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Significant improvement of taxane production in suspension cultures of Taxus chinensis by sucrose feeding strategy
Process Biochemistry 35 (1999) 479 – 483 www.elsevier.com/locate/procbio
Significant improvement of taxane production in suspension cultures of Taxus chinensis by sucrose feeding strategy H.Q. Wang, J.T. Yu, J.J. Zhong * State Key Laboratory of Bioreactor Engineering, East China Uni6ersity of Science and Technology, 130 Meilong Road, Shanghai 200237, China Received 3 February 1999; received in revised form 12 April 1999; accepted 10 July 1999
Abstract The effects of both initial sucrose concentration and sucrose feeding on cell growth and accumulation of taxane diterpene (taxuyunnanine C) in suspension cultures of Taxus chinensis were investigated in detail. For the initial sucrose concentrations of 20, 30, 40 and 50 g/l, the cell growth was repressed by the later two high levels (i.e. 40 and 50 g/l of initial sucrose) with a relatively longer lag phase. Combination of initial low sucrose concentration (20 g/l) and subsequent sucrose feeding during cultivation (fed-batch culture) improved the cell growth, and a final cell concentration of over 27 g dry cells/l was successfully achieved after 23 days of cultivation. The time course of the taxane formation was related to the cell growth profile. For example, in the cell cultivation with under 30 g/l of initial sucrose, the specific taxane production (content) was relatively low when the cells were under active growth, but sharply increased up to 14 mg/g DW when the cells approached the stationary phase. In the batch cultures with an increase in initial sucrose concentration from 20 to 50 g/l, both the taxane content and productivity were decreased. In the fed-batch cultures, a relatively high taxane content was obtained at the end of cultivation; both a very high taxane production of 274.4 mg/l and a high productivity of 9.3 mg/(l day) were successfully achieved by feeding 20 g/l of sucrose (on day 7) to the cell cultures. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: Taxus chinensis; Sucrose feeding strategy; Taxane production; Plant cell suspension culture; Secondary metabolite
1. Introduction Since the discovery of paclitaxel (Taxol®) and its clinical anticancer activity, paclitaxel and related taxane compounds have attracted great attention around the world. More than 100 new taxanes have now been isolated and identified in Taxus spp., and the diterpenoid family is still expanding. Taxanes can be used as starting materials for semi-synthesis and further modifications for useful compounds and new taxane drugs are under active exploration [1]. Production of taxanes by cell cultures of Taxus spp. is viewed as a promising alternative to plant extraction. Until now, many scientists have focused on cell line selection (for fast growth or higher taxane content),
* Corresponding author. Tel.: +86-21-64252091; fax: + 86-2164253904. E-mail address: [email protected] (J.J. Zhong)
medium modification, precursor addition, elicitation, gas phase composition, culture kinetics, cell immobilization, and continuous cultivation of Taxus spp. cells [2–8]. In plant cell cultures, metabolite productivity is a key factor in determining its process efficiency and commercial potential. A relatively high sucrose concentration can be used to increase cell density and volumetric productivity of secondary metabolites. An increase of sucrose concentration in the medium above the normally used 2–3% level was reported to stimulate the production of taxol and related taxanes in a static study on Taxus bre6ifolia cell cultures [9]. In this paper, the kinetics of both the growth and taxane accumulation by Taxus chinensis cells were investigated in detail under various initial sucrose concentrations. Furthermore, based on our newly obtained information, an efficient feeding strategy was formulated to enhance taxane productivity in the cell suspension cultures.
0032-9592/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 9 9 ) 0 0 0 9 4 - 1
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2. Materials and methods
2.1. Plant materials and cell cultures Suspension cells of T. chinensis were cultured in Murashige and Skoog medium supplemented with 0.5 mg 6-benzyladenine/l, 0.2 mg 2,4-dichlorophenoxyacetic acid/l, 0.5 mg naphthaleneacetic acid/l, 100 mg ascorbic acid/l, and 30 g sucrose/l. The cells were subcultured at an interval of 2 weeks in a 250-ml Erlenmeyer flask containing 50 ml medium on a rotary shaker in darkness, 110 rpm and 25°C [10]. In this work, the initial sucrose concentrations were set at 20, 30, 40 and 50 g/l. Furthermore, other cultures with 20 g/l of initial sucrose concentration, were also fed with a dense sucrose solution to make the total amount of sucrose supply identical to 30, 40 and 50 g/l. Before inoculation, the subcultured cells from a number of flasks were thoroughly mixed to provide homogeneous cell inocula to each flask. In all cases, the inoculation density was controlled at 7.0 g DW/l.
