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Enhanced catharanthine production in catharanthus roseus cell cultures by combined elicitor treatment in shake flasks and bioreactors
Enzyme and Microbial Technology 28 (2001) 673– 681
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Enhanced catharanthine production in Catharanthus roseus cell cultures by combined elicitor treatment in shake flasks and bioreactors Jian Zhao*,1 Wei-Hua Zhu, Qiu Hu Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 P.R. China Received 7 September 2000; received in revised form 8 January 2001; accepted 30 January 2001
Abstract Chemical and fungal elicitors were added to Catharanthus roseus cell suspension cultures so as to improve the production of indole alkaloids. A synergistic effect on alkaloid accumulation was observed in C. roseus cell cultures when treated with some combined elicitors of fungal preparations and chemicals. Among them, the combination of tetramethyl amminium bromide and Aspergillum niger mycelial homogenate gave the highest ajmalicine yield (63 mg l⫺1) and an improved catharanthine accumulation (17 mg l⫺1). The combined elicitors of malate and sodium alginate resulted in the highest catharanthine yield (26 mg l⫺1) and a high ajmalicine accumulation (41 mg l⫺1) in the cell cultures. Based on the synergistic effect of malate and sodium alginate, a process with enhanced catharanthine production in Catharanthus roseus cell cultures was developed in shake flasks and a bioreactor. After 10 days of culture, 25 mg l⫺1, 32 mg l⫺1 and 22 mg l⫺1 catharanthine yield were obtained in 500-ml flasks, 1000-ml flasks and in a 20-l airlift bioreactor, respectively. Upon malate-alginate combining treatments, peroxidase, catalase and superoxide dismutase activities decreased in elicited cells but phenylalanine ammonia lyase and lipoxygenase activities increased dramatically. That suggests a typical defense responses took place in the combined elicitors-treated cell cultures. Furthermore, the combined elicitors also caused a significant increase of malondialdehyde level in cell cultures, which suggests a serious lipid peroxidation occurred in the elicited cell cultures. Comparison of these results suggests that malate and alginate combining treatment also stimulates defense responses, such as lipid peroxidation, in all C. roseus culture processes and this may mediate the indole alkaloid production via jasmonate pathway. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Catharanthine; Catharanthus roseus; Cell culture; Combined elicitor; Bioreactor; Lipid peroxidation
1. Introduction Catharanthus roseus cell cultures have been studied extensively but the clinically important anti-cancer medicine, vinblastine, still cannot be produced by this technique [1]. Therefore, a semisynthesis method of chemically coupling vindoline with catharanthine to form vinblastine was successfully exploited [2]. This provides a novel and efficient way to commercially produce vinblastine since vindoline is abundant in C. roseus plants and catharanthine can reach high levels in C. roseus cell cultures [3]. Therefore, catharanthine production by plant cell cultures has been investigated for many years [3,4,5,6], including employment of fungal and chemical elicitors. Until now the production of * Corresponding author. Tel.: ⫹81-92-6422990; fax: ⫹81-926423078. E-mail address: [email protected] (J. Zhao). †Present address: Laboratory of Wood Chemistry, Department of Forest Products, Kyushu University, 812-8581 Fukuoka, Japan.
catharanthine in cell cultures was still too low to be industrialized, further study on the methodology and mechanism of the improvement of indole alkaloid production is therefore very necessary and of great importance. Selection of efficient elicitors and optimization of culture process are among the most important and practical ways to an improved performance since a high-alkaloid-yielding cell line may genetically unstable and difficult to maintain. We have screened various fungal and chemical elicitors for the improvement of indole alkaloid production in C. roseus cell cultures, and some effective elicitors have been found [5,7,8,9]. Currently we tried some combinations of chemicals and fungal elicitors in the C. roseus cell cultures in order to further improve alkaloid production and their utilizations at various scales. On the other hand, some studies on fungal elicitor-treated C. roseus cell cultures suggest that lipid peroxidation, octadecanoid pathway, and other elicitor signaling pathways may be involved in fungal elicitor-induced indole alkaloid production [7,10,11]. In order to develop a more profound understanding of alkaloid pro-
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duction in these elicitor treated cell cultures, we have also analyzed some physiological and biochemical changes during these culture processes.
2. Materials and methods 2.1. Plant cell culture and combined elicitor treatment Catharanthus roseus cell cultures CR6B and CR8C were established and maintained in our laboratory as described previously [8]. All cell cultures were maintained in MS medium [12] supplemented with ␣-naphthaleneacetic acid (NAA) 2 mg l⫺1, indole acetic acid (IAA) 2 mg l⫺1, kinetin 0.1 mg l⫺1 and sucrose 30 g l⫺1. The 500-ml Erlenmeyer flasks containing 200 ml cultures (including 10 g fresh cells) were incubated on a rotary shaker (130 rpm) at 23°C ⫾ 2°C in the dark. Subculture in the same scale was carried out weekly to maintain the cell cultures. Chemical agents (malate, tetramethyl ammonium bromide and NaCl) were dissolved in small volume of water (pH 5.6) and filtersterilized before addition. Mannitol and sodium alginate were prepared in the maintenance medium at high concentrations and autoclaved at 15 pound for 25 min. The Aspergillum niger mycelium homogenate was prepared as described previously [7]. For testing the combined elicitors, 8-day-old CR6B cells (50 g l⫺1) were mixed with various combined elicitors and incubated for 3 days of before harvested. The growth rate or index is calculated by using an equation: growth index (rate) ⫽ Dry weighttreatment (harvest)/ Dry weightcontrol (inoculum) ⫻ 100%. 2.2. Culture process in shake-flasks and bioreactor About 60 g l⫺1 fresh weight of CR8C cells were inoculated into 400 ml of the maintenance medium in 1000-ml Erlenmeyer flasks for biomass production. After one week, the cells were collected and inoculated using 60 g fresh weight l⫺1 of inoculum into either a 250-ml flask, 500-ml flask, 1000-ml flask or 20-l airlift bioreactor with working volumes of 100 ml, 200 ml, 300 ml and 16 l, respectively. The alkaloid production medium was MS medium supplemented with NAA 1 mg l⫺1, kinetin 0.1 mg l⫺1 and sucrose 50 g l⫺1. Flask culture conditions were the same as described above. 20-l airlift circle bioreactor (BIOSTART威 E, B. Braun, Germany) was operated as following culture conditions: 0.7 l min⫺1 of an air flow rate (0.05 vvm) at 22°C ⫾ 2°C in darkness. After 7 days all cultures were treated with combined elicitors of malate (40 mg l⫺1) and sodium alginate (1.5% w/v) for 3 days. The harvested cells were used for enzyme activity assay and alkaloid analysis. 2.3. Enzyme activity and lipid peroxidation assay Fresh cells were frozen and homogenized with extraction buffer in a motor on an ice bath with a pestle. The enzyme
