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High stable production of taxol in elicited synchronous cultures of Taxus chinensis cells
Process Biochemistry 38 (2002) 207 /210 www.elsevier.com/locate/procbio
High stable production of taxol in elicited synchronous cultures of Taxus chinensis cells Long-Jiang Yu *, Wen-Zhi Lan, Wen-Min Qin, Hui-Bi Xu School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, Hubei, People’s Republic of China Received 12 June 2001; received in revised form 24 February 2002; accepted 28 February 2002
Abstract Elicitation was an efficient method for enhancing taxol production in asynchronous cultures of Taxus chinensis cells but high production of taxol was unstable in 250 ml Erlenmeyer flask and a 10 l working volume stirred bioreactor. This situation could be remedied using two synchronization methods, double phosphate starvation and low temperature treatment. Low temperature treatment could not only maintain stable high taxol production, but also further enhance taxol production compared with elicited asynchronous cultures. This resulted in the greatest taxol production of 27 mg l 1, being about 2 and 11 times higher than in asynchronous cultures and in elicited asynchronous cultures, respectively. Taxol production in a stirred bioreactor was less than in flasks, but changes of taxol production in asynchronous cultures and synchronous cultures in a stirred bioreactor were similar to those in 250 ml flask. Therefore, high stable taxol production in elicited synchronous cultures of T. chinensis cells was amenable to scaleup. The possible relationship between stable production of taxol and cell cycle phase is discussed. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Taxus chinensis ; Taxol; Elicitation; Synchronization; Cell cycle phase; Stable production
1. Introduction Taxol, a novel diterpenoid secondary product from the bark of yew species, has been approved as an efficient anti-cancer drug. In vitro cell culture has been viewed as a promising alternative to whole plant extraction for obtaining taxol and related taxane compounds [1]. At present, many Taxus spp. have been explored for this purpose but with limited commercial success. Low yield of taxol is one of the constraints. Our group has established many methods to improve production of taxol [2 /5], and found combined elicitation is one of the most effective methods for this purpose [4]. Unfortunately, the high production of taxol is unstable in cell suspension cultures of Taxus chinensis . Secondary metabolite synthesis is often correlated with cell proliferation in plant tissue culture [6,7], and there are many systems in which production of specific
* Corresponding author. Fax: /86-27-8754-0184. E-mail address: [email protected] (L.-J. Yu).
metabolites occurs during certain cell cycle phases [8]. Even some metabolites, such as terpenoids, are necessary for cell cycle progress [9,10]. Therefore, terpenoid production is in close relation to the cell cycle. However, in suspension cultures of plant cells, the cell cycle phase of individual cells is different [11], which probably causes unstable production of terpenoid. To our knowledge, so far there have been no reports of maintaining terpenoid production stable by regulating cell cycle phase. If cell populations are known to be homogeneous with all cells cycling at equal rates, the fraction of cells in a particular cell cycle phase is a direct indication of the relative duration of that phase [11]. Synchronous cultures may be induced by many ways, such as physical methods [12], nutrition starvation [13] and chemical inhibitors [14], and can effectively realize the homogeneous cell cycle phase. In this document, we apply two methods, namely, double phosphate starvation and low temperature treatment, to establish synchronous cultures of T. chinensis cells, and then elicit synchronous cultures with combined elicitation in order to obtain high stable production of taxol. Further, we investigate
0032-9592/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 2 ) 0 0 0 6 9 - 9
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L.-J. Yu et al. / Process Biochemistry 38 (2002) 207 /210
the changes of taxol production in a 15 l bioreactor to test if the method is applicable in large-scale cultures.
