factors (TFs). Using STR gene as an simplied example to show transcription factors, like ORCA3 binding to GCC box, activate STR gene expression, STR further is targeted into vacuole, where it catalyzes strictosidine biosynthesis from cytosolic tryptamine (Trp) and precursor of from plastid-derived gera-
niol, which converted into geraniol-10-hydroxylate by tonoplast-localized P450: geraniol 10-hydroxylase (GH). Strictosidine is transported to ER and converted by ERlocalized strictosidine glucosidase (SG) into cathenamine, which is translocated to the cytosol, where it is further modied to synthesize ajmalicine and catharanthine. Strictosidine-derived tabersonine was further modied into intermediates that could be taken up into plastids, where it modied into precursors for vindoline biosynthesis. The nal two steps of vindoline biosynthesis were carried out in the cytoplasm. These monoindole alkaloids are taken up into vacuoles, most probably by the action of MRP-type ABC transporters or ABC transporter-like proteins. Inside the vacuole, vindoline and catharanthine are coupled by peroxidases into bisindole alkaloids whereas ajmalicine is converted into serpentine. Under some circumstances (such as elicitation), these indole alkaloids were exported into the cytosol and then further secreted into the medium, probably by the action of MDRtype ABC transporters. The bioreactor processing of C. roseus cell cultures consists of numerous cell factories for production of indole alkaloids
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hydrodynamic shear forces in the surrounding uid (Leckie et al. 1991a; Schlatmann et al. 1995a, b). However, various experiments suggest that shear force effects on C. roseus cell cultures are relatively not such a serious problem in normal operation (Leckie et al. 1991b, c; Schlatmann et al. 1994, 1995b). C. roseus suspension cell cultures tend to adhere to the walls of the culture vessels and accumulate at the headspace, particularly at high cell densities. A series of studies on processing C. roseus cell cultures in 3-l and 15-l turbine stirred tanks and other bioreactors have provided many useful data for understanding of the issues mentioned above (Leckie et al. 1991b, c; Kargi et al. 1990; Kargi and Potts 1991; Schlatmann et al. 1993, 1994, 1995a, b). Gas regime The importance of gas components in the headspace of culture vessels has long been recognized. Early studies showed that a limited oxygen supply to C. roseus cell culture incubated in the 4-l stirred tank bioreactor caused a reduced biomass accumulation, but also a high gassing rate in the bioreactor reduced biomass production (Pareilleux and Vinas 1983). Gases like oxygen, carbon dioxide, and ethylene accumulate in plant cell culture systems. Additional oxygen supply is essential for energy generation and various metabolic pathways in the bioreactor process. Carbon dioxide is the main metabolic gas component produced by plant cells. Ethylene is a gas borne phytohormone that is necessary for development and growth, as well as defense response. There are other unknown gaseous factors also affecting cell growth, biomass and secondary metabolite production in plant cell cultures. Recently about 76 volatile components were identied from C. roseus plants, including alkanes, alcohol, aldehydes, ketones, fatty acids, fatty acid esters, terpenoids and phenylpropanoids (Brun et al. 2001). It is very likely that in vitro C. roseus cell cultures also generate some of these volatiles, which accumulate in the headspace and affect the cell culture process and production of biomass and indole alkaloids. Particularly, some aldehydes, ketones, fatty acids, and fatty acid esters derived from oxylipin biosynthesis pathways may
have interesting effects that researchers have not investigated yet (Zhao et al. 2005a). Catharanthus roseus cell cultures scaled up in simple stirred bioreactors often have much lower biomass accumulation and indole alkaloid production than in shake asks. It was shown that gas composition and shear forces are the main reasons for this difference (Schlatmann et al. 1993). Recirculation of exhaust gases in the stirred bioreactor partly restored the biomass and indole alkaloid production in a stirred tank reactor or bubble column, suggesting that exhaust gases play an essential role in biomass and indole alkaloid production (Schlatmann et al. 1993). Therefore, effects of gas regime on the culture process were extensively studied in C. roseus cell cultures in various bioreactors. In a 15-l turbine stirred bioreactor, ajmalicine production in high dissolved oxygen (DO2) conditions (80% of air saturation) was 5-fold higher than that in low DO2 (15% of air saturation) (Schlatmann et al. 1994). A linear relationship between DO2 and ajmalicine production was observed in DO2 between 29% of air saturation and 43% of air saturation (Schlatmann et al. 1993). A high-density cell culture produced much lower ajmalicine than a low- density cell culture (with high DO2), but an increase in DO2 to high-density cell culture could not restore the ajmalicine production (Schlatmann et al. 1994). During two-stage culture for optimal growth and alkaloid production, aeration rate for the growth stage was usually set as 228300 l/h, stirrer speed at minimum of 200 rpm; in that case, DO2 was about 40% (Schlatmann et al. 1994). According to Pareilleux and Vinas (1983), the critical dissolved oxygen concentration for C. roseus cell suspension is 0.05 mmol/l (20 % of air saturation). The respiration rate was measured to be around 0.150.3 mmol/g cells/h (without oxygen limitation for cell cultures) (Pareilleux and Vinas, 1983). The optimal value for oxygen mass transfer coefcient KLa for C. roseus cell cultures in bioreactor ranges between 15 and 20 h1. However, different optimal KLa values were found for growth and alkaloid production in a 12.5-l stirred tank bioreactor: KLa for serpentine production is 16.0 h1 and for ajmalicine are 4.5 h1 (Leckie et al. 1991a). High KLa values caused increased aggregation of the cultures,
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depressed biomass yields, and altered patterns of alkaloid accumulation (Leckie et al. 1991a). The KLa for optimum biomass production were among 4.5 h1 and 12.5 h1 (Leckie et al. 1991a). Smith et al. (1990) developed a bioreactor system to determine and control dissolved concentrations of oxygen and carbon dioxide (DCO2) at constant shear force. It was shown that DO2 set at 50% of air saturation and DCO2 set as 20 mbar at starting point could keep the culture system in long-term balance (Smith et al. 1990). Diaz et al. (1996) studied the partial pressures of dissolved oxygen and dissolved carbon dioxide in a bioreactor. Doran (1998) reported a new technology to improve oxygen delivery to hairy root cultures by using membrane tubing aeration and peruorocarbons. For catharanthine production in a 2-l jar bioreactor with 300 g cells as inoculums, an increase in DO2 (8 ppm) signicantly enhanced catharanthine production from 180 mg/l/week to 230 mg/l/week. In the same bioreactor, Schlatmann et al. (1994) showed that with certain levels of DO2, more CO2 promoted biomass accumulation and ajmalicine production. Aeration affects CO2 supply in cell cultures. Both higher dissolved CO2 caused by too high airow rate and lower DCO2 caused by too low airow rate are not good for C. roseus cell growth (Ducos and Pareilleux 1986; Hegarty et al. 1986). Rheology in cell culture Rheological studies on plant cell suspension cultures provided important information for improvement of the cell culture process. Studies including viscosity, aeration, mass transfer, shear stress and cell growth, as well as metabolite production in plant cell cultures can provide insights into problems of the bioreactor process. Rheological effects of treatments with e.g., alginate, and sugar or with osmotic regulators such as sorbitol and mannitol on biomass and indole alkaloid production are very clear (Zhao et al. 2000c; Zhao et al. 2001a). Even with rigorously mixing, DO2 and DCO2 are signicantly reduced, yet accumulation of secondary metabolites was not changed much, suggesting specic rheological effects on alkaloid biosynthesis. Rheological