extraction buffer was 0.05 M Na-phosphate buffer (pH 7.0) containing insoluble polyvinylpyrrolidone 1% w/v, sucrose 0.25 M, EDTA 5 mM, dithiothreitol 5 mM and MgCl2 5 mM. The homogenate was filtered through 4-layers of nylon cloth and the filtrate was centrifuged at 13,000 g for 15 min at 4°C. The supernatant was used for enzyme assays. Catalase activity was assayed by measuring adsorbance of H2O2 at 240 nm and peroxidase activity was assayed using guaiacol and H2O2 as substrates [13]. Superoxide dismutase activity was assayed according to the method of Giannoplitis and Ries [14]. Lipoxygenase activity was assayed using linoleic acid as the substrate [15]. Phenylalanine ammonia lyase (PAL) activity was assayed by measuring cinnamic acid adsorbance at 290 nm [16]. Protein was determined by the Bradford method with bovine serum albumin as standard. Malondialdehyde was extracted and measured by thiobarbituric acid reaction method [17]. 2.4. Alkaloid extraction and determination Alkaloid extraction and determination was carried out as previously described [18]. Dry cells powder was extracted twice with methanol, and the extraction solutions were combined and concentrated under vacuum. The residues dissolved in water were quickly extracted with ethyl acetate phase three times after the pH of aqueous solution was adjusted to 10. The alkaloids in the medium were quickly extracted three times with ethyl acetate after adjustment of the pH of the medium to 10. The ethyl acetate extractions were combined and concentrated on vacuum. The residues were dissolved in 1 ml of methanol and analyzed by HPLC. HPLC analysis conditions: Shimadzu LC-6A apparatus with Shimadzu C-R3A integrator and SPD-6A UV-detector monitoring at 280 nm; Nucleosil 5 C-18 column (250 ⫻ 4.6 mm, 5 m); Samples were eluted with a mobile phase of methanol/acetonitrile/0.025 M ammonium acetate/triethylamine (15:40:45:0.1 v/v) at a flow rate of 1 ml/min. Alkaloids were identified by TLC, co-elution and HPLC-Diode Array Detection (Hewlett-Packard 1100 LC-DAD HP) compared with standards.
3. Results 3.1. Effects of combined elicitors on alkaloid accumulation Our previous studies have demonstrated that employment of some chemicals and osmotic stresses could significantly improve indole alkaloid production in C. roseus cell cultures [5,8,9]. It is reported that some treatments of combined elicitors could result in synergistic improving effects on indole alkaloid production [19,20], we thus tested the effects of combined elicitors of those chemicals and fungal preparations on indole alkaloid production. As shown in Fig. 1, although single chemical treatment gave a high
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Fig. 1. Biomass and alkaloid production in C. roseus suspension cells (A) and culture medium (B) by various combined chemicals or fungal elicitor. Eight-day-old CR6B cell cultures were treated with combined fungal elicitor or chemicals, such as Aspergillum niger mycelium homogenate (Asp, 20 g carbohydrate equivalent ml⫺1), tetramethyl ammonium bromide (TeB, 100 mg l⫺1), malate (MA, 40 mg l⫺1), sodium alginate (Alg, 1.5% w/v) and mannitol (Man, 300 mM), sodium chloride (Na, 6 g l⫺1). Control received the same volume of acetate buffer or maintenance medium. Data were expressed as the mean of triplicate experiments ⫾ SD.
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alkaloid yield [5,8,9], after combined with some other chemicals or fungal elicitors, not all combined elicitors generated a synergistic effect on alkaloid production. For example, tetramethyl ammonium bromide (TeB) combined with NaCl only stimulated a less alkaloid production (26 mg l⫺1) than TeB treatment alone (70 mg l⫺1). The combination of Aspergillum niger elicitor (Asp) and malate gave less indole alkaloid accumulation (27 mg l⫺1) than single treatment alone (47 mg l⫺1 and 62 mg l⫺1, respectively). But Asp elicitor (giving 24 mg l⫺1 of ajmalicine) combined with TeB caused the greatest ajmalicine production (73 mg/l), which was about 4-fold higher than the control. Malate (giving 21 mg l⫺1 of catharanthine production) combined with sodium alginate (giving 22 mg l⫺1 of catharanthine production) also stimulated the highest catharanthine production (28 mg/l), about 6.5-fold greater than the control. These two combined elicitors synergistically promoted greater alkaloid production than each single elicitor. The reasons why these combined elicitors gave a diverse of effects on indole alkaloid production are still not known yet. Because each chemical or fungal elicitor stimulates indole alkaloid accumulation by different means, the mechanism for every combining treatment result may be more complicated since it depends on the interactions between physiological effects caused by two treatments. An improved ajmalicine production in a bioreactor process has been developed by using the combined elicitors of Asp and TeB [7]. 3.2. Flask and bioreactor processes for indole alkaloid production CR8C cell line consisted of yellowish and compact multicells aggregates cultures, which produce a relatively high level of catharanthine [8]. Like CR6B cells, CR8C cells were also sensitive to elicitor, and a significantly improved catharanthine and ajmalicine production was obtained in the CR8C cultures as treated with malate and sodium alginate combined in bolt flasks and bioreactor. Fig. 2 and Fig. 3 show that by day 7 only low levels of total alkaloids (about 17 mg l⫺1) were produced. But after addition of combined elicitors malate and alginate on day 7 a significant increase in total alkaloid production, about 2.5- to 3.5-fold higher than the control cultures, was observed. Such an improvement effect obtained in different scale cultures may suggest reproducible elicitation effect of the combined elicitors. As the flasks became larger an increase in alkaloid and biomass accumulation was observed after elicitation. As shown in Table 1, after 10 days of incubation, biomass yield of control cultures in 500-ml flask reached 14 g l⫺1, but the biomasses of combined elicitor-treated cultures in 250-ml, 500-ml and 1000-ml flasks were obviously less than that of the control because of the elicitation. 20-l airlift bioreactor process produced a less biomass and alkaloid production than 500-ml and 1000-ml flasks. Comparing the flasks and bioreactor process shows that 1000-ml flask process gave
the highest biomass and total alkaloid production, whereas 20-l bioreactor process produced the lowest biomass and all alkaloid production, which were almost similar to 250-ml flask process (Fig. 2 and Fig. 3). The low biomass accumulation in the bioreactor may be a result of cell damage caused by shear force of strong airflow because a browning color of the CR8C cells after transferring was observed in bioreactor process. Although using the same bioreactor under the identical operating conditions, CR8C cell cultures in the bioreactor were not damaged so seriously as CR6B cell cultures, which browned over a long time before the recovery of cell growth. CR8C cells seemed to be more resistant against the shear force caused by airflow, therefore, the color of cell cultures slightly browned after 1 days of transfer, and subsequently recovered to yellow. However, this may influence growth of the cell cultures. The kinetics of alkaloid accumulation in the bioreactor showed a lower alkaloid production than in 500 ml and 1000 ml flask process, even after elicitation. These results may be due to various differences between flask and bioreactor processes, such as gas regime, nutriment supply and shear force, which significantly affect biomass and secondary metabolite production. These points have been demonstrated extensively in non-elicited cell cultures [21,22,23]. But how about the gas regime in elicited cell cultures in flask or bioreactor processes remains unknown. 3.3. Alkaloid production in shake-flask and bioreactor The data presented in Table 1 and Table 2 show the final alkaloid production in all processes. The improved ajmalicine production in 250-ml, 500 ml, 1000 ml flasks and the 20-l bioreactor were about 2.6-, 3.1-, 3.5- and 2.2-fold greater than the control, respectively while catharanthine production reached 2.6-, 3.1-, 3.9- and 2.7-fold of the control, respectively. The major portions of these alkaloids (about 78% of the total ajmalicine and 81% of total catharanthine) were released into the medium. That could be of great convenience for the recovery of indole alkaloids from cell cultures. Unusually after transferring the cell culture to bioreactor, a significantly decreased alkaloid production is often observed; this may be due to improper culture conditions, such as nutrient supply, gas factor and other unknown reasons, in bioreactors [1,2,7,22,23]. Although in current study, bioreactor process also gave the decreased ajmalicine and catharanthine production as compared with flask processes, the volumetric productivity was significantly higher than the control. The accumulation rate for ajmalicine and catharanthine were 2.7 mg l⫺1 and 2.2 mg l⫺1 per day, respectively, which were relatively high. Therefore using the combined elicitor for short time culture may be a practical way for industrial alkaloid production since this strategy could greatly improve alkaloid production but shorten the culture period, and therefore reduce the process cost.