Methyl jasmonate was dissolved in some ethanol and then diluted in water to make 1% content. 2.4. Elicitation experiments in flasks and bioreactor
2. Materials and methods 2.1. Suspension culture T. chinensis cells, initiated from zygote embryos, are maintained in MS medium supplemented with 3% sucrose and 10 mmol l 1 a-naphthalene acetic acid, and kept in the dark at 259/1 8C. For taxol production, about 10 g fresh weight cells were transferred to 100 ml production medium in 250 ml Erlenmyers flasks. The production medium was modified Gamborg’s B5 liquid medium according to Zhang et al. [4] except sucrose concentration was 30 g l 1. The flasks were shaken at 1259/5 rpm in the dark at 259/1 8C. 2.2. Synchronization Synchronous cultures of T. chinensis were achieved by double phosphate starvation and low temperature treatment. 2.2.1. Double phosphate starvation The procedure established for synchronization was according to the method previously described by Amina et al. [13] with modification. Fifteen-day-old cells were transferred to production medium without PO34 and cultured for 25 days, and then filtered cells were transferred to new production medium without PO34 and cultured for 25 days again. Phosphate is required in nucleic metabolism for cell cycle progression, and the cell cycle in most cells is considered to be arrested just after the M phase by the secondary phosphate starvation [13]. 2.2.2. Low temperature treatment The procedure established for synchronization was according to the method previously described by Wang et al. [12] with minor modification. Twenty-five-day-old cells were kept in the production medium at 4 8C for 72 h, then incubated at 25 8C for 24 h. After incubation, the cells were transferred to fresh medium and cultured for 24 h at 25 8C, then kept at 4 8C for 72 h. Low temperature treatment blocks the formation of spindle and most cells are blocked at M phase.
Synchronous cultures by double phosphate starvation or low temperature treatment, and asynchronous cultures were collected and inoculated using 100 g fresh weight per liter of production medium in 250 ml flasks and 15 l stirred bioreactors with working volumes of 100 ml and 10 l, respectively. Therefore, there were three types of initial cell cycle phase (ICCP): ICCP in synchronous cultures by double phosphate starvation was main G1 phase was marked as (GP-ICCP). ICCP in synchronous cultures by low temperature treatment in main M phase was marked as (MP-ICCP). ICCP in asynchronous cultures in various phases was marked as (VP-ICCP). Flask culture conditions were the same as described above. The stirred bioreactor with a takensample vial, designed by our laboratory, was columnar in shape and 25 cm diameter. Mixing was accomplished using spiral blade impellers at agitation rate of 60 rpm. Temperature was controlled at 259/1 8C for all runs. After 8 days, a combination of 60 mmol l 1 methyl jasmonate and 50 mg l 1 fungal elicitor was added into cultures. Samples were taken for assaying taxol production at timed intervals. 2.5. Assay of taxol Cell growth was determined by dry weight (DW) after cells were lyophilized to constant weight. Dry samples (0.2 g) are extracted with methanol chloride (1:1, v/v). The CH2Cl2 phase was separated from the aqueous phase and then evaporated in a rotary evaporator equipped with a condenser for solvent recovery. The residue was resuspended in 2 ml methanol and centrifuged at 8000 /g for 5 min. The supernatant was analyzed for taxol production by HPLC [4]. Samples of 5 ml from the free media are extracted with 2 ml CH2Cl2 for three times. The combined CH2Cl2 fraction was dried and redissolved in 2 ml methanol, then centrifuged for HPLC analysis [4]. Taxol production in the sample is the combination of taxol in cells and medium.
3. Results 3.1. Taxol production in flask
2.3. Elicitor preparation The fungus was an endophytic strain of Aspergillus niger , which was isolated from the inner bark of T. chinensis tree. The preparation of fungal elicitor was according to the method described by Zhang et al. [5].