effects also dramatically inuence the bioreactor process when C. roseus cell cultures are cultured at high density. High-density cell culture can improve volumetric productivity of plant secondary metabolites. High-density cell cultures together with other treatments to initiate biosynthesis of target secondary metabolites could considerably improve the productivity (Zhao et al. 2001a; Zhong 2001). However, a bioreactor process of high-density cell cultures may result in lower indole alkaloid productivity in part due to the decreased DO2 and nutrient limitation because of the decreased mass transfer (Schlatmann et al. 1994, 1995c). The signicantly reduced mass transfer is mainly due to the low dynamics in the high-density cell culture. Increasing DO2 indeed can partly recover ajmalicine productivity (Schlatmann et al. 1994, 1995c). Bioreactor process monitoring An important part of cell culture process is to monitor the biomass concentration of the plant cells or even secondary metabolites over the growth cycle, since it is essential to know how culture cells are growing and how target metabolites are accumulated. Bioreactor processing of plant cell cultures represents a physically, chemically, and biologically dynamic system, in which different levels of interactions are ongoing: cells with their environment, cells with cells, between subcellular organelles, and cells with endogenous molecules in the culture. To obtain as many details as possible about changes in these variables is a prerequisite for optimizing and controlling the bioreactor process. Shear forces often exert negative effects on plant cell growth and secondary metabolite accumulation, quantitatively determining shear forces and their effects on plant cell cultures can be done by using special ow and shearing devices such as corvette-type apparatus, recirculating ow capillary, and submerged jet (Zhong, 2001). The shear damage effects can also be detected by measuring O2 uptake rate, cell growth rate, conductometry, osmotic pressure, and O2/CO2 concentrations in the bioreactor inlet and outlet gases. Many important parameters, such as KLa for O2 and CO2, mixing conditions, DO2, DCO2, and viscosity
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of cell culture always change during a bioreactor process of plant cell cultures due to cell growth and death. For example, KLa values were found to increase by about 1020% compared to their corresponding initial KLa, but as the cell density increased, KLa ultimately decreased (Ho et al. 1995). Biomass also decreased with KLa, probably due to the hydrodynamic forces at impeller speeds of 100325 rpm as at the aeration rate of 0.43 vvm no oxygen starvation was observed (Ho et al. 1995). Kinetic monitors for above parameters are usually present in various commercial bioreactors and are important to evaluate the culture system and establishing a mathematical model for autocontroling the culture system. On-line monitoring of an established bioreactor process based on a computer-aided monitoring system was applied to a plant cell process (Zhong 2001). Komaraiah et al. (2004) developed multisensor array gas sensors to continuously monitor changes of the gas regime in plant cell cultures. Analyzing the multiarray responses using two pattern recognition methods, principal component analysis and articial neural networks showed that plant cell suspension cultures can generate volatile emissions and these emissions may be detected using electronic noses sensor arrays. Analysis of these process variables in turn can predict the biomass concentration and the secondary metabolite production (Komaraiah et al. 2004). Recently, a multiwavelength uorescence probe was tested for in situ monitoring of Eschscholtzia californica and C. roseus cell cultures (Hisiger and Jolicoeur 2005). Using endogenous uorophores with different excitation and emission peaks of plant (secondary) metabolites, this probe can be used to monitor NAD(P)H as marker of cell activity and riboavins for cell concentration and growth. The real-time production of tryptophan, tryptamine, ajmalicine and serpentine could also be monitored with this probe (Hisiger and Jolicoeur 2005). This provides a very useful tool for the control and optimization of plant cell processes. Except for monitoring the whole culture system, assaying enzyme activity and detecting gene expression of C. roseus cell cultures in bioreactors also are important to understand the kinetic process. Previous studies have already provided insight in the enzyme activities in C. roseus cell cultures
grown in a bioreactor under different cell-densities and sugar concentrations (Schlatmann et al. 1995a, b). It was reasoned that shear stress, special gas regime, high pressure, and other unknown factors in bioreactor-processed C. roseus cell cultures may change expression of some genes critical for indole alkaloid production. Mathematical model and process control Mathematical models describing the bioreactor process are essential tools for designing, optimizing, scaling up, and auto-controlling the bioreactor operation and cell culture process. Several mathematical models for plant cell cultures were developed (Bailey and Nicholson 1989; Bailey and Nicholson 1990; De Gunst et al. 1990). A basic structured kinetic model was established and used for batch tobacco suspension cultures, regarding structural component production, secondary metabolite synthesis and cellular respiration (Hooker and Lee 1992). Models for utilization of nutrients by C. roseus cell cultures were also established, e.g., a bioreactor system for controlling dissolved concentrations of both DO2 and DCO2 simultaneously (Smith et al. 1990); an unstructured mathematical model in glucose limited chemostats showed a linear relation between specic glucose uptake, oxygen consumption, and carbon dioxide production as a function of the growth rate (van Gulik et al. 1992). Furthermore, they developed a structured mathematical model for the description of the kinetics of growth and intracellular accumulation of glucose and phosphate, as a function of glucose and phosphate supply; this structured model well described the growth of C. roseus cell suspensions (van Gulik et al. 1993). More recently, based on the description of metabolic events during the production stage, a simple structured model for maintenance, biomass formation, and ajmalicine production by non-dividing C. roseus cells was established (Schlatmann et al. 1999). This model describes stoichiometry of biomass (including two parts, active biomass and storage carbohydrates) and ajmalicine production kinetics of non-dividing C. roseus cells in the second stage of a two-stage batch process. It provides a satisfactory description of the results even though ajmalicine production did