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Fig. 2. Time-course of biomass (A) and total alkaloids (B) accumulation in C. roseus cell cultures of in 250-ml flasks, 500-ml flasks, 1000-ml flasks and a 20-l airlift bioreactor using combined elicitor treatment. The arrow showed seven-day-old CR8C cell cultures were treated with a combined elicitor of malate (40 mg l⫺1) and sodium alginate (1.5% w/v) for 3 days. Control (in 500-ml flask) only received acetate buffer. Total alkaloids refer to sum of ajmalicine, serpentine and catharanthine recovered from the cells and the medium.
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Fig. 3. Ajmalicine (A) and catharanthine (B) production in C. roseus cell cultures in shake flasks and a bioreactor as a function of a combined elicitor of malate and sodium alginate. The arrow shows that seven-day-old CR8C cell cultures were treated with a combined elicitor of malate (40 mg l⫺1) and sodium alginate (1.5% w/v) for 3 days. Control (in 500-ml flask) only received acetate buffer.
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Table 1 Biomass and alkaloid production in C. roseus cells upon a combined elicitor treatment in various volumetric shake flasks and a 20-l airlift bioreactor Culture process
Harvest biomass (g l⫺1)
Control 250-ml flask 500-ml flask 1000-ml flask 20-l bioreactor
14.1 ⫾ 1.0 9.6 ⫾ 0.9 10.3 ⫾ 1.2 11.6 ⫾ 1.0 8.5 ⫾ 0.8
Tryptamine and alkaloid accumulation in cells (mg g⫺1 dry cells) Tryptamine
Serpentine
Ajmalicine
Catharanthine
0.34 ⫾ 0.03 0.99 ⫾ 0.05 1.19 ⫾ 0.09 1.05 ⫾ 0.08 1.89 ⫾ 0.13
0.11 ⫾ 0.01 0.21 ⫾ 0.02 0.24 ⫾ 0.03 0.28 ⫾ 0.03 0.19 ⫾ 0.01
0.30 ⫾ 0.02 0.68 ⫾ 0.05 0.82 ⫾ 0.08 0.84 ⫾ 0.05 0.75 ⫾ 0.05
0.19 ⫾ 0.01 0.43 ⫾ 0.03 0.51 ⫾ 0.04 0.57 ⫾ 0.04 0.36 ⫾ 0.03
Seven-day-old CR8C cell cultures were treated with a combined elicitor of malate (40 mg⫺1) and sodium alginate (1.5% w/v) for 3 days. All data were expressed as the mean of triplicate experiments ⫾ SD. Control culture (in 500-ml flask) received acetate buffer only.
3.4. Changes of tryptamine content and lipid peroxidation Like the ajmalicine production process with fungal elicitor and tetramethyl ammonium bromide combined treatment, a higher tryptamine level was observed in the alginate and malate treated cell cultures, particularly those grown in the bioreactor (Table 1). That result suggests that tryptamine level may be an indicator of stress in C. roseus cell cultures. Previous studies demonstrated that ungelled alginate could stimulate plant cells to generate some defense-like responses, such as culture browning, increased alkaloid levels [8,24] and an increase in defense enzyme activity, including chitinase, catalase and 5⬘-phosphodiesterase [25]. Therefore, we assayed the activities of some defense enzyme and oxygen-radical-scavenging enzymes, as well as lipid peroxidation to further study changes in these processes. In all alginate and malate combination treated cell cultures, PAL activities were increased, at least 3-fold higher than the control, which was similar to fungal elicitor treated CR6B cell cultures (Table 3). Peroxidase, superoxide dismutase and catalase activities in elicited cells obviously decreased, especially, catalase activities of all alginatemalate treated cells in 250-ml, 500-ml flasks and 20-l bioreactor processes were approximately 30% of the control. The phenomena were contrary to those in elicited-CR6B cell cultures in the similar courses [7], and this may be due to the release of some enzymes to the medium. However, similar to the previously reported [7], lipoxygenase activity and malondialdehyde level significantly increased upon the alginate and malate treatment (Table 3). About 3-fold higher
of lipoxygenase activity and 8-fold higher of malondialdehyde level than the control were obtained in alginate and malate treated cells in flask and bioreactor processes. These results suggested that in combined elicitor-treated cell cultures lipid peroxidation or membrane damage occurred. This may cause an increasing permeability of the plasma membrane, through which some enzymes or alkaloids were released to the medium. More importantly, the lipoxygenase-mediated lipid peroxidation not only overproduces malondialdehyde, but also generates jasmonate, which can activate some key genes involved in indole alkaloid biosynthesis [10,26]. This perhaps was one of acting mechanisms for the improving effects of the combined elicitors of sodium alginate and malate. This is also in agreement with a previous conclusion [7].