Taxol production in asynchronous cultures (VPICCP) and two synchronous cultures (MP-ICCP and GP-ICCP) on the 15th day were higher than that on the tenth day, but taxol productions in the three cultures were different (Fig. 1). On the 15th day, taxol produc-
L.-J. Yu et al. / Process Biochemistry 38 (2002) 207 /210
Fig. 1. Effects of elicitation and synchronization on taxol production in cell suspension cultures of T. chinensis . Elicitors were incorporated in 8-day-old cultures. Values are means of five individual experiments and error bars represent standard errors. Symbols: VP-ICCP, synchronous cultures whose ICCP is various phase; MP-ICCP, synchronous cultures by low temperature treatment whose ICCP is M phase; GP-ICCP, synchronous cultures by double phosphate starvation whose ICCP is G1 phase; VP-ICCP/E, VP-ICCP treated with 60 mmol l 1 methyl jasmonate and 50 mg l 1 fungal elicitor; MP-ICCP/ E, MP-ICCP treated with 60 mmol l 1 methyl jasmonate and 50 mg l 1 fungal elicitor; GP-ICCP/E, GP-ICCP treated with 60 mmol l 1 methyl jasmonate and 50 mg l 1 fungal elicitor.
tion in MP-ICCP and GP-ICCP was 4 and 2 mg l 1, respectively, while taxol production in VP-ICCP was 2.5 mg l 1, being between that in MP-ICCP and that in GP-ICCP. Previous studies have demonstrated that treatment of combined elicitors could result in synergistic improving effects on taxol production [3 /5], and the effects of combined treatment of methyl jasmonate and fungal elicitor on taxol production in synchronous cultures of T. chinensis cells were tested. Cells from VP-ICCP, MPICCP and GP-ICCP showed different responses to the elicitors (Fig. 1). After elicitation, taxol production in MP-ICCP was the highest, at 27 mg l 1 being 2 and 3.5 times higher than that in VP-ICCP and GP-ICCP, respectively. Further, the fluctuating range of taxol production of individual experiments in asynchronous cultures (VP-ICCP) was greater than that in synchronous cultures (MP-ICCP and GP-ICCP). 3.2. Taxol production in bioreactor The time course of taxol production in cultures of T. chinensis cells is demonstrated in Fig. 2. Taxol productions in asynchronous cultures and synchronous cultures increased gradually in the process of the experiment. However, under the condition of elicitation, there were significant differences in taxol production between asynchronous cultures and synchronous cultures after 4 days. Taxol production in elicited asynchronous cultures reached a maximum on the 10th day. Taxol production in two elicited synchronous cultures reached a maximum on the 12th day, and taxol production in
209
Fig. 2. Time courses of taxol production in cell suspension cultures of T. chinensis . Elicitors were incorporated in 8-day-old cultures. Values are means of three individual experiments and error bars represent standard errors. Symbols, VP-ICCP, synchronous cultures whose ICCP was various phase; MP-ICCP, synchronous cultures by low temperature treatment whose ICCP is M phase; GP-ICCP, synchronous cultures by double phosphate starvation whose ICCP is G1 phase; VP-ICCP/E, VP-ICCP treated with 60 mmol l 1 methyl jasmonate and 50 mg l 1 fungal elicitor; MP-ICCP/E, MP-ICCP treated with 60 mmol l 1 methyl jasmonate and 50 mg l 1 fungal elicitor; GPICCP/E, GP-ICCP treated with 60 mmol l 1 methyl jasmonate and 50 mg l 1 fungal elicitor.
MP-ICCP was greatest, at 24 mg l 1, being almost 2 and 3.5 times higher than in VP-ICCP and GP-ICCP. In general, taxol production in the bioreactor was lower than in flasks, but the changes of taxol production in asynchronous cultures and synchronous cultures in bioreactor were similar to those in asynchronous cultures and synchronous cultures in flasks (Figs. 1 and 2).