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not t well due to accumulation of inhibiting gaseous metabolites (Schlatmann et al. 1999). Continuous culture is preferred for the development of mathematical models, because of the potential of steady state conditions. However, no good mathematical model is established yet to describe the important model of the bioreactor process. Establishing a well-tting mathematical model for a plant cell culture process is difcult due to the uncertainty and the nonlinear nature of the bioprocess. Recently articial intelligence methods were used for the design and control of microbial processes, this knowledge-based methodology can also be used for plant cell culture (Zhong 2001). Recently Leduc et al. (2006) developed a kinetic metabolic model describing C. roseus hairy root growth and nutrition conditions. The model used intracellular nutrients and energy shuttles to describe metabolic regulation, providing an efcient tool for estimating the growth rate. A suitable metabolic model should be made on the basis of measurements of many metabolic pathways. High-density culture Calculation of cost-effectiveness of a plant cell culture process for secondary metabolite production has shown that only with a high biomass it is possible to reach a low price, which means that one has to reach maximal biomass by high-density culture to obtain a cost-effective production of secondary metabolites (Verpoorte et al. 2002). But high-density cell culture of C. roseus was shown to have 5-fold lower productivity than the low-density culture, due to low DO2 and other factors (Moreno et al. 1993a; Schlatmann et al. 1995c), optimizing culture conditions such as DO2 may increase the volumetric productivity of indole alkaloids. At a density of about 2030 g/l cell cultures have only 510% free medium, which makes it difcult to recover anything from the medium, consequently, the products must be extracted from the biomass (Verpoorte 2002). Other plant cell cultures have been grown in highdensity for producing useful secondary metabolites (Zhong 2001). It was shown that high-density cell cultures gave much higher productivity of target secondary metabolites, particularly in com-
bination with other strategies, thus corresponding bioreactor congurations have been developed for high density cultures, such as a double helicalribbon impeller reactor and maxblend fermentor (Jolicoeur et al. 1992; Yokoi et al. 1993; Zhong 2001; Zhao et al. 2000a, 2001a). It thus seems that high-density culture could be best for production of intracellular-accumulated secondary metabolites in combination with stimulation strategies. In a two-stage culture strategy, high-density cultures can be treated at the second stage in many ways to obtain the target secondary metabolites, such as elicitation, immobilization, two-phase and continuous cultivation (including semi-continuous and fed-batch cultures). Continuous culture It was suggested that continuous culture of plant cells might be an economical process for commercial production of secondary metabolites by plant cell cultures. The continuous culture technique would enable cell cultures in suitable bioreactors to continuously synthesize and release secondary metabolites for long time, upon feeding nutrients, precursors, or stimulation substances. However, due to the technical and practical limitations on keeping cell viability and stability, sterile operation of bioreactors, and some other factors, such processes have not been realized and cost calculations show that the costs will be higher than in case of a fed-batch culture (Verpoorte et al. 2002; Zhong 2001). However, for modeling the secondary metabolite production the continuous cultures are an important research model. Pareilleux and Vinas (1984) rst tried continuous culture of C. roseus cells for indole alkaloid production. Van Gulik et al. (1992, 1993) studied C. roseus cell cultures in stirred tank bioreactors operated in batch and continuous modes. Comparison of stoichiometry of C. roseus growth in steady-state glucose limited chemostats and dynamic conditions showed that they are very different (van Gulik et al. 1992). The chemostat culture technique is useful to obtain reliable data on the stoichiometry of the growth of plant cells in a stirred bioreactor. Several other groups have also studied growth kinetics, stoichiometry, and modeling of the growth of suspension-cultured plant
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cells by using semi continuous or fed-batch cultures to achieve steady-state growth (for a review, see Zhong 2001). The results suggest that removal of indole alkaloids away from cells facilitates the metabolic ux to production of more alkaloids due to deletion of feedback inhibition. This strategy could be important for efcient up scaling of plant cell cultures as both productivity and recovery of secondary metabolites are improved. A continuous culture process that adapts this strategy might be an economical way for production of plant secondary metabolites although in such a system high-density biomass is not possible as free medium is required for the extraction of these compounds. The improved productivity per unit of biomass has a tradeoff in terms of a decrease in volumetric productivity. The optimum balance between the two must be determined to come to the true commercial production system. Two-phase culture Two-phase cultivation systems of plant cells have been developed to improve production of secondary metabolites. Both liquidliquid and liquidsolid systems have been used to concentrate secondary metabolites from the cells and the medium into the second phase. Introduction of such an additional phase is not only of interest for in-situ extraction and prevention of degradation, but also may enhance the metabolic ux toward desired products by reducing the feedback inhibition by removal of the products from their biosynthesis site (intracellular compartments). Based on this theory, different two-phase culture systems were developed for plant cell cultures. Byun and Pedersen (1994) used a two-phase airlift bioreactor in combination with elicitation for a signicantly enhanced production of benzophenanthridine alkaloids in cell suspensions of E. californica. The use of Amberlite XAD-7 resin in C. roseus cell cultures can dramatically improve both volumetric productivity and recovery of indole alkaloids (Brodelius and Pedersen 1993; Payne et al. 1998; Lee-Parsons and Shuler 2002). The alkaloids are absorbed on the resin, which dramatically improves the production of alkaloids. Tikhomiroff et al. (2002) tried C. roseus hairy root cultures in a two-liquid-phase bioreac-