4. Discussion There always exists a possibility in theory to synergistically improve the secondary metabolite production by treatment of the cell cultures with some combined elicitors, if these elicitors could enhance the stimulating effect each other by related mechanism(s). However, due to the complicated mechanisms for these treatment effects of combined elicitors, the optimal effects on target compound production may not be archived just by simply adding of each single elicitor, even these single treatment were in their optimal doses, treatment time or other conditions. For example, in malate and alginate combination treatment, al-
Table 2 Biomass and alkaloid production in C. roseus cells upon the combined elicitors treatment in various volumetric shake flasks and a 20-l airlift bioreactor Culture process
pH value of medium
Control 250-ml flask 500-ml flask 1000-ml flask 20-l bioreactor
6.8 ⫾ 0.3 6.1 ⫾ 0.4 6.4 ⫾ 0.3 6.1 ⫾ 0.2 5.8 ⫾ 0.4
Alkaloid production in medium (mg l⫺1) Serpentine
Ajmalicine
Catharanthine
2.9 ⫾ 0.02 3.8 ⫾ 0.04 4.2 ⫾ 0.03 4.4 ⫾ 0.03 5.7 ⫾ 0.05
8.56 ⫾ 0.06 25.9 ⫾ 2.1 31.8 ⫾ 3.3 35.0 ⫾ 3.5 21.5 ⫾ 2.1
5.5 ⫾ 0.04 16.9 ⫾ 1.2 20.1 ⫾ 1.8 25.2 ⫾ 2.1 19.4 ⫾ 1.5
Seven-day-old CR8C cell cultures were treated with a combined elicitor of malate (40 mg l⫺1) and sodium alginate (1.5% w/v) and harvested after 3 days of treatment. Control (in 500-ml flask) received acetate buffer only. All data were expressed as the mean of triplicate experiments ⫾ SD.
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Table 3 Changes in enzyme activities and malondialdehyde content of C. roseus cell cultures in various volumetric shake flasks and a 20-l airlift bioreactor processes Enzymes tested
Control
250-mlflask
500-ml flask
1000-ml flask
20-l bioreactor
phenylalanine ammonia lyase (Units mg⫺1 protein) Peroxidase (⌬OD470 mg⫺1 protein min⫺1) Superoxide dismutase (Units mg⫺1 protein) Catalase (mol H2O2 mg⫺1 protein min⫺1) Lipoxygenase (OD234 mg⫺1 protein min⫺1) Malondialdehyde (nmol g⫺1 fresh cells)
63 ⫾ 5.4 44 ⫾ 3.8 78 ⫾ 5.9 6.5 ⫾ 0.6 6 ⫾ 0.5 14 ⫾ 1.2
198 ⫾ 18 34 ⫾ 4.1 53 ⫾ 5.8 2.1 ⫾ 0.1 17 ⫾ 1.2 109 ⫾ 9.5
219 ⫾ 21 38 ⫾ 3.2 62 ⫾ 6.0 2.3 ⫾ 0.2 17 ⫾ 1.7 99 ⫾ 8.4
210 ⫾ 18 35 ⫾ 3.5 62 ⫾ 5.8 4.3 ⫾ 0.3 14 ⫾ 1.2 117 ⫾ 10.2
200 ⫾ 21 42 ⫾ 4.0 42 ⫾ 3.2 1.7 ⫾ 0.1 19 ⫾ 1.5 157 ⫾ 12.8
Seven-day-old CR8C cell cultures were treated by a combined elicitor of malate (40 mg l⫺1) and sodium alginate (1.5% w/v) for 3 days. Control (in 500-ml flask) received acetate buffer. All data were expressed as the mean of triplicate experiments ⫾ SD. Enzyme activity units: one unit activity of phenylalanine ammonia lyase was defined as 0.01 of changes in ⌬OD290 per hour; one enzyme unit of superoxide dismutase was defined as the enzyme amount for 50% of inhibition on nitroblue tetrazolium reduction per min.
though both the malate and alginate treatments were in their own optimal dose and addition time, after combination, they may not stimulate the maximum indole alkaloid production because of their interactions. Therefore, optimizing the combined elicitor treatment is still needed by using proper method. In spite of this, our current study, together with previous results [7], suggests that a bioreactor process combined with elicitation may be a practical means for indole alkaloid production although the performance of the model system still need to be improved. Recently it is reported that an acetate/mevalonate pathway and triose phosphate/pyruvate pathway may be involved in indole alkaloid biosynthetic pathway in C. roseus cell cultures [27]. Exogenous malate may inhibit tricarboxylic acid cycle by a negative feedback control, or exogenous malate can be transformed to pyruvate by a NADP⫹-malate enzyme. Therefore, addition of malate may stimulate indole alkaloid accumulation by increasing pyruvate pool or directing acetyl-CoA and pyruvate flux to monoterpenoid indole alkaloid biosynthesis pathway. Furthermore, additions of malate and alginate will certainly change the gas regime in the treated-cell cultures. Although the roles of the dissolved oxygen, CO2 and ethylene concentrations on alkaloid accumulation of elicited or non-elicited C. roseus cell cultures in flasks and bioreactor processes were not analyzed here because of the limited conditions, we could think that there are some differences in the gas regime between different culture systems, such as between the normal and malate-alginate treated cell cultures. Since it is suggested that the ungelled alginate causes a high viscosity, low agitation efficiency and low dissolved oxygen concentration but an significantly increased alkaloid accumulation in cell cultures [28]. Therefore, certain levels of dissolved oxygen needed by non-elicited cell cultures for alkaloid accumulation may not be critically needed by the elicited cell cultures for alkaloid accumulation, especially in current culture processes. The elicited cells may generate more ethylene but less CO2 than the non-elicited cells, and the situations in non-elicited cell cultures were exactly contrary to that. If really so, using the combined elicitor, increasing
cell density and optimizing the elicitor treatment and other culture conditions will further improve bioreactor performance.
Acknowledgments This investigation was supported by A Doctoral Program from Chinese Ministry of Education and funds from Chinese Academy of Medical Sciences and Peking Union Medical College. We would like to thank Dr. Frank DiCosmo (Toronto University, Canada) for kind gifts of vindoline, catharanthine and serpentine standards.