2.2. Determination of cell weight The cells from a sampled shake flask were washed with a large amount of distilled water, and filtrated under vacuum. The cells were then weighed as fresh weight. The fresh cells were dried at 50°C to a constant weight, when the dry cell weight was determined. Finally, the cell concentrations based on fresh and dry cell weights were calculated.
[10]. After 2 years of subcultures, taxol accumulation was found to be unstable and finally became undetectable. On the other hand, in HPLC analysis, an unknown elutant with a large peak was detected and isolated and identified by IR, MR and NMR. Its structure was determined to be 2a,5a,10b,14b-tetra-acetoxy-4(20),11-taxadiene (taxuyunnanine C), which was the same as that from the roots of Taxus yunnanensis as reported in 1994 by Zhang et al. [13].
3. Results and discussion
3.1. Effect of initial sucrose concentration on cell growth and sugar consumption As shown in Fig. 1A and B, in the presence of 20 and 30 g/l initial sucrose concentration, the cells entered their growth phase from day 3 with a specific growth rate of 0.12 per day. At high initial sucrose concentrations of 40 and 50 g/l, extended lag phases of 6 and 9 days were observed, respectively. After that, the cells grew very rapidly. The growth rate [(max. cell mass− initial cell mass)/(initial cell mass)/(time)] was 0.10 per day, similar to that under 20 and 30 g/l initial sucrose concentration (Table 1). The final dry cell weight accumulated in the cultures was significantly affected by the initial sucrose level in the medium. The maximum dry biomass obtained was 13.9 (on day 9), 17.0 (on day 14),
2.3. Measurement of medium nutrients Residual sugar concentration was determined as reported by Hodge and Hofreiter [11]. Inorganic phosphate concentration in media was analysed according to the ascorbic acid method [12].
2.4. Taxane extraction and analysis For taxane extraction, 200 mg of powdered dry cells was soaked in 3 ml methanol for 2 days, then ultrasonicated twice for 40 min. The extracts were dried at 25°C. The residue was dissolved by 2 ml dichloromethane and 2 ml distilled water. After sufficient mixing, the mixture was centrifuged at 4000 rpm for 5 min. The organic phase was collected and dried at 25°C, then dissolved in 0.5 ml HPLC grade methanol for HPLC analysis. A reverse-phase column (Whatman Pentafluorophenyl, 5 mm, 250 × 4.6 mm) was used. The mobile phase consisted of acetonitrile and water (40:60, v/v), and the flow rate was 1 ml/min. Taxanes were monitored at a wavelength of 227 nm [10]. In our previous cultivation of T. chinensis cells, taxol was detected although with a low content in the cells
Fig. 1. Dynamic profiles of fresh and dry mass accumulation and sugar consumption in suspension cultures of T. chinensis. A, B, C: cells cultured at four different initial sucrose concentrations, 20 g/l,
30 g/l, 40 g/l, 50 g/l; D, E, F: cultures started with 20 g/l sucrose concentration, and fed sucrose at day 7, + 10 g/l, +20 g/l, +30 g/l. Each value is an average of two to three samples, and the bar in the figure represents the standard error.
H.Q. Wang et al. / Process Biochemistry 35 (1999) 479–483
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Table 1 Effect of initial sucrose concentration and sucrose feeding on cell growth rate (GR), cell yield (Yx/s), maximum taxane production (Pr) and productivity (Pv) in suspension cultures of T. chinensis a Initial sucrose concentration (g/l)
GR (d−1) Yx/s (g g−1) Pr (mg/l) Pv (mg l−1 d−1) a
Sucrose feed (g/l) at day 7
20
30
40
50
10
20
30
0.11 0.36 168.3 7.7
0.11 0.35 206.7 7.3
0.10 0.38 147.4 4.8
0.10 0.41 209.0 5.1
0.10 0.35 192.6 9.4
0.11 0.39 274.4 9.3
0.13 0.41 263.1 7.8
GR is calculated as: (max. cell mass−initial cell mass)/(initial cell mass)/(time).