tor, which was designed to extract indole alkaloids with silicon oil. This two-phase culture system could efciently absorb tabersonine and lochnericine and thereby increase the production of the two indole alkaloids by 100400% and 14200%, respectively, without signicantly affecting the availability of nutrients and hairy root growth. In combination with elicitation by jasmonic acid, specic production of all indole alkaloids including non-silicon oil-absorbed serpentine further increased. A polyurethane foam draft tube as the immobilizing matrix was applied to an airlift bioreactor to carry out a two-phase C. roseus culture. The bioreactor was connected to a neutral polymeric resin column to absorb indole alkaloids. The total secreted indole alkaloids reached 380 mg/l, most of the intracellular alkaloid produced by C. roseus cells was secreted into the medium (Yuan et al. 1999). The volumetric oxygen transfer coefcient KLa in cell cultures processed in an organic solvent two-phase culture system, as well as rheological properties of the system, was studied regarding the effects of organic solvent, agitation speed, and aeration rate (Wu et al. 2000). Such data are of interest for developing optimal conditions for growth and secondary metabolite production.
Strategies to improve the productivity of the cell factory Feasibility of the commercial production of a valuable secondary metabolite by plant cell cultures largely depends on the economics of the production process, which in turn depends on productivity. Economics for a bioreactor process for a plant cell culture producing indole alkaloids was calculated (Verpoorte et al. 1999), showing that current productivity of ajmalicine (maximum 0.3g/l) (Verpoorte et al. 2002) is not enough for an economical feasible large-scale production. Since the low productivity of indole alkaloids in C. roseus cell cultures is one of the obstacles towards commercial production, extensive efforts are made to overcome the biological limitation. Selection or creation of new high-alkaloid-yield cell lines, eliciting C. roseus cell cultures, precursor-feeding, or metabolic engineering of biosyn-
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thetic pathways are potential strategies to improve indole alkaloid productivity. The whole idea is to create high-indole alkaloid-yield cell lines by stable genetic over-expression of the expected biosynthetic pathways or inhibiting competitive pathways, to improve bioreactor performance by to-the target eliciting, precursor feeding, optimizing growth and production of engineered cell cultures, and to release indole alkaloids into medium and recover them efciently by processing technologies. Selection and creation of high-alkaloid-yield cell lines Catharanthus roseus cell cultures often are heterogeneous and are composed of low-alkaloidyielding, high-alkaloid-yielding, as well as nonalkaloid-producing cells. Like bacteria colony isolation, i.e., selection of cell lines with suitable and uniform genetic, biochemical, and physiological characteristics, is an important approach to improve productivity of target secondary metabolites. Researchers used radioimmunoassay and uorescence assay methods to screen cell lines to obtain high-yield cell lines. UV- and radioactiveirradiation, as well as other mutagenic treatments have been applied to further widen the genetic resources (for review, see Moreno et al. 1995). The random mutagenesis and the laborious and time-consuming screening are rather like a lottery with an unpredictable outcome, therefore now the preferred strategy is to genetically engineer cells in a clearly targeted approach. Advances in understanding the biosynthesis and its regulation resulting in the cloning of a number of genes involved in the pathway are the basis of metabolic engineering. Developments in the eld of molecular biology have greatly facilitated the unraveling of plant metabolism. To date many successful examples of improved secondary metabolite production by genetic modications have been reported (for a review, see Verpoorte and Memelink 2002). Elicitation of indole alkaloid biosynthesis Elicitation of cell cultures with various abiotic and biotic elicitors or signal molecules often
results in a dramatic increase in yield of certain secondary metabolites, probably due to the defense role of these secondary metabolites. Production of indole alkaloids is also induced by biotic or abiotic stresses. Examples are many: salt stress using NaCl and KCl, osmotic stress using sorbitol, mannitol (Moreno et al. 1995; Zhao et al. 2000c), polyethylene glycol, polyvinyl pyrrolidone, and sodium alginate (Aoyagi et al. 1998; Zhao et al. 2000c), metal stress with sodium orthovanadate, vanadyl sulphate and some rare earth elements (Zhao et al. 2000b), stimulation with various chemicals (Zhao et al. 2000d; 2001c), fungal elicitors and hormones (Namdeo et al. 2000; Zhao et al. 2001d; El-Sayed and Verpoorte 2004). Application of the elicitation to C. roseus cell cultures not only improves indole alkaloid biosynthesis in short time, but causes also excretion of the products into the medium. Combination of two or more elicitors that can synergistically induce metabolic uxes towards indole alkaloids even further improves the productivity of target compounds and performance of bioreactor processing. A C. roseus cell line was cultured in a 14-l bioreactor with 80% decrease in total alkaloid production compared to the shake ask culture, but combined osmotic stress with 1 mM trans-cinnamic acid treatment restored the original alkaloid amounts (Godoy-Hernandez et al. 2000). A combined elicitor treatment with an Aspergillus niger mycelium and tetramethylammonium bromide resulted in much higher ajmalicine production in a 20-l airlift bioreactor (Zhao et al. 2000a). In addition, a synergistic effect on indole alkaloid accumulation was observed in C. roseus cell cultures when treated with combined elicitation with malate and sodium alginate, resulting in a higher catharanthine yield in asks and a 20-l airlift bioreactor compared with control (Zhao et al. 2001a). Treatment of C. roseus cells with ABA induced catharanthine and ajmalicine accumulation. In a 30-l airlift bioreactor, 8.3 mg/l ABA was added to 7-day-old C. roseus cell culture and 82.25 mg/l of catharanthine can be obtained after three days of further culture (Smith et al., 1987). The effect of a combination of treatments was nicely illustrated for taxane diterpenes production that was dramatically increased when suspension cultures of
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Taxus chinensis were treated with MeJA, sucrose feeding, and ethylene exposure (Dong and Zhong 2002). Precursor feeding and metabolic ux distribution The indole alkaloids derive from precursors from two biosynthetic pathways, the terpenoid pathway and shikimate pathway. By feeding precursors an improved production of indole alkaloids can be achieved. Commonly, precursors from the shikimate pathway such as tryptophan or tryptamine and precursors from the terpenoid pathway such as loganin, loganic acid, and secologanin are used to effectively promote indole alkaloid production. Of the iridoid precursors, loganin is most efciently incorporated into indole alkaloids (Moreno et al. 1993b; Whitmer et al. 1998a, 2002a, b). Precursor feeding time usually signicantly affects feeding outcomes. It is concluded that feeding cells in exponential growth phase (Moreno et al. 1993b; Silvestrini et al. 2002) with precursors gives the maximum outcome, probably due to active uptake and biosynthesis capability of cell cultures at this stage. There are many other branches in the terpenoid and the shikimate pathway leading to the production of other compounds, which compete for the precursor pools with indole alkaloid production. Inhibiting production of these undesirable compounds may enhance precursor ow to indole alkaloids. For example, trans-cinnamic acid (inhibitor of phenolic compound biosynthesis), phenobarbital, an inducer of Cytochrome P450 enzymes like geraniol-10-hydroxylase, or an inhibitor of Cytochrome P450s, all affect production of indole alkaloids with predicted results (Contin et al. 1999). Fed with tryptophan in a 20-l airlift bioreactor, C. roseus cell suspension produced 0.31 mg/g dry mass ajmalicine after 14 d of cultivation (Fulzele and Heble 1994). Fed with stemmadenine C. roseus cell cultures accumulated more catharanthine, tabersonine and condylocarpine, suggesting that stemmadenine is an intermediate in the pathway to catharanthine and tabersonine (El-Sayed et al. 2004). Recent feeding studies on elicited cells or transgenic cell lines clearly showed that the terpenoid pathway is