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[19] Kargi F, Ganapathi B. Effects of precursor stimulating agents on formation of indole alkaloids by C. roseus (periwinkle) cells. Enzyme Microb Technol 1991;13:643–7. [20] Godoy-Hernandez G, Loyola-Vargas VM. Effect of fungal homogenetes, enzyme inhibitors and osmotic stress on alkaloid content of Catharanthus roseus cell suspension cultures. Plant Cell Rep 1991; 10:537– 40. [21] Lee CWT, Shuler ML. Different flask closures alter gas phase composition and ajmalicine production in Catharanthus roseus cell suspensions. Biotechnol Tech 1991;5:173– 8. [22] Schiel O, Berlin J. Large scale fermentation and alkaloid production of cell suspension cultures of Catharanthus roseus. Plant Cell Tiss Org Cult 1987;8:153– 62. [23] Kargi F, Rosenbergm MZ. Plant cell bioreactors: present status and future trends. Biotechnol Prog 1987;3:1– 8. [24] Villegas M, Leon R, Brodelius PE. Effects of alginate and immobilization by entrapment in alginate on benzophenanthridine alkaloid production in cell suspension cultures of Eschscholtzia californica. Biotechnol Lett 1999;21:49 –55. [25] Aoyagi H, Yasuhira J, Tanaka H. Alginate promotes production of various enzymes by Catharanthus roseus cells. Plant Cell Rep 1998; 17:243–7. [26] Menke FL, Champion A, Kijne JW, Memelink J. A novel jasmonateand elicitor-responsive element in the periwinkle secondary metabolite biosynthetic gene Str interacts with a jasmonate- and elicitorinducible AP2-domain transcription factor, ORCA2. EMBO J 1999; 18:4455– 63. [27] Contin A, van der Heijden R, Lefeber AWM, Verpoorte R. The iridoid glucoside secologanin in derived from the novel triose phosphate/pyruvate pathway in a Catharanthus roseus cell culture. FEBS Lett 1998;434:413– 6. [28] Gontier E, Sangwan BS, Barbotin JN. Effects of calcium, alginate, and calcium alginate immobilization on growth and tropane alkaloid levels of a stable suspension cell line of Datura innoxia Mill. Plant Cell Rep 1994;13:533– 6.
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Enhanced catharanthine production in Catharanthus roseus cell cultures by combined elicitor treatment in shake flasks and bioreactors Jian Zhao*,1 Wei-Hua Zhu, Qiu Hu Institute of Materia Medica, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing, 100050 P.R. China Received 7 September 2000; received in revised form 8 January 2001; accepted 30 January 2001
Abstract Chemical and fungal elicitors were added to Catharanthus roseus cell suspension cultures so as to improve the production of indole alkaloids. A synergistic effect on alkaloid accumulation was observed in C. roseus cell cultures when treated with some combined elicitors of fungal preparations and chemicals. Among them, the combination of tetramethyl amminium bromide and Aspergillum niger mycelial homogenate gave the highest ajmalicine yield (63 mg l⫺1) and an improved catharanthine accumulation (17 mg l⫺1). The combined elicitors of malate and sodium alginate resulted in the highest catharanthine yield (26 mg l⫺1) and a high ajmalicine accumulation (41 mg l⫺1) in the cell cultures. Based on the synergistic effect of malate and sodium alginate, a process with enhanced catharanthine production in Catharanthus roseus cell cultures was developed in shake flasks and a bioreactor. After 10 days of culture, 25 mg l⫺1, 32 mg l⫺1 and 22 mg l⫺1 catharanthine yield were obtained in 500-ml flasks, 1000-ml flasks and in a 20-l airlift bioreactor, respectively. Upon malate-alginate combining treatments, peroxidase, catalase and superoxide dismutase activities decreased in elicited cells but phenylalanine ammonia lyase and lipoxygenase activities increased dramatically. That suggests a typical defense responses took place in the combined elicitors-treated cell cultures. Furthermore, the combined elicitors also caused a significant increase of malondialdehyde level in cell cultures, which suggests a serious lipid peroxidation occurred in the elicited cell cultures. Comparison of these results suggests that malate and alginate combining treatment also stimulates defense responses, such as lipid peroxidation, in all C. roseus culture processes and this may mediate the indole alkaloid production via jasmonate pathway. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Catharanthine; Catharanthus roseus; Cell culture; Combined elicitor; Bioreactor; Lipid peroxidation
1. Introduction Catharanthus roseus cell cultures have been studied extensively but the clinically important anti-cancer medicine, vinblastine, still cannot be produced by this technique [1]. Therefore, a semisynthesis method of chemically coupling vindoline with catharanthine to form vinblastine was successfully exploited [2]. This provides a novel and efficient way to commercially produce vinblastine since vindoline is abundant in C. roseus plants and catharanthine can reach high levels in C. roseus cell cultures [3]. Therefore, catharanthine production by plant cell cultures has been investigated for many years [3,4,5,6], including employment of fungal and chemical elicitors. Until now the production of * Corresponding author. Tel.: ⫹81-92-6422990; fax: ⫹81-926423078. E-mail address: [email protected] (J. Zhao). †Present address: Laboratory of Wood Chemistry, Department of Forest Products, Kyushu University, 812-8581 Fukuoka, Japan.
catharanthine in cell cultures was still too low to be industrialized, further study on the methodology and mechanism of the improvement of indole alkaloid production is therefore very necessary and of great importance. Selection of efficient elicitors and optimization of culture process are among the most important and practical ways to an improved performance since a high-alkaloid-yielding cell line may genetically unstable and difficult to maintain. We have screened various fungal and chemical elicitors for the improvement of indole alkaloid production in C. roseus cell cultures, and some effective elicitors have been found [5,7,8,9]. Currently we tried some combinations of chemicals and fungal elicitors in the C. roseus cell cultures in order to further improve alkaloid production and their utilizations at various scales. On the other hand, some studies on fungal elicitor-treated C. roseus cell cultures suggest that lipid peroxidation, octadecanoid pathway, and other elicitor signaling pathways may be involved in fungal elicitor-induced indole alkaloid production [7,10,11]. In order to develop a more profound understanding of alkaloid pro-
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duction in these elicitor treated cell cultures, we have also analyzed some physiological and biochemical changes during these culture processes.