4. Discussion Unlike most well-dispersed microbial and animal cell suspensions, plant cell suspension are characterized by a high degree of heterogeneity. There can also be significant variation between individual cells in their ability to growth, their rates of growth and their cell cycle activity, and their participation in secondary metabolic synthesis [8,15]. Hall et al. reported that only about 10% of the cell population in suspended Catharanthus cells was found to accumulate anthocyanin, and variations in total anthocyanin content between cultures were due primarily to differences in the anthocyanin within those cells [15]. Taxol production in GP-ICCP was lower than that in VP-ICCP, while taxol production in MP-ICCP was higher than that in VP-ICCP. Thus, taxol biosynthesis in different cell cycle phases was different. Therefore, population-average procedures for measuring production levels of taxol, which were universally accepted, are inaccurate and unavoidably cause unstable
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L.-J. Yu et al. / Process Biochemistry 38 (2002) 207 /210
production of taxol. Fortunately, this situation can be remedied using synchronization methods. In flasks and the bioreactor, the fluctuating range of taxol production in synchronous cultures (MP-ICCP or GP-ICCP) is less than that in asynchronous cultures. Therefore, synchronizing cultures of T. chinensis will be in favor of maintaining stable taxol production. Elicitors are compounds of biotic or abiotic origin that can stimulate taxol production [3 /5]. In the present study, the same phenomenon of enhanced taxol production in flask and bioreactor after elicitation were noted (Figs. 1 and 2). However, the high taxol production in elicited asynchronous cultures was not stable. Applying low temperature treatment to cultures does not only maintain high production of taxol stable, but also further enhance taxol production compared with that in elicited asynchronous cultures. This was possible due to the following reasons. (a) Terpenoid biosynthesis is necessary for cell cycle progress [9]. (b) Terpenoid biosynthetic enzymes are involved in the cell cycle [10]. (c) The synthesis of secondary metabolites in plants is widely believed to be part of the defence responses of plants to pathogenic attack. (d) Defense-related genes activated by elicitors are correlated with repression of cell cycle-related genes [16]. Therefore, applying suitable synchronization methods and elicitors on cultures of T. cheninsis cell will be favorable of stable high taxol production in T. chinensis cells.
Acknowledgements This work was supported by the Ministry of Education (2000 year excellent youth teacher fund). The authors wish to thank NCI for presenting taxol standard sample.
References [1] Jaziri M, Zhiri A, Guo YW, Dupon JP, Shimonura K, Hamada H, Vanhaelen M, Home`s J. Taxus sp. cell, tissue and organ cultures as alternative sources for taxoids production: a literature survey. Plant Cell Tissue and Organ Culture 1996;46:59 /75. [2] Mei XG, Lu MB, Yu LJ, Hu DW. Kinetics of taxol biosynthesis in bioreactors. Medicinal Chemistry Research 1996;4:256 /63.
[3] Zhang CH, Mei XG, Liu L, Yu LJ. Enhanced paclitaxel production induced by the combination of elicitors in cell suspension cultures of Taxus chinensis . Biotechnology Letters 2000;22:1561 /4. [4] Wu JY, Wang CG, Mei XG. Stimulation of taxol production and excretion in Taxus spp. cell cultures by rare chemical lanthanum. Journal of Biotechnology 2001;85:67 /73. [5] Zhang CH, Xu HB. Improved paclitaxel production by in situ extraction and elicitation in cell suspension cultures of Taxus chinensis . Biotechnology Letters 2001;23:189 /93. [6] Komamine A, Sakuta M, Hirose M, Hirano H, Takagi T, Ozeki Y. Regulation of secondary metabolism in relation to growth and differentiation. In: Kurz WGW, editor. Primary and Secondary Metabolism of Plant Cell Cultures II. Berlin: Springer, 1989:49 / 52. [7] Yeoman MM, Lindsey K, Miedzybrodzka MB, McLaachlan WR. Accumulation of secondary products as a facet of differentiation in plant and tissue cultures. In: Yeoman MM, Truman DES, editors. Differentiation In Vitro. Cambridge: Cambridge University Press, 1982:65 /82. [8] Maki H, Ando S, Kodama