limiting the production of indole alkaloids, feeding both tryptamine and loganin the cell factory can even produce much higher levels of alkaloids. This proves that the capacity of the cell factory for producing alkaloids in fact is much higher than the actual production (Whitmer et al. 1998a; 2002a, b). Release and recovery of indole alkaloids Excretion of the secondary metabolites into the medium makes recovery of these chemicals much easier. However, there is only a minor portion of indole alkaloids released into the culture medium, with improved production of indole alkaloids most of the synthesized indole alkaloids are intracellularly stored. The lack of appropriate methods to release secondary metabolites into the medium is a problem for developing an industrial process in which extraction from the medium is desired. Techniques that cause continuous production and release of secondary metabolites from plant cells without decreasing their viability and biosynthesis capability could be of interest. From studies on immobilized and two-phase C. roseus cell cultures researchers have established some methods for releasing indole alkaloids (Brodelius and Pedersen 1993; Payne et al. 1998). These include chemical permeabilization, elicitation, oxygen or phosphate limitation, pH gradient variation, pressure or heat shock, ultrasonication, and electropermeabilization. Brodelius and Pedersen (1993) tested several permeabilizing agents such as DMSO and Triton-X-100 on C. roseus cell cultures and found that they effectively released indole alkaloids but also reduced cell viability. In situ extraction of indole alkaloids from cell cultures with neutral resins, on the other hand, was shown to stimulate production of indole alkaloids (see above) (Brodelius and Pedersen 1993; Payne et al. 1998; Lee-Parsons and Shuler 2002). Also some other release methods stimulate the production of plant secondary metabolites, for example, various elicitors, ultrasonication (Wu and Ge 2004), and electropermeabilization (Yang et al. 2003). Therefore release of indole alkaloids from C. roseus cell cultures is an important strategy to improve overall productivity. Based on the ionic
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properties of the indole alkaloids, electropermeabilization of C. roseus cell membranes and simultaneous electrophoric transport and collection of indole alkaloids was explored (Yang et al. 2003). Both batch and continuous electrophoretic tubular membrane reactors were developed for simultaneous release, transport, and collection of ionic metabolic products, and validated by using C. roseus and Beta vulgaris cell cultures for positively and negatively charged plant secondary metabolites (Yang et al. 2003). Promising results were obtained with the application of an oscillatory electrical eld, it appeared to improve production of secondary metabolites while retaining high cell viability (Yang et al. 2003).
Signal transduction to indole alkaloid biosynthesis Since various biotic and abiotic elicitation methods have been found that successfully stimulated net indole alkaloid production in various C. roseus cultures, investigation of the signal transduction pathway(s) involved in the response to elicitation will be of great importance (for a review, see Zhao et al. 2005a). Studies of signal transduction and regulatory mechanisms underlying the induction of the biosynthesis of indole alkaloids by elicitation with yeast extract or MeJA lead to identication of several transcription factors that control the alkaloid biosynthetic genes (Memelink et al. 2001). Overexpression of such transcription factors might be used to turn on a complete pathway. Studies on elicitation revealed various signaling components that mediate elicitor-or other stress-induced indole alkaloid production (Zhao et al. 2005a). Ca2+, reactive oxygen species, and nitric oxide are found to be components in the elicitor signaling pathway leading to indole alkaloid production (Zhao et al. 2001b; Xu et al. 2005). Elicitor induction of endogenous jasmonate biosynthesis was proved to be an important step in mediating the elicitor-induced strictosidine synthase (STR) and tryptophan decarboxylase (TDC) gene expression and indole alkaloid production. Different protein kinases may be activated upstream and downstream of jasmonate biosynthesis (Menke et al. 1999). Calcium ions inux is identied as a prerequisite for fungal elicitor-induced oxidative stress, jasmonate biosynthesis, and indole alkaloid production (Zhao et al. 2001b; Pauw et al. 2004; Lee-Parsons and Erturk 2005). Even though H2O2 was shown to stimulate biosynthesis of secondary metabolites, it is still a question whether H2O2 is a signaling compound for indole alkaloid biosynthesis (Zhao et al. 2001b; Pauw et al. 2004). In addition, nitric oxide burst was also observed in elicitor-induced C. roseus cell cultures and mediates catharanthine production (Xu et al. 2005). Plant cell cultures in bioreactors often suffer from oxidative stress due to improper culture conditions such as oxygen supply, mechanical damages, and nutrition imbalance even without elicitation. Such oxidative stress can exert multiple effects on
Metabolic engineering of the cell factory for indole alkaloid production Metabolic engineering requires detailed knowledge of the various metabolic pathways in an organism. This knowledge comes from studies on the enzymes involved in the pathway, their characterization and measuring their activity and determining its regulation. Based on this enzymes can be selected as targets for cloning of the gene and subsequent engineering of the cellular biosynthetic machinery, e.g., to increase the metabolic ux to desired secondary products and thus improve the productivity of the plant or plant cell culture for the target metabolite. This goal can be achieved either by redirecting metabolic uxes by overexpressing the target pathway; suppressing other pathways that compete with the target pathway for precursor pools; suppressing catabolic pathways of the product of interest; or any combination of these. In other words, metabolic engineering is expected to control as many closely related metabolic pathways as possible to achieve maximal effects. Metabolic engineering provides completely new perspectives with great potential for production of important plant secondary metabolites. Despite the limited knowledge on the metabolic pathways and genes involved, the available information and materials obtained from studies on C. roseus have already provided the tools for starting the metabolic engineering of the alkaloid biosynthesis.