2. Materials and methods 2.1. Plant cell culture and combined elicitor treatment Catharanthus roseus cell cultures CR6B and CR8C were established and maintained in our laboratory as described previously [8]. All cell cultures were maintained in MS medium [12] supplemented with ␣-naphthaleneacetic acid (NAA) 2 mg l⫺1, indole acetic acid (IAA) 2 mg l⫺1, kinetin 0.1 mg l⫺1 and sucrose 30 g l⫺1. The 500-ml Erlenmeyer flasks containing 200 ml cultures (including 10 g fresh cells) were incubated on a rotary shaker (130 rpm) at 23°C ⫾ 2°C in the dark. Subculture in the same scale was carried out weekly to maintain the cell cultures. Chemical agents (malate, tetramethyl ammonium bromide and NaCl) were dissolved in small volume of water (pH 5.6) and filtersterilized before addition. Mannitol and sodium alginate were prepared in the maintenance medium at high concentrations and autoclaved at 15 pound for 25 min. The Aspergillum niger mycelium homogenate was prepared as described previously [7]. For testing the combined elicitors, 8-day-old CR6B cells (50 g l⫺1) were mixed with various combined elicitors and incubated for 3 days of before harvested. The growth rate or index is calculated by using an equation: growth index (rate) ⫽ Dry weighttreatment (harvest)/ Dry weightcontrol (inoculum) ⫻ 100%. 2.2. Culture process in shake-flasks and bioreactor About 60 g l⫺1 fresh weight of CR8C cells were inoculated into 400 ml of the maintenance medium in 1000-ml Erlenmeyer flasks for biomass production. After one week, the cells were collected and inoculated using 60 g fresh weight l⫺1 of inoculum into either a 250-ml flask, 500-ml flask, 1000-ml flask or 20-l airlift bioreactor with working volumes of 100 ml, 200 ml, 300 ml and 16 l, respectively. The alkaloid production medium was MS medium supplemented with NAA 1 mg l⫺1, kinetin 0.1 mg l⫺1 and sucrose 50 g l⫺1. Flask culture conditions were the same as described above. 20-l airlift circle bioreactor (BIOSTART威 E, B. Braun, Germany) was operated as following culture conditions: 0.7 l min⫺1 of an air flow rate (0.05 vvm) at 22°C ⫾ 2°C in darkness. After 7 days all cultures were treated with combined elicitors of malate (40 mg l⫺1) and sodium alginate (1.5% w/v) for 3 days. The harvested cells were used for enzyme activity assay and alkaloid analysis. 2.3. Enzyme activity and lipid peroxidation assay Fresh cells were frozen and homogenized with extraction buffer in a motor on an ice bath with a pestle. The enzyme
extraction buffer was 0.05 M Na-phosphate buffer (pH 7.0) containing insoluble polyvinylpyrrolidone 1% w/v, sucrose 0.25 M, EDTA 5 mM, dithiothreitol 5 mM and MgCl2 5 mM. The homogenate was filtered through 4-layers of nylon cloth and the filtrate was centrifuged at 13,000 g for 15 min at 4°C. The supernatant was used for enzyme assays. Catalase activity was assayed by measuring adsorbance of H2O2 at 240 nm and peroxidase activity was assayed using guaiacol and H2O2 as substrates [13]. Superoxide dismutase activity was assayed according to the method of Giannoplitis and Ries [14]. Lipoxygenase activity was assayed using linoleic acid as the substrate [15]. Phenylalanine ammonia lyase (PAL) activity was assayed by measuring cinnamic acid adsorbance at 290 nm [16]. Protein was determined by the Bradford method with bovine serum albumin as standard. Malondialdehyde was extracted and measured by thiobarbituric acid reaction method [17]. 2.4. Alkaloid extraction and determination Alkaloid extraction and determination was carried out as previously described [18]. Dry cells powder was extracted twice with methanol, and the extraction solutions were combined and concentrated under vacuum. The residues dissolved in water were quickly extracted with ethyl acetate phase three times after the pH of aqueous solution was adjusted to 10. The alkaloids in the medium were quickly extracted three times with ethyl acetate after adjustment of the pH of the medium to 10. The ethyl acetate extractions were combined and concentrated on vacuum. The residues were dissolved in 1 ml of methanol and analyzed by HPLC. HPLC analysis conditions: Shimadzu LC-6A apparatus with Shimadzu C-R3A integrator and SPD-6A UV-detector monitoring at 280 nm; Nucleosil 5 C-18 column (250 ⫻ 4.6 mm, 5 m); Samples were eluted with a mobile phase of methanol/acetonitrile/0.025 M ammonium acetate/triethylamine (15:40:45:0.1 v/v) at a flow rate of 1 ml/min. Alkaloids were identified by TLC, co-elution and HPLC-Diode Array Detection (Hewlett-Packard 1100 LC-DAD HP) compared with standards.
3. Results 3.1. Effects of combined elicitors on alkaloid accumulation Our previous studies have demonstrated that employment of some chemicals and osmotic stresses could significantly improve indole alkaloid production in C. roseus cell cultures [5,8,9]. It is reported that some treatments of combined elicitors could result in synergistic improving effects on indole alkaloid production [19,20], we thus tested the effects of combined elicitors of those chemicals and fungal preparations on indole alkaloid production. As shown in Fig. 1, although single chemical treatment gave a high
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Fig. 1. Biomass and alkaloid production in C. roseus suspension cells (A) and culture medium (B) by various combined chemicals or fungal elicitor. Eight-day-old CR6B cell cultures were treated with combined fungal elicitor or chemicals, such as Aspergillum niger mycelium homogenate (Asp, 20 g carbohydrate equivalent ml⫺1), tetramethyl ammonium bromide (TeB, 100 mg l⫺1), malate (MA, 40 mg l⫺1), sodium alginate (Alg, 1.5% w/v) and mannitol (Man, 300 mM), sodium chloride (Na, 6 g l⫺1). Control received the same volume of acetate buffer or maintenance medium. Data were expressed as the mean of triplicate experiments ⫾ SD.
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alkaloid yield [5,8,9], after combined with some other chemicals or fungal elicitors, not all combined elicitors generated a synergistic effect on alkaloid production. For example, tetramethyl ammonium bromide (TeB) combined with NaCl only stimulated a less alkaloid production (26 mg l⫺1) than TeB treatment alone (70 mg l⫺1). The combination of Aspergillum niger elicitor (Asp) and malate gave less indole alkaloid accumulation (27 mg l⫺1) than single treatment alone (47 mg l⫺1 and 62 mg l⫺1, respectively). But Asp elicitor (giving 24 mg l⫺1 of ajmalicine) combined with TeB caused the greatest ajmalicine production (73 mg/l), which was about 4-fold higher than the control. Malate (giving 21 mg l⫺1 of catharanthine production) combined with sodium alginate (giving 22 mg l⫺1 of catharanthine production) also stimulated the highest catharanthine production (28 mg/l), about 6.5-fold greater than the control. These two combined elicitors synergistically promoted greater alkaloid production than each single elicitor. The reasons why these combined elicitors gave a diverse of effects on indole alkaloid production are still not known yet. Because each chemical or fungal elicitor stimulates indole alkaloid accumulation by different means, the mechanism for every combining treatment result may be more complicated since it depends on the interactions between physiological effects caused by two treatments. An improved ajmalicine production in a bioreactor process has been developed by using the combined elicitors of Asp and TeB [7]. 3.2. Flask and bioreactor processes for indole alkaloid production CR8C cell line consisted of yellowish and compact multicells aggregates cultures, which produce a relatively high level of catharanthine [8]. Like CR6B cells, CR8C cells were also sensitive to elicitor, and a significantly improved catharanthine and ajmalicine production was obtained in the CR8C cultures as treated with malate and sodium alginate combined in bolt flasks and bioreactor. Fig. 2 and Fig. 3 show that by day 7 only low levels of total alkaloids (about 17 mg l⫺1) were produced. But after addition of combined elicitors malate and alginate on day 7 a significant increase in total alkaloid production, about 2.5- to 3.5-fold higher than the control cultures, was observed. Such an improvement effect obtained in different scale cultures may suggest reproducible elicitation effect of the combined elicitors. As the flasks became larger an increase in alkaloid and biomass accumulation was observed after elicitation. As shown in Table 1, after 10 days of incubation, biomass yield of control cultures in 500-ml flask reached 14 g l⫺1, but the biomasses of combined elicitor-treated cultures in 250-ml, 500-ml and 1000-ml flasks were obviously less than that of the control because of the elicitation. 20-l airlift bioreactor process produced a less biomass and alkaloid production than 500-ml and 1000-ml flasks. Comparing the flasks and bioreactor process shows that 1000-ml flask process gave