H, Komamine A. Polyamines and the cell cycle of Catharanthus roseus cells in culture. Plant Physiology 1991;96:1008 /13. [9] Andrea H, Fischt I, Bach TJ. Differential interaction of branchspecific inhibitors of isoprenoid biosynthesis with cell cycle progression in tobacco BY-2 cells. Physiologia Plantarum 2000;110:342 /9. [10] Qian DQ, Zhou DF, Ju R, Cramer CL, Yang ZB. Protein farnesyltransferase in plants: molecular characterization and involvement in cell cycle control. Plant Cell 1996;8:2387 /794. [11] Yanpaisan W, King NJC, Donran PM. Analysis of cell cycle activity and population dynamics in heterogeneous plant cell suspension using flow cytometry. Biotechnology and Bioengineering 1998;58:515 /28. [12] Wang AS, Philips RL. Synchronization of suspension cultures cell. In: Vasil LK, editor. Cell Culture and Somatic Cell Genetics of Plants I. Academic Press, 1984:175 /81. [13] Amino S, Fujimura T, Komanine A. Synchrony induced by double phosphate starvation in a suspension culture of Catharanthus roseus . Physiologia Plantarum 1983;59:393 /6. [14] Planchais S, Glab N, Inze´ D, Bergounioux C. Chemical inhibitors: a tool for plant cell cycle studies. FEBS Letters 2000;476:78 / 83. [15] Hall RD, Yeoman MM. Intercellular and intercultural heterogeneity in secondary metabolite accumulation in cultures of Catharanthus roseus following cell line selection. Journal of Experimental Botany 1987;38:1391 /8. [16] Logemann E, Wu SC, Schro¨der J, Schemezer E, Somssich IE, Hahlbrock K. Gene activation by UV light, fungal elicitor or fungal infection in Petroselinum crispum is correlated with repression of cell cycle-related genes. Plant Journal 1995;8:865 / 76.
High stable production of taxol in elicited synchronous cultures of Taxus chinensis cells Long-Jiang Yu *, Wen-Zhi Lan, Wen-Min Qin, Hui-Bi Xu School of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, Hubei, People’s Republic of China Received 12 June 2001; received in revised form 24 February 2002; accepted 28 February 2002
Abstract Elicitation was an efficient method for enhancing taxol production in asynchronous cultures of Taxus chinensis cells but high production of taxol was unstable in 250 ml Erlenmeyer flask and a 10 l working volume stirred bioreactor. This situation could be remedied using two synchronization methods, double phosphate starvation and low temperature treatment. Low temperature treatment could not only maintain stable high taxol production, but also further enhance taxol production compared with elicited asynchronous cultures. This resulted in the greatest taxol production of 27 mg l 1, being about 2 and 11 times higher than in asynchronous cultures and in elicited asynchronous cultures, respectively. Taxol production in a stirred bioreactor was less than in flasks, but changes of taxol production in asynchronous cultures and synchronous cultures in a stirred bioreactor were similar to those in 250 ml flask. Therefore, high stable taxol production in elicited synchronous cultures of T. chinensis cells was amenable to scaleup. The possible relationship between stable production of taxol and cell cycle phase is discussed. # 2002 Elsevier Science Ltd. All rights reserved. Keywords: Taxus chinensis ; Taxol; Elicitation; Synchronization; Cell cycle phase; Stable production
1. Introduction Taxol, a novel diterpenoid secondary product from the bark of yew species, has been approved as an efficient anti-cancer drug. In vitro cell culture has been viewed as a promising alternative to whole plant extraction for obtaining taxol and related taxane compounds [1]. At present, many Taxus spp. have been explored for this purpose but with limited commercial success. Low yield of taxol is one of the constraints. Our group has established many methods to improve production of taxol [2 /5], and found combined elicitation is one of the most effective methods for this purpose [4]. Unfortunately, the high production of taxol is unstable in cell suspension cultures of Taxus chinensis . Secondary metabolite synthesis is often correlated with cell proliferation in plant tissue culture [6,7], and there are many systems in which production of specific
* Corresponding author. Fax: /86-27-8754-0184. E-mail address: [email protected] (L.-J. Yu).