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plant cell growth and secondary metabolism (Zhao et al. 2000a; Zhao et al. 2001a; Zhao et al. 2005b). Reactive oxygen species generated during oxidative stress may be the main factors for these effects. Recently a new group of jasmonate-like oxylipins generated in tobacco under H2O2 stress was identied as inducers of plant secondary metabolite accumulation (Thoma et al. 2003). Almost all responses to elicitation and physiological conditions inducing indole alkaloid biosynthesis may be mediated by the jasmonate pathway. Cytosolic Ca2+ spiking is required for fungal elicitor-induced jasmonate biosynthesis (Menke et al. 1999; Pauw et al. 2004). Auxin suppression of STR and TDC expression and indole alkaloid production is also due to suppression of endogenous jasmonate biosynthesis. Removal of auxins from C. roseus cell cultures and exposure of the cell cultures to kinetins may require intracellular Ca2+ spiking to resume indole alkaloid biosynthesis, and most probably recovery of jasmonate biosynthesis (Gantet et al. 1998). The cytokinin-induced alkaloid biosynthesis is mediated by a two-component system, like the cytokinin signaling pathway in Arabidopsis (Papon et al. 2003). How jasmonate signaling mediate elicitation as an integral signal to induce indole alkaloid biosynthesis is not well known yet. However, several transcriptional factors have been found that are specically induced by elicitors or MeJA. These transcription factors bind to promoter regions of STR and TDC (Memelink et al. 2001; van der Fits and Memelink 2000). Biosynthetic and metabolic pathway, gene and enzyme characterization The biosynthesis of indole alkaloids has been extensively studied and many details about enzymes and genes involved have been reported, yet many genes involved in the secologanin and catharanthine biosynthesis remain unknown (Verpporte et al. 1997; van der Heijden et al. 2004). Except for several transcription factors for the elicitor and jasmonate signaling, other regulatory mechanisms are not known, e.g., intra- and intercellular metabolite transport, enzyme or regulatory factor trafcking, compartmentation,
and degradation of indole alkaloids (catabolic pathways) (St-Pierre et al. 1999; Memelink et al. 2001; van der Heijden et al. 2004). Despite that so far only a limited number of biosynthetic genes are available, metabolic engineering of the indole alkaloid biosynthesis has generated already some interesting results (van der Heijden et al. 2004). Inter- and intra-cellular transport of indole alkaloids and trafcking of proteins Numerous studies have shown that biosynthesis and storage of plant secondary metabolites take place in different subcellular organelles of the cells. Indole alkaloid biosynthesis and storage involve multiple machineries in different cellular and subcellular compartments. Firstly it was shown that many different cell types are implicated in vindoline formation and storage: earlier steps for tryptamine and secologanin biosynthesis occur at epidermis cells, and later steps for vindoline formation take place at mesophyll, idioblast, or laticifer cells (Murata and De Luca 2005; Mahroug et al. 2006). Secondly it was found that, different subcellular compartments are involved in indole alkaloid biosynthesis, these include at least the plastids, endoplasmic reticulum, and vacuole. Therefore, the assembly of the indole alkaloid biosynthesis pathway requires not only appropriate enzyme trafcking but also efcient transport of substrates and metabolites. Transport of biosynthetic intermediates and metabolic products in and out of the chloroplasts and vacuoles through endomembranes inside the cells or from cell to cell through the plasma membrane are essential parts of the biosynthesis (St-Pierre et al. 1999; Yazaki 2005). Recent studies have revealed that different ABC transporter and H+-antiporters are involved in these functions. For example, efux of berberine from C. japonica cells is nished by one type of ABC transporter, a multidrug resistance protein (MDR), CjMDR1 (Yazaki 2005). The barley secondary metabolite saponarin is imported into barley vacuoles by a proton-motive forced H+transporter but into Arabidopsis vacuoles by an ABC transporter (Frangne et al. 2002). These studies provide insights and tools for production of plant secondary metabolites. Most recent
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studies using an in vitro vacuolar uptake assay and pharmacological methods have shown that inux and efux of alkaloids and its precursors through the vacuolar membrane or C. roseus cell plasma membrane involves different types of ABC transporters (Roytrakul 2004). Roytrakuls study indicated that ajmalicine, catharanthine, and vindoline could be taken up into the vacuole by a subfamily of ABC transporters, the multidrug resistance associated proteins (MRP); while strictosidine and secologanin could be taken up into the vacuoles by ABC transporter-like proteins; and tryptamine uptake into the vacuole may involve H+-antiporters (Roytrakul 2004). All of these alkaloids could be secreted from the vacuole by MDR-type transporters (Roytrakul 2004). Since these ABC transporters and H+-antiporters are less specic in substrate preference and highly regulated (Yazaki 2005); their activity denitely affects production of indole alkaloids in C. roseus cell cultures. Overexpression of indole alkaloidbiosynthesis genes in tobacco cells showed that strictosidine generated by transgenic tobacco cell cultures fed with secologanin and tryptamine are exported into the medium and not stored in the vacuole (Hallard et al. 1997; Verpoorte, unpublished results). Apparently every plant species has different selective transport systems. Further study on the transport of precursors, intermediates and indole alkaloids may lead to discovery of new strategies that could facilitate the production of certain indole alkaloids. Metabolic engineering of transport of related metabolites can improve overall production of indole alkaloids in C. roseus cell cultures. Protein trafcking involved in plant secondary metabolism is not well understood until recent years, but is thought to play an important regulatory role. Characterization of CaaX-prenyltransferases from C. roseus cell cultures supported this idea (Courdavault et al. 2005). Prenyltransferases are a group of heterodimeric enzymes that can modify and re-localize many regulatory proteins such as transcription factors and signal components, and membrane-associated enzymes or membrane trafcking proteins. RNAi suppression of subunits of the protein dramatically inhibits expression of early steps of indole alkaloids and blocks indole alkaloid production,