the highest biomass and total alkaloid production, whereas 20-l bioreactor process produced the lowest biomass and all alkaloid production, which were almost similar to 250-ml flask process (Fig. 2 and Fig. 3). The low biomass accumulation in the bioreactor may be a result of cell damage caused by shear force of strong airflow because a browning color of the CR8C cells after transferring was observed in bioreactor process. Although using the same bioreactor under the identical operating conditions, CR8C cell cultures in the bioreactor were not damaged so seriously as CR6B cell cultures, which browned over a long time before the recovery of cell growth. CR8C cells seemed to be more resistant against the shear force caused by airflow, therefore, the color of cell cultures slightly browned after 1 days of transfer, and subsequently recovered to yellow. However, this may influence growth of the cell cultures. The kinetics of alkaloid accumulation in the bioreactor showed a lower alkaloid production than in 500 ml and 1000 ml flask process, even after elicitation. These results may be due to various differences between flask and bioreactor processes, such as gas regime, nutriment supply and shear force, which significantly affect biomass and secondary metabolite production. These points have been demonstrated extensively in non-elicited cell cultures [21,22,23]. But how about the gas regime in elicited cell cultures in flask or bioreactor processes remains unknown. 3.3. Alkaloid production in shake-flask and bioreactor The data presented in Table 1 and Table 2 show the final alkaloid production in all processes. The improved ajmalicine production in 250-ml, 500 ml, 1000 ml flasks and the 20-l bioreactor were about 2.6-, 3.1-, 3.5- and 2.2-fold greater than the control, respectively while catharanthine production reached 2.6-, 3.1-, 3.9- and 2.7-fold of the control, respectively. The major portions of these alkaloids (about 78% of the total ajmalicine and 81% of total catharanthine) were released into the medium. That could be of great convenience for the recovery of indole alkaloids from cell cultures. Unusually after transferring the cell culture to bioreactor, a significantly decreased alkaloid production is often observed; this may be due to improper culture conditions, such as nutrient supply, gas factor and other unknown reasons, in bioreactors [1,2,7,22,23]. Although in current study, bioreactor process also gave the decreased ajmalicine and catharanthine production as compared with flask processes, the volumetric productivity was significantly higher than the control. The accumulation rate for ajmalicine and catharanthine were 2.7 mg l⫺1 and 2.2 mg l⫺1 per day, respectively, which were relatively high. Therefore using the combined elicitor for short time culture may be a practical way for industrial alkaloid production since this strategy could greatly improve alkaloid production but shorten the culture period, and therefore reduce the process cost.
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Fig. 2. Time-course of biomass (A) and total alkaloids (B) accumulation in C. roseus cell cultures of in 250-ml flasks, 500-ml flasks, 1000-ml flasks and a 20-l airlift bioreactor using combined elicitor treatment. The arrow showed seven-day-old CR8C cell cultures were treated with a combined elicitor of malate (40 mg l⫺1) and sodium alginate (1.5% w/v) for 3 days. Control (in 500-ml flask) only received acetate buffer. Total alkaloids refer to sum of ajmalicine, serpentine and catharanthine recovered from the cells and the medium.
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Fig. 3. Ajmalicine (A) and catharanthine (B) production in C. roseus cell cultures in shake flasks and a bioreactor as a function of a combined elicitor of malate and sodium alginate. The arrow shows that seven-day-old CR8C cell cultures were treated with a combined elicitor of malate (40 mg l⫺1) and sodium alginate (1.5% w/v) for 3 days. Control (in 500-ml flask) only received acetate buffer.
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Table 1 Biomass and alkaloid production in C. roseus cells upon a combined elicitor treatment in various volumetric shake flasks and a 20-l airlift bioreactor Culture process
Harvest biomass (g l⫺1)
Control 250-ml flask 500-ml flask 1000-ml flask 20-l bioreactor
14.1 ⫾ 1.0 9.6 ⫾ 0.9 10.3 ⫾ 1.2 11.6 ⫾ 1.0 8.5 ⫾ 0.8
Tryptamine and alkaloid accumulation in cells (mg g⫺1 dry cells) Tryptamine
Serpentine
Ajmalicine
Catharanthine
0.34 ⫾ 0.03 0.99 ⫾ 0.05 1.19 ⫾ 0.09 1.05 ⫾ 0.08 1.89 ⫾ 0.13
0.11 ⫾ 0.01 0.21 ⫾ 0.02 0.24 ⫾ 0.03 0.28 ⫾ 0.03 0.19 ⫾ 0.01
0.30 ⫾ 0.02 0.68 ⫾ 0.05 0.82 ⫾ 0.08 0.84 ⫾ 0.05 0.75 ⫾ 0.05
0.19 ⫾ 0.01 0.43 ⫾ 0.03 0.51 ⫾ 0.04 0.57 ⫾ 0.04 0.36 ⫾ 0.03
Seven-day-old CR8C cell cultures were treated with a combined elicitor of malate (40 mg⫺1) and sodium alginate (1.5% w/v) for 3 days. All data were expressed as the mean of triplicate experiments ⫾ SD. Control culture (in 500-ml flask) received acetate buffer only.
3.4. Changes of tryptamine content and lipid peroxidation Like the ajmalicine production process with fungal elicitor and tetramethyl ammonium bromide combined treatment, a higher tryptamine level was observed in the alginate and malate treated cell cultures, particularly those grown in the bioreactor (Table 1). That result suggests that tryptamine level may be an indicator of stress in C. roseus cell cultures. Previous studies demonstrated that ungelled alginate could stimulate plant cells to generate some defense-like responses, such as culture browning, increased alkaloid levels [8,24] and an increase in defense enzyme activity, including chitinase, catalase and 5⬘-phosphodiesterase [25]. Therefore, we assayed the activities of some defense enzyme and oxygen-radical-scavenging enzymes, as well as lipid peroxidation to further study changes in these processes. In all alginate and malate combination treated cell cultures, PAL activities were increased, at least 3-fold higher than the control, which was similar to fungal elicitor treated CR6B cell cultures (Table 3). Peroxidase, superoxide dismutase and catalase activities in elicited cells obviously decreased, especially, catalase activities of all alginatemalate treated cells in 250-ml, 500-ml flasks and 20-l bioreactor processes were approximately 30% of the control. The phenomena were contrary to those in elicited-CR6B cell cultures in the similar courses [7], and this may be due to the release of some enzymes to the medium. However, similar to the previously reported [7], lipoxygenase activity and malondialdehyde level significantly increased upon the alginate and malate treatment (Table 3). About 3-fold higher
of lipoxygenase activity and 8-fold higher of malondialdehyde level than the control were obtained in alginate and malate treated cells in flask and bioreactor processes. These results suggested that in combined elicitor-treated cell cultures lipid peroxidation or membrane damage occurred. This may cause an increasing permeability of the plasma membrane, through which some enzymes or alkaloids were released to the medium. More importantly, the lipoxygenase-mediated lipid peroxidation not only overproduces malondialdehyde, but also generates jasmonate, which can activate some key genes involved in indole alkaloid biosynthesis [10,26]. This perhaps was one of acting mechanisms for the improving effects of the combined elicitors of sodium alginate and malate. This is also in agreement with a previous conclusion [7].