metabolites occurs during certain cell cycle phases [8]. Even some metabolites, such as terpenoids, are necessary for cell cycle progress [9,10]. Therefore, terpenoid production is in close relation to the cell cycle. However, in suspension cultures of plant cells, the cell cycle phase of individual cells is different [11], which probably causes unstable production of terpenoid. To our knowledge, so far there have been no reports of maintaining terpenoid production stable by regulating cell cycle phase. If cell populations are known to be homogeneous with all cells cycling at equal rates, the fraction of cells in a particular cell cycle phase is a direct indication of the relative duration of that phase [11]. Synchronous cultures may be induced by many ways, such as physical methods [12], nutrition starvation [13] and chemical inhibitors [14], and can effectively realize the homogeneous cell cycle phase. In this document, we apply two methods, namely, double phosphate starvation and low temperature treatment, to establish synchronous cultures of T. chinensis cells, and then elicit synchronous cultures with combined elicitation in order to obtain high stable production of taxol. Further, we investigate
0032-9592/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 0 2 ) 0 0 0 6 9 - 9
208
L.-J. Yu et al. / Process Biochemistry 38 (2002) 207 /210
the changes of taxol production in a 15 l bioreactor to test if the method is applicable in large-scale cultures.
Methyl jasmonate was dissolved in some ethanol and then diluted in water to make 1% content. 2.4. Elicitation experiments in flasks and bioreactor
2. Materials and methods 2.1. Suspension culture T. chinensis cells, initiated from zygote embryos, are maintained in MS medium supplemented with 3% sucrose and 10 mmol l 1 a-naphthalene acetic acid, and kept in the dark at 259/1 8C. For taxol production, about 10 g fresh weight cells were transferred to 100 ml production medium in 250 ml Erlenmyers flasks. The production medium was modified Gamborg’s B5 liquid medium according to Zhang et al. [4] except sucrose concentration was 30 g l 1. The flasks were shaken at 1259/5 rpm in the dark at 259/1 8C. 2.2. Synchronization Synchronous cultures of T. chinensis were achieved by double phosphate starvation and low temperature treatment. 2.2.1. Double phosphate starvation The procedure established for synchronization was according to the method previously described by Amina et al. [13] with modification. Fifteen-day-old cells were transferred to production medium without PO34 and cultured for 25 days, and then filtered cells were transferred to new production medium without PO34 and cultured for 25 days again. Phosphate is required in nucleic metabolism for cell cycle progression, and the cell cycle in most cells is considered to be arrested just after the M phase by the secondary phosphate starvation [13]. 2.2.2. Low temperature treatment The procedure established for synchronization was according to the method previously described by Wang et al. [12] with minor modification. Twenty-five-day-old cells were kept in the production medium at 4 8C for 72 h, then incubated at 25 8C for 24 h. After incubation, the cells were transferred to fresh medium and cultured for 24 h at 25 8C, then kept at 4 8C for 72 h. Low temperature treatment blocks the formation of spindle and most cells are blocked at M phase.
Synchronous cultures by double phosphate starvation or low temperature treatment, and asynchronous cultures were collected and inoculated using 100 g fresh weight per liter of production medium in 250 ml flasks and 15 l stirred bioreactors with working volumes of 100 ml and 10 l, respectively. Therefore, there were three types of initial cell cycle phase (ICCP): ICCP in synchronous cultures by double phosphate starvation was main G1 phase was marked as (GP-ICCP). ICCP in synchronous cultures by low temperature treatment in main M phase was marked as (MP-ICCP). ICCP in asynchronous cultures in various phases was marked as (VP-ICCP). Flask culture conditions were the same as described above. The stirred bioreactor with a takensample vial, designed by our laboratory, was columnar in shape and 25 cm diameter. Mixing was accomplished using spiral blade impellers at agitation rate of 60 rpm. Temperature was controlled at 259/1 8C for all runs. After 8 days, a combination of 60 mmol l 1 methyl jasmonate and 50 mg l 1 fungal elicitor was added into cultures. Samples were taken for assaying taxol production at timed intervals. 2.5. Assay of taxol Cell growth was determined by dry weight (DW) after cells were lyophilized to constant weight. Dry samples (0.2 g) are extracted with methanol chloride (1:1, v/v). The CH2Cl2 phase was separated from the aqueous phase and then evaporated in a rotary evaporator equipped with a condenser for solvent recovery. The residue was resuspended in 2 ml methanol and centrifuged at 8000 /g for 5 min. The supernatant was analyzed for taxol production by HPLC [4]. Samples of 5 ml from the free media are extracted with 2 ml CH2Cl2 for three times. The combined CH2Cl2 fraction was dried and redissolved in 2 ml methanol, then centrifuged for HPLC analysis [4]. Taxol production in the sample is the combination of taxol in cells and medium.