suggesting that proper targeting of some regulatory proteins is essential for regulation and expression of indole alkaloid biosynthesis genes (Courdavault et al. 2005). Manipulating biosynthetic genes Genetic modication results showed that the production of indole alkaloids in C. roseus cell cultures was not affected by the overexpression of TDC (Canel et al. 1998; Whitmer et al. 1998a, b; Goddijn et al. 1995; Whitmer et al. 2002b). However, feeding with tryptophan and more loganin (6.4 mM) resulted in about 400 mg/l of total indole alkaloids including strictosidine, ajmalicine, serpentine, catharanthine and tabersonine as major components. On the other hand, overexpression of STR resulted in a higher production of total indole alkaloids at a level of almost 300 mg/l (Whitmer et al. 2002a). Like other high-yield cell lines, genetic or epigenetic instability of secondary metabolite biosynthesis also occurs in transgenic lines. The levels of indole alkaloids in STRtransgenic cell lines decreased gradually after years of subculture maintenance, but the high capacity of indole alkaloid production can be restored by precursor (like loganin) feeding (Whitmer et al. 2003). These results not only suggest the feasibility of metabolic engineering for indole alkaloid production, but also indicate that other unknown regulatory mechanisms exist to control the metabolic uxes. Results also suggests that there seems to be a physiological barrier for the production of more than about 400 mg/l, as feeding larger amounts of precursors does not lead to further increase of alkaloids. The regulation includes probably substrate and product trafcking, compartmentation and metabolism, since transgenic cells still kept high enzyme activity. The results from transgenic tobacco and Cinchona ofcinalis hairy roots expressing the TDC and STR genes also point to the importance of subcellular trafcking and storage for production of secondary metabolites (Hallard et al. 1997; Verpoorte et al. 2002). Metabolic engineering was also tried on C. roseus hairy root cultures. Overexpression of a truncated hamster 3-hydroxy-3-methylglutarylCoA reductase, a key enzyme in mevalonate/
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acetate pathway for terpenoid biosynthesis, in C. roseus hairy roots was reported to result in an increased production of ajmalicine and catharanthine or serpentine compared with control (Ayora-Talavera et al. 2002). Transgenic hairy root cultures of C. roseus were reported with a glucocorticoid-inducible promoter controlling an Arabidopsis feedback-resistant anthranilate synthase a-subunit (ASa) or b-subunit (Asb) (Hughes et al. 2004a). The transgenic hairy roots produced more than 20-fold higher levels of tryptophan or tryptamine under certain induction conditions compared with control whereas most indole alkaloids were not signicantly altered with the exception of lochnericine, which increased 81% after a 3-day induction period (Hughes et al. 2004a). Furthermore, glucocorticoid inducible expression of TDC alone or in combination with ASa in transgenic C. roseus hairy root cultures showed an increased TDC activity after induction (Hughes et al. 2004b). The induced TDC line showed no signicant increase in tryptamine level but 129% increase in serpentine production, whereas TDC-ASa line showed 6-fold increase in tryptamine level but no increase in serpentine production (Hughes et al. 2004b). Secologanin biosynthesis limitation in such transgenic hairy roots was conrmed (Peebles et al. 2006). In feeding 1-deoxy-D-xylulose to ASa-overexpressing hairy root line, an increase of 125% in hoerhammericine was observed, while loganin feeding increased catharanthine by 45%. In feeding loganin to the ASa- and ASb-overexpressing hairy root line, increases of 26% in catharanthine, 84% in ajmalicine, 119% in lochnericine, and 225% in tabersonine were observed (Peebles et al. 2006). Manipulating transcription factors It is generally recognized that signal transduction pathways, controlling the biosynthetic genes of secondary metabolites, do so via transcription factors, which activate or suppress biosynthetic pathways (for review, see Zhao et al. 2005a). Identication and subsequent manipulation of these transcription factors becomes very attractive for metabolic engineering of plant secondary metabolites (Gantet and Memelink 2002). The
idea has been validated for various plant secondary metabolite pathways (for review, see Gantet and Memelink 2002). Searching transcription factors that bind to the promoter region of the STR gene led to the identication of two important AP2/ERF family transcription factors ORCA2 and ORCA3, which are specically induced by jasmonate and thought to mediate jasmonate-induced STR and TDC expression and indole alkaloid production (van der Fits and Memelink 2000). Ectopic expression of ORCA3 in C. roseus cultured cells resulted in an increased expression of several indole alkaloid-biosynthetic genes, and an almost 3 fold increased indole alkaloid production upon feeding of loganin if compared with control (van der Fits and Memelink 2000). Because of the difculty to overexpress multiple biosynthetic genes at the same time and the facts that one transcription factor may regulate many functional-related secondary metabolism genes, metabolic engineering of secondary metabolism by manipulating transcription factors can be very efcient and successful. However, multiple targets and low specicity of transcription factors may also bring problems, e.g., if competitive pathways are induced as well. Heterologous construction of metabolic pathway Since microorganism cultures have been successfully scaled up for production of various pharmaceuticals, the overexpression of plant or mammalian pathways was attempted in microorganisms such as yeasts and bacteria. Geerlings et al. (1999, 2001) have made a pioneering effort in yeast by using C. roseus genes STR and SG (strictosidine glucosidase). Functional enzymes were expressed and found in the culture medium and the cells, respectively. Upon feeding of secologanin and tryptamine, yeast produced 2g/l of strictosidine in the medium, and after releasing SG enzyme by breaking the yeast cells strictosidine was converted into cathenamine by the action of SG. By feeding the juice of Symphoricarpus albus berries, which are rich in sugar and secologanin, strictosidine and cathenamine can be produced. Bacterially expressed STR from Rauvola serpentina can functionally synthesize strictosidine after
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immobilization to a matrix and adding precursors (Shen et al. 1998). Heterologous construction of a plant metabolic pathway in well-engineered microorganisms such as bacteria or yeasts certainly is a great idea, yet it can be a long-term project to clone and functionally express all metabolic genes and overcome metabolic trafcking problems. Recently a preliminary trial was carried out for reconstructing biosynthesis pathway of the highly valuable anticancer diterpenoid taxol in yeast cells by over-expression of 5 sequential pathway genes (Dejong et al. 2006). Eight single genes could be functionally expressed in yeast (Jennewein et al. 2005), however, when 5 of these genes were expressed in a single yeast cells, only metabolites from the rst two steps were detected (Dejong et al. 2006), suggesting enzyme or protein trafcking problems. These studies are important to identify such problems and eventually nd solutions to overcome them. A success was made recently on production of antimalarial sesquiterpene precursor artemisinic acid in yeast. The heterologously constructed metabolic pathway by expressing amorphadiene synthase and a cytochrome P450 monooxygenase from Artemisia annua in yeast generated up to 100 mg/l of artemisinic acid (Ro et al. 2006). This engineered yeast shows a potential for further yield optimization and industrial scale-up. Upscale of transgenic cell cultures Obviously volumetric productivity of some of these transgenic C. roseus cell lines overexpressing TDC or STR is very high and they may have great potential for being used in a larger scale bioreactor process to produce indole alkaloids. But they all need to be fed with high concentrations of tryptophan and loganin, which are also costly. Ratio of cost and effect for the production processes using these transgenic cell lines may be high in a fed-batch bioreactor. Actually the productivity of transgenic lines with feeding is still too low for a commercial process. Co-overexpression of TDC and STR in a C. roseus cell line may further improve the productivity of indole alkaloids. However, since insufcient precursor supply is the major problem in these transgenic cell lines, a strategy may be necessary to increase