4. Discussion There always exists a possibility in theory to synergistically improve the secondary metabolite production by treatment of the cell cultures with some combined elicitors, if these elicitors could enhance the stimulating effect each other by related mechanism(s). However, due to the complicated mechanisms for these treatment effects of combined elicitors, the optimal effects on target compound production may not be archived just by simply adding of each single elicitor, even these single treatment were in their optimal doses, treatment time or other conditions. For example, in malate and alginate combination treatment, al-
Table 2 Biomass and alkaloid production in C. roseus cells upon the combined elicitors treatment in various volumetric shake flasks and a 20-l airlift bioreactor Culture process
pH value of medium
Control 250-ml flask 500-ml flask 1000-ml flask 20-l bioreactor
6.8 ⫾ 0.3 6.1 ⫾ 0.4 6.4 ⫾ 0.3 6.1 ⫾ 0.2 5.8 ⫾ 0.4
Alkaloid production in medium (mg l⫺1) Serpentine
Ajmalicine
Catharanthine
2.9 ⫾ 0.02 3.8 ⫾ 0.04 4.2 ⫾ 0.03 4.4 ⫾ 0.03 5.7 ⫾ 0.05
8.56 ⫾ 0.06 25.9 ⫾ 2.1 31.8 ⫾ 3.3 35.0 ⫾ 3.5 21.5 ⫾ 2.1
5.5 ⫾ 0.04 16.9 ⫾ 1.2 20.1 ⫾ 1.8 25.2 ⫾ 2.1 19.4 ⫾ 1.5
Seven-day-old CR8C cell cultures were treated with a combined elicitor of malate (40 mg l⫺1) and sodium alginate (1.5% w/v) and harvested after 3 days of treatment. Control (in 500-ml flask) received acetate buffer only. All data were expressed as the mean of triplicate experiments ⫾ SD.
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Table 3 Changes in enzyme activities and malondialdehyde content of C. roseus cell cultures in various volumetric shake flasks and a 20-l airlift bioreactor processes Enzymes tested
Control
250-mlflask
500-ml flask
1000-ml flask
20-l bioreactor
phenylalanine ammonia lyase (Units mg⫺1 protein) Peroxidase (⌬OD470 mg⫺1 protein min⫺1) Superoxide dismutase (Units mg⫺1 protein) Catalase (mol H2O2 mg⫺1 protein min⫺1) Lipoxygenase (OD234 mg⫺1 protein min⫺1) Malondialdehyde (nmol g⫺1 fresh cells)
63 ⫾ 5.4 44 ⫾ 3.8 78 ⫾ 5.9 6.5 ⫾ 0.6 6 ⫾ 0.5 14 ⫾ 1.2
198 ⫾ 18 34 ⫾ 4.1 53 ⫾ 5.8 2.1 ⫾ 0.1 17 ⫾ 1.2 109 ⫾ 9.5
219 ⫾ 21 38 ⫾ 3.2 62 ⫾ 6.0 2.3 ⫾ 0.2 17 ⫾ 1.7 99 ⫾ 8.4
210 ⫾ 18 35 ⫾ 3.5 62 ⫾ 5.8 4.3 ⫾ 0.3 14 ⫾ 1.2 117 ⫾ 10.2
200 ⫾ 21 42 ⫾ 4.0 42 ⫾ 3.2 1.7 ⫾ 0.1 19 ⫾ 1.5 157 ⫾ 12.8
Seven-day-old CR8C cell cultures were treated by a combined elicitor of malate (40 mg l⫺1) and sodium alginate (1.5% w/v) for 3 days. Control (in 500-ml flask) received acetate buffer. All data were expressed as the mean of triplicate experiments ⫾ SD. Enzyme activity units: one unit activity of phenylalanine ammonia lyase was defined as 0.01 of changes in ⌬OD290 per hour; one enzyme unit of superoxide dismutase was defined as the enzyme amount for 50% of inhibition on nitroblue tetrazolium reduction per min.
though both the malate and alginate treatments were in their own optimal dose and addition time, after combination, they may not stimulate the maximum indole alkaloid production because of their interactions. Therefore, optimizing the combined elicitor treatment is still needed by using proper method. In spite of this, our current study, together with previous results [7], suggests that a bioreactor process combined with elicitation may be a practical means for indole alkaloid production although the performance of the model system still need to be improved. Recently it is reported that an acetate/mevalonate pathway and triose phosphate/pyruvate pathway may be involved in indole alkaloid biosynthetic pathway in C. roseus cell cultures [27]. Exogenous malate may inhibit tricarboxylic acid cycle by a negative feedback control, or exogenous malate can be transformed to pyruvate by a NADP⫹-malate enzyme. Therefore, addition of malate may stimulate indole alkaloid accumulation by increasing pyruvate pool or directing acetyl-CoA and pyruvate flux to monoterpenoid indole alkaloid biosynthesis pathway. Furthermore, additions of malate and alginate will certainly change the gas regime in the treated-cell cultures. Although the roles of the dissolved oxygen, CO2 and ethylene concentrations on alkaloid accumulation of elicited or non-elicited C. roseus cell cultures in flasks and bioreactor processes were not analyzed here because of the limited conditions, we could think that there are some differences in the gas regime between different culture systems, such as between the normal and malate-alginate treated cell cultures. Since it is suggested that the ungelled alginate causes a high viscosity, low agitation efficiency and low dissolved oxygen concentration but an significantly increased alkaloid accumulation in cell cultures [28]. Therefore, certain levels of dissolved oxygen needed by non-elicited cell cultures for alkaloid accumulation may not be critically needed by the elicited cell cultures for alkaloid accumulation, especially in current culture processes. The elicited cells may generate more ethylene but less CO2 than the non-elicited cells, and the situations in non-elicited cell cultures were exactly contrary to that. If really so, using the combined elicitor, increasing
cell density and optimizing the elicitor treatment and other culture conditions will further improve bioreactor performance.
Acknowledgments This investigation was supported by A Doctoral Program from Chinese Ministry of Education and funds from Chinese Academy of Medical Sciences and Peking Union Medical College. We would like to thank Dr. Frank DiCosmo (Toronto University, Canada) for kind gifts of vindoline, catharanthine and serpentine standards.
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