20.8 (on day 20) and 26.3 g/l (on day 26) at an initial sucrose concentration of 20, 30, 40 and 50 g/l, respectively (Fig. 1B). Compared to the case of 30 g initial sucrose/l, the apparent cell yield over sugar (Yx/ s) was also relatively high at a high initial sucrose concentration of 50 g/l (i.e. 0.41 vs. 0.35 g g − 1) (Table 1). In the case of Vitis 6inifera cell cultures, both a lag phase and a reduced cell concentration were observed under a relatively high sucrose concentration of 50 g/l [14]. For cell cultures of Coleus blumei, a high initial sucrose concentration of 60 g/l led to a high biomass accumulation without an obvious lag phase [15]. With suspension cultures of Perilla frutescens, the growth rate increased with an increase of initial sucrose level up to 60 g/l in the medium [16]. It is clear that initial sucrose concentration is important to the growth of plant cells and its effect is dependent on a specific cell line. The time profile of sugar consumption is shown in Fig. 1C. In all cases, almost all the medium sugar was exhausted during the monitored cultivation period. Sugar consumption under 40 and 50 g/l of initial sucrose level was characterised with two phases as separated on day 12 (for the former case) and 15 (for the later case). This time corresponded to the onset of rapid cell growth in each case. In both cases, the sugar consumption rate in the second phase (days 12–21 and days 15 – 26) was much faster than that in the first phase (days 0 – 12 and days 0–15), i.e. 2.6 versus 1.2 g sugar/(l day). The cell yield (Yx/s) in the second phase was still higher than that in the first phase. For example, at an initial sucrose concentration of 50 g/l, the cell yield was 0.14 and 0.62 g g − 1 in the first and second phase, respectively. Medium phosphate was taken up by the cells at a very fast rate (data not shown). For the cases of low initial sucrose concentrations (20 and 30 g/l), phosphate was depleted by day 5, but its exhaustion was delayed to day 9 and 12 in the cultures with high initial sucrose concentrations of 40 and 50 g/l, respectively.
3.2. Effect of initial sucrose concentration on taxane production The dynamic profile of taxane content of the cells was dependent on a different initial sucrose concentration in medium (Fig. 2A). At a relatively high initial sucrose concentration of 50 g/l, a significant increase of taxane accumulation in the cells was observed during the lag phase; after that, the taxane content showed a decrease. However, at the end of cultivation, it was increased again from 5 mg/g DW on day 23 to 8.3 mg/g DW on day 29. A similar taxane accumulation pattern was observed in cells in media containing 40 g/l initial sucrose. Although the increase of taxane content in cells during the lag phase was less significant, its final value (on day 26) was comparable to that under 50 g/l of initial sucrose. At the low initial sucrose concentrations of 20 and 30 g/l, the taxane content showed a decreasing trend during the exponential growth phase (days 3–7 and days 3–12, respectively). When growth approached stationary phase, taxane accumulation in-
Fig. 2. Time courses of content and production of taxuyunnanine C in suspension cultures of T. chinensis. A, B: cells cultured at four different initial sucrose concentrations, 20 g/l, 30 g/l, 40 g/l, 50 g/l; C, D: cultures started with 20 g/l sucrose concentration, and fed sucrose at day 7, +10 g/l, +20 g/l, +30 g/l. Each value is an average of two to three samples, and the bar in the figure represents the standard error.