these precursor pools by activating upstream genes or directly manipulating a key upstream gene in the biosynthesis pathway for terpenoid precursors. On the other hand, of the many biosynthetic genes involved in indole alkaloid biosynthesis, overexpression of only two or three genes may not be effective to promote overall productivity. From metabolomics point of view, there are many trade-offs at different levels among metabolic pathways such as direction and rate of metabolic uxes or size, location, and distribution of precursor pools to ensure normal cellular processes and physiological functions (Stephanopoulos 1999). Such metabolic balances are essential for plant cells; any imbalance in metabolic uxes could hamper growth or even be lethal (Stephanopoulos 1999; Manzano et al. 2004). The instability of high-alkaloid-yield cell lines obtained from selection or genetic manipulation may arise from an unbalanced metabolic uxes. High levels of indole alkaloids and/or feedback inhibition could initiate regulatory mechanisms minimizing the overuse of precursors by the alkaloid biosynthetic pathway and slowly come back to the balance of the whole metabolic network. However, here the biochemical engineering of the process may solve these problems, as for example feedback inhibition can be overcome in two-phase systems (see above). The limited achieves in metabolic engineering of indole alkaloid production reect our limited knowledge about metabolic pathways and their regulations. New technologies in genomics, proteomics, and metabolomics will lead to an indepth understanding of the biosynthesis and metabolic pathways for the indole alkaloid and the regulatory mechanisms (Verpoorte and Memelink 2002; Choi et al. 2004; Jacobs et al. 2005; Zhao et al. 2006; Rischer et al. 2006). Networks drawn on the basis of these gene-togene, protein-to-protein, protein-to-metabolite, and metabolite-to-metabolite collections will help to gain a whole view of indole alkaloid biosynthesis and metabolism, as well as their regulations. Perspectives In theory, the productivity of the plant cell factory for natural compounds should be unlimited.
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453 roseus (L) G Don and its conversion into serpentine. Planta 183:170177 Brodelius P, Pedersen H (1993) Increasing secondary metabolite production in plantcell culture by redirecting transport. Trends Biotechnol 11:3036 ` Brun G, Bessiere JM, Dijoux-Franca MG, David B, Mariotte AM (2001) Volatile components of Catharanthus roseus (L) G Don (Apocynaceae). Flavour Fragrance J 16:116119 Byun SY, Pedersen H (1994) Two-phase airlift fermentor operation with elicitation for the enhanced production of benzophenanthridine alkaloids in cell suspensions of Escherichia californica. Biotechnol Bioeng 44:14 20 Canel C, Lopez-Cardoso MI, Whitmer S, van der Fits L, Pasquali G, van der Heijden R, Hoge JHC, Verpoorte R (1998) Effects of over-expression of strictosidine synthase and tryptophan decarboxylase on alkaloid production by cell cultures of Catharanthus roseus. Planta 205:414419 Choi YH, Tapias EC, Kim HK, Lefeber AW, Erkelens C, Verhoeven JT, Brzin J, Zel J, Verpoorte R (2004) Metabolic discrimination of Catharanthus roseus leaves infected by phytoplasma using 1H-NMR spectroscopy and multivariate data analysis. Plant Physiol 135:23982410 Contin A, Collu G, van der Heijden R, Verpoorte R (1999) The effects of phenobarbital and ketoconazole on the alkaloid biosynthesis in Catharanthus roseus cell suspension cultures. Plant Physiol Biochem 37:139144 Courdavault V, Thiersault M, Courtois M, Gantet P, Oudin A, Doireau P, St-Pierre B, Giglioli-Guivarch N (2005) CaaX-prenyltransferases are essential for expression of genes involvedin the early stages of monoterpenoid biosynthetic pathway in Catharanthus roseus cells. Plant Mol Biol 57:855870 de Gunst MCM, Harkes PAA, Val J, Zwet WR, Libbenga KR (1990) Modeling the growth of a batch culture of plant cells: a corpuscular approach. Enzyme Microb Technol 12:6171 Dejong JM, Liu Y, Bollon AP, Long RM, Jennewein S, Williams D, Croteau RB (2006) Genetic engineering of taxol biosynthetic genes in Saccharomyces cerevisiae. Biotechnol Bioeng 93:212224 Diaz C, Dieu P, Feuillerat C, Lelong P, Salome M (1996) Simultaneous adaptive predictive control of the partial pressures of dissolved oxygen (pO2) and dissolved carbon dioxide (pCO2) in a laboratory-scale bioreactor. J Biotechnol 52:135150 Dong J, Zhong JJ (2002) Enhanced taxane productivity in bioreactor cultivation of Taxus chinensis cells by combining MeJA elicitation, sucrose feeding and ethylene incorporation. Enzyme Microb Technol 31:116121 Doran PM (1998) Application of membrane tubing aeration and peruocarbon to improve oxygen delivery to hairy root cultures. Biotechnol Prog 14:479486 Ducos JP, Pareilleux A (1986) Effect of aeration rate and inuence of carbon dioxide partial pressure in large-
Actually, the main limitation for commercial production of indole alkaloids by C. roseus cell cultures remains the low level of alkaloid production of the cells in the bioreactor. In the past years biochemical engineering approaches has resulted in optimized conditions for growth and production in the bioreactor. These experiences with the increased knowledge about the cell factory itself and perspectives of metabolic engineering eventually will lead to considerably higher productivity, but it will involve a combination of methods. Close collaboration between the biochemical engineers, metabolic engineers and cell biologists is required to reach this goal.
Acknowledgements We thank guest editor to give us the chance to express and exchange our ideas with this research community. Although there are many other excellent publications on bioreactor processing Catharanthus roseus cell culture and related aspects, we regret not be able to cite them due to space limitation.
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2.manipulating Production in Bio Reactor - Verpoorte

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Phytochem Rev (2007) 6:435457 DOI 10.

Phytochem Rev (2007) 6:435457

Phytochem Rev (2007) 6:435457

Phytochem Rev (2007) 6:435457

Phytochem Rev (2007) 6:435457

Phytochem Rev (2007) 6:435457

ajmalicine catharanthine vindoline ABC tra nsp orter alkaloids

strictosidine STR TFs

Tr p JA signalling signal t ra n s d u c t i o n [Ca2+]cyt spiking Elicitation

ajmalicine catharanthine vindoline

vindoline precursors t ran spo rter JA biosynthesis

Phytochem Rev (2007) 6:435457

Phytochem Rev (2007) 6:435457

Phytochem Rev (2007) 6:435457

Phytochem Rev (2007) 6:435457

Phytochem Rev (2007) 6:435457

Phytochem Rev (2007) 6:435457

Phytochem Rev (2007) 6:435457

Phytochem Rev (2007) 6:435457

Phytochem Rev (2007) 6:435457

Phytochem Rev (2007) 6:435457

Phytochem Rev (2007) 6:435457

Phytochem Rev (2007) 6:435457

Phytochem Rev (2007) 6:435457

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