3. Results 3.1. Taxol production in flask
2.3. Elicitor preparation The fungus was an endophytic strain of Aspergillus niger , which was isolated from the inner bark of T. chinensis tree. The preparation of fungal elicitor was according to the method described by Zhang et al. [5].
Taxol production in asynchronous cultures (VPICCP) and two synchronous cultures (MP-ICCP and GP-ICCP) on the 15th day were higher than that on the tenth day, but taxol productions in the three cultures were different (Fig. 1). On the 15th day, taxol produc-
L.-J. Yu et al. / Process Biochemistry 38 (2002) 207 /210
Fig. 1. Effects of elicitation and synchronization on taxol production in cell suspension cultures of T. chinensis . Elicitors were incorporated in 8-day-old cultures. Values are means of five individual experiments and error bars represent standard errors. Symbols: VP-ICCP, synchronous cultures whose ICCP is various phase; MP-ICCP, synchronous cultures by low temperature treatment whose ICCP is M phase; GP-ICCP, synchronous cultures by double phosphate starvation whose ICCP is G1 phase; VP-ICCP/E, VP-ICCP treated with 60 mmol l 1 methyl jasmonate and 50 mg l 1 fungal elicitor; MP-ICCP/ E, MP-ICCP treated with 60 mmol l 1 methyl jasmonate and 50 mg l 1 fungal elicitor; GP-ICCP/E, GP-ICCP treated with 60 mmol l 1 methyl jasmonate and 50 mg l 1 fungal elicitor.
tion in MP-ICCP and GP-ICCP was 4 and 2 mg l 1, respectively, while taxol production in VP-ICCP was 2.5 mg l 1, being between that in MP-ICCP and that in GP-ICCP. Previous studies have demonstrated that treatment of combined elicitors could result in synergistic improving effects on taxol production [3 /5], and the effects of combined treatment of methyl jasmonate and fungal elicitor on taxol production in synchronous cultures of T. chinensis cells were tested. Cells from VP-ICCP, MPICCP and GP-ICCP showed different responses to the elicitors (Fig. 1). After elicitation, taxol production in MP-ICCP was the highest, at 27 mg l 1 being 2 and 3.5 times higher than that in VP-ICCP and GP-ICCP, respectively. Further, the fluctuating range of taxol production of individual experiments in asynchronous cultures (VP-ICCP) was greater than that in synchronous cultures (MP-ICCP and GP-ICCP). 3.2. Taxol production in bioreactor The time course of taxol production in cultures of T. chinensis cells is demonstrated in Fig. 2. Taxol productions in asynchronous cultures and synchronous cultures increased gradually in the process of the experiment. However, under the condition of elicitation, there were significant differences in taxol production between asynchronous cultures and synchronous cultures after 4 days. Taxol production in elicited asynchronous cultures reached a maximum on the 10th day. Taxol production in two elicited synchronous cultures reached a maximum on the 12th day, and taxol production in
209
Fig. 2. Time courses of taxol production in cell suspension cultures of T. chinensis . Elicitors were incorporated in 8-day-old cultures. Values are means of three individual experiments and error bars represent standard errors. Symbols, VP-ICCP, synchronous cultures whose ICCP was various phase; MP-ICCP, synchronous cultures by low temperature treatment whose ICCP is M phase; GP-ICCP, synchronous cultures by double phosphate starvation whose ICCP is G1 phase; VP-ICCP/E, VP-ICCP treated with 60 mmol l 1 methyl jasmonate and 50 mg l 1 fungal elicitor; MP-ICCP/E, MP-ICCP treated with 60 mmol l 1 methyl jasmonate and 50 mg l 1 fungal elicitor; GPICCP/E, GP-ICCP treated with 60 mmol l 1 methyl jasmonate and 50 mg l 1 fungal elicitor.