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creased sharply to a level of 14 mg/g DW within 5 days. Fig. 2B showed the time courses of total taxane production in cell cultures under the various initial sucrose concentrations. In all cases, the total taxane amount showed a significant increase in the later part of cultivation due to its higher content and/or much higher biomass level. Under a high initial sucrose concentration of 50 g/l, although the taxane content was relatively low, its highest volumetric production (209.0 mg/l) was achieved on day 29 due to a relatively high biomass concentration in this case. It is clear that raising the cell culture density was effective in the improving taxane production by T. chinensis cells. The significance of biomass accumulation was also claimed in other Taxus cell cultures [2]. On the other hand, for taxane productivity, with an increase in initial sucrose concentration from 20 to 50 g/l, this was decreased from 7.7 to 5.1 mg/(l day) due to the long time of cultivation for the later case (Table 1). At low initial sucrose concentrations, the taxane accumulation pattern seemed to be antagonistic with cell growth. Taxane content decreased in the exponential growth phase, while a large amount of taxane accumulated when the growth was approaching or had entered its stationary phase. In cell cultures of T. baccata, Srinivasan et al. also reported that taxol was produced exclusively during a period of declining growth rate [7]. At high initial sucrose concentrations, the increase of taxane content during the lag phase may be due to the effects of high osmotic pressure, as claimed by Zhang et al. in suspension cultures of Panax notoginseng for production of ginseng saponin [17]. In cell cultures with a relatively high initial sucrose concentration, when the cell growth entered stationary phase, taxane was not synthesised as much as under a low initial sucrose concentration. The above information indicates that the effect of initial sucrose level on the kinetics of taxane formation is quite complicated. In cell cultures of T. bre6ifolia, more than 60 g/l of sucrose in a production medium was claimed to be desirable for production of taxol and related taxane (cephalomannine) [9]. Since only a static result was presented in that study, it was not clear how an initial sucrose concentration affected the kinetics of both cell growth and taxol synthesis. The positive effects of a relatively high initial sucrose concentration on metabolite production were also reported in a number of other plant cell cultures [15 – 18].
3.3. Effect of sucrose feeding on cell growth and sugar consumption From the above experiments, it is clear that a shorter lag phase, higher taxane content, and higher taxane productivity were achieved at a low initial sucrose concentration (e.g. 20 g/l), while the final biomass level and total taxane production titre were high under a high
initial sucrose concentration (e.g. 50 g/l). Thus, it is logical for us to consider a series of new cell cultures with low initial sucrose concentration (20 g/l) combined with sucrose feeding at a later stage of cultivation in order to enhance both the specific production (content) and productivity of taxanes while maintaining a high final cell density in the cell cultures. As shown in Fig. 1D and E, cells grown in 20 g/l initial sucrose concentration with sucrose feeding on day 7 displayed a similar growth pattern to those of batch cultures under low initial sucrose concentrations. In the case of feeding 30 g sucrose/l on day 7, the cell growth seemed to be repressed again during the period of days 7–12. This was assumed to be due to the osmotic shock caused by a relatively high medium sucrose concentration (about 42 g/l) immediately after sucrose feeding. Compared to that in batch cultures with equal amounts of total sugar (40 and 50 g/l), the growth rates in the corresponding two fed-batch cultures increased by 10– 30% (Table 1). The reason was that a shorter time was required to reach a relatively high biomass level in the later case. For example, at day 15, 19.2 and 16.5 g DW/l were reached by feeding 20 and 30 g/l of sucrose, respectively, while only 15.6 and 9.84 g/l were reached for initial sucrose concentrations of 40 and 50 g/l, respectively. The maximum dry weights were comparable to those of batch cultures. They were 16.9 (at day 14), 22.3 (at day 20) and 27.2 g/l (at day 23) for sucrose feed of 10, 20 and 30 g/l, respectively. The medium sucrose fed at day 7 was also consumed rapidly in a similar way to that of batch cultures at low initial sucrose concentrations (Fig. 1F). In all cases, the time of maximum growth reached corresponded to medium sugar depletion.