MP-ICCP was greatest, at 24 mg l 1, being almost 2 and 3.5 times higher than in VP-ICCP and GP-ICCP. In general, taxol production in the bioreactor was lower than in flasks, but the changes of taxol production in asynchronous cultures and synchronous cultures in bioreactor were similar to those in asynchronous cultures and synchronous cultures in flasks (Figs. 1 and 2).
4. Discussion Unlike most well-dispersed microbial and animal cell suspensions, plant cell suspension are characterized by a high degree of heterogeneity. There can also be significant variation between individual cells in their ability to growth, their rates of growth and their cell cycle activity, and their participation in secondary metabolic synthesis [8,15]. Hall et al. reported that only about 10% of the cell population in suspended Catharanthus cells was found to accumulate anthocyanin, and variations in total anthocyanin content between cultures were due primarily to differences in the anthocyanin within those cells [15]. Taxol production in GP-ICCP was lower than that in VP-ICCP, while taxol production in MP-ICCP was higher than that in VP-ICCP. Thus, taxol biosynthesis in different cell cycle phases was different. Therefore, population-average procedures for measuring production levels of taxol, which were universally accepted, are inaccurate and unavoidably cause unstable
210
L.-J. Yu et al. / Process Biochemistry 38 (2002) 207 /210
production of taxol. Fortunately, this situation can be remedied using synchronization methods. In flasks and the bioreactor, the fluctuating range of taxol production in synchronous cultures (MP-ICCP or GP-ICCP) is less than that in asynchronous cultures. Therefore, synchronizing cultures of T. chinensis will be in favor of maintaining stable taxol production. Elicitors are compounds of biotic or abiotic origin that can stimulate taxol production [3 /5]. In the present study, the same phenomenon of enhanced taxol production in flask and bioreactor after elicitation were noted (Figs. 1 and 2). However, the high taxol production in elicited asynchronous cultures was not stable. Applying low temperature treatment to cultures does not only maintain high production of taxol stable, but also further enhance taxol production compared with that in elicited asynchronous cultures. This was possible due to the following reasons. (a) Terpenoid biosynthesis is necessary for cell cycle progress [9]. (b) Terpenoid biosynthetic enzymes are involved in the cell cycle [10]. (c) The synthesis of secondary metabolites in plants is widely believed to be part of the defence responses of plants to pathogenic attack. (d) Defense-related genes activated by elicitors are correlated with repression of cell cycle-related genes [16]. Therefore, applying suitable synchronization methods and elicitors on cultures of T. cheninsis cell will be favorable of stable high taxol production in T. chinensis cells.
Acknowledgements This work was supported by the Ministry of Education (2000 year excellent youth teacher fund). The authors wish to thank NCI for presenting taxol standard sample.
References [1] Jaziri M, Zhiri A, Guo YW, Dupon JP, Shimonura K, Hamada H, Vanhaelen M, Home`s J. Taxus sp. cell, tissue and organ cultures as alternative sources for taxoids production: a literature survey. Plant Cell Tissue and Organ Culture 1996;46:59 /75. [2] Mei XG, Lu MB, Yu LJ, Hu DW. Kinetics of taxol biosynthesis in bioreactors. Medicinal Chemistry Research 1996;4:256 /63.
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