3.4. Effect of sucrose feeding on taxane production Fig. 2C and D show the dynamic profiles of the specific production (i.e. content) and total production of taxuyunnanine C under sucrose feeding. The patterns of taxane formation were similar for all three cases of feeding experiments (Fig. 2C). The taxane content was relatively low and stable (around 6 mg/g DW) during the exponential growth phase. After that, a significant increase in taxane formation was observed after the beginning of the slowing-down phase of the cell growth in the cases of feeding 10 (at day 12), 20 (at day 21) and 30 g/l of sucrose (at day 24). At the end of cultivation, a high taxane content of 14 mg/g DW was reached in all cases. Compared to that in batch cultures under high initial sucrose concentrations (40 and 50 g/l), the increase of taxane content in the later stage of cultivation was much more significant in the experiments with a sucrose feed of 20 and 30 g/l. The final amount of total taxane produced was 192.6 (at day 14), 274.4 (at day 23) and 263.1 mg/l (at day 26) for the cases with sucrose feeding of 10, 20 and 30 g/l, respectively (Fig. 2D and
H.Q. Wang et al. / Process Biochemistry 35 (1999) 479–483
Table 1). For taxane productivity, it was higher in feeding experiments than in their corresponding batch cultures. The highest productivity of 9.3 mg/(l day) was successfully achieved in the both cases of feeding 10 and 20 g sucrose/l (Table 1). Compared with that at high initial sucrose concentrations, it is evident that sucrose feeding improved both taxane production (Fig. 2D vs. Fig. 2B) and productivity (Table 1). The manipulation of medium sucrose level based on the characteristics of cell growth and metabolite production was very important and effective for optimal cell growth and metabolite production. For example, Zhang et al. reported a successful medium sucrose manipulation for hyper-production of saponin and polysaccharide in suspension cultures of Panax notoginseng [18]. In the present case, both the initial sucrose concentration and the total amount of medium sucrose were important for the enhancement of both production and productivity of taxuyunnanine C. In conclusion, a relatively high initial sucrose concentration above 30 g/l could increase the cell density, but decreased taxane productivity in suspension cultures of T. chinensis. Taxane accumulation was significantly increased when growth approached its stationary phase. The operation of a cell culture at a low initial sucrose concentration (20 g/l) in combination with sucrose feeding at a later stage could effectively improve cell growth and both the production and productivity of taxane. The above results are useful for the understanding of the sucrose effect in Taxus cell cultures and for largescale cultivation of plant cells for production of anticancer taxane diterpenes.
Acknowledgements This work was supported by National Natural Science Foundation of China (No. 29606003). We also thank Z.W. Pan and Y.Z. Zhang for their helpful assistance during the experiments.
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[2] Ketchum REB, Gibson DM, Greenspan Gallo L. Media optimisation for maximum biomass production in cell cultures of Pacific yew. Plant Cell Tissue Org Cult 1995;42:185 – 93. [3] Kawamura M, Shigeoka T, Tahara M, Takami M, Ohashi H, Akita M, et al. Efficient selection of cells with high taxol content from heterogeneous Taxus cell suspensions by magnetic or fluorescent antibodies. Seibutsu-kogaku Kaishi 1998;76:3–7. [4] Fett-Neto AG, Melanson SJ, Sakata K, DiCosmo F. Improved growth and taxol yield in developing calli of Taxus cuspidata by medium composition modification. Bio/Technology 1993;11:731 – 4. [5] Mirjalili N, Linden JC. Gas phase composition effects on suspension cultures of Taxus cuspidata. Biotechnol Bioeng 1995;48:123 – 32. [6] Yukimune Y, Tabata H, Higashi Y, Hara Y. Methyl jasmonateinduced overproduction of paclitaxel and baccatin III in Taxus cell suspension cultures. Nat Biotechnol 1996;14:1129 –32. [7] Srinivasan V, Pestchanker L, Moser S, Hirasuna TJ, Taticek RA, Shuler ML. Taxol production in bioreactors: kinetics of biomass accumulation, nutrient uptake, and taxol production by cell suspensions of Taxus baccata. Biotechnol Bioeng 1995;47:666 – 76. [8] Seki M, Ohzora C, Takeda M, Furusaki S. Taxol (Paclitaxel) production using free and immobilised cells of Taxus cuspidata. Biotechnol Bioeng 1997;53:214 – 9. [9] Kim JH, Yun JH, Hwang YS, Byun SY, Kim DI. Production of taxol and related taxanes in Taxus bre6ifolia cell cultures: effect of sugar. Biotechnol Lett 1995;17:101 – 6. [10] Wang HQ, Zhong JJ, Yu JT. Enhanced production of taxol in suspension cultures of Taxus chinensis by controlling inoculum size. Biotechnol Lett 1997;19:353 – 5. [11] Hodge JE, Hofreiter BT. Determination of Reducing Sugars and Carbohydrate, In: Whistler RL, Walfrom ML, editors. Methods in Carbohydrate Chemistry: New York: Academic Press, 1962. p. 380 – 94. [12] Chen PS, Toribara TY, Warner H. Microdetermination of phosphorus. Anal Chem 1956;28:1756 – 8. [13] Zhang HJ, Takeda Y, Minami Y, Yoshida K, Matsumoto T, Xiang W, Mu Q, Sun HD. Three new taxanes from the roots of Taxus yunnanensis, Chem. Lett. 1994;5:957 – 960. [14] Cormier F, Crevier HA, Do CB. Effects of sucrose concentration on the accumulation of anthocyanins in grape (Vitis 6inifera) cell suspension. Can J Bot 1990;68:1822 – 5. [15] Gertlowski C, Petersen M. Influence of the carbon source on growth and rosmarinic acid production in suspension cultures of Coleus blumei. Plant Cell Tissue Org Cult 1993;34:183 –90. [16] Zhong JJ, Yoshida T. High-density cultivation of Perilla frutescens cell suspensions for anthocyanin production: effects of sucrose concentration and inoculum size. Enzyme Microb Technol 1995;17:1073 – 9. [17] Zhang YH, Zhong JJ, Yu JT. Effect of osmotic pressure on cell growth and saponin polysaccharide production by suspension cultures of Panax notoginseng. Biotechnol Lett 1995;17:1347–50. [18] Zhang YH, Zhong JJ, Yu JT. Enhancement of ginseng saponin production in suspension cultures of Panax notoginseng: manipulation of medium sucrose. J Biotechnol 1996;51:49 – 56.
Significant improvement of taxane production in suspension cultures of Taxus chinensis by sucrose feeding strategy H.Q. Wang, J.T. Yu, J.J. Zhong * State Key Laboratory of Bioreactor Engineering, East China Uni6ersity of Science and Technology, 130 Meilong Road, Shanghai 200237, China Received 3 February 1999; received in revised form 12 April 1999; accepted 10 July 1999
Abstract The effects of both initial sucrose concentration and sucrose feeding on cell growth and accumulation of taxane diterpene (taxuyunnanine C) in suspension cultures of Taxus chinensis were investigated in detail. For the initial sucrose concentrations of 20, 30, 40 and 50 g/l, the cell growth was repressed by the later two high levels (i.e. 40 and 50 g/l of initial sucrose) with a relatively longer lag phase. Combination of initial low sucrose concentration (20 g/l) and subsequent sucrose feeding during cultivation (fed-batch culture) improved the cell growth, and a final cell concentration of over 27 g dry cells/l was successfully achieved after 23 days of cultivation. The time course of the taxane formation was related to the cell growth profile. For example, in the cell cultivation with under 30 g/l of initial sucrose, the specific taxane production (content) was relatively low when the cells were under active growth, but sharply increased up to 14 mg/g DW when the cells approached the stationary phase. In the batch cultures with an increase in initial sucrose concentration from 20 to 50 g/l, both the taxane content and productivity were decreased. In the fed-batch cultures, a relatively high taxane content was obtained at the end of cultivation; both a very high taxane production of 274.4 mg/l and a high productivity of 9.3 mg/(l day) were successfully achieved by feeding 20 g/l of sucrose (on day 7) to the cell cultures. © 1999 Elsevier Science Ltd. All rights reserved. Keywords: Taxus chinensis; Sucrose feeding strategy; Taxane production; Plant cell suspension culture; Secondary metabolite
1. Introduction Since the discovery of paclitaxel (Taxol®) and its clinical anticancer activity, paclitaxel and related taxane compounds have attracted great attention around the world. More than 100 new taxanes have now been isolated and identified in Taxus spp., and the diterpenoid family is still expanding. Taxanes can be used as starting materials for semi-synthesis and further modifications for useful compounds and new taxane drugs are under active exploration [1]. Production of taxanes by cell cultures of Taxus spp. is viewed as a promising alternative to plant extraction. Until now, many scientists have focused on cell line selection (for fast growth or higher taxane content),
* Corresponding author. Tel.: +86-21-64252091; fax: + 86-2164253904. E-mail address: [email protected] (J.J. Zhong)
medium modification, precursor addition, elicitation, gas phase composition, culture kinetics, cell immobilization, and continuous cultivation of Taxus spp. cells [2–8]. In plant cell cultures, metabolite productivity is a key factor in determining its process efficiency and commercial potential. A relatively high sucrose concentration can be used to increase cell density and volumetric productivity of secondary metabolites. An increase of sucrose concentration in the medium above the normally used 2–3% level was reported to stimulate the production of taxol and related taxanes in a static study on Taxus bre6ifolia cell cultures [9]. In this paper, the kinetics of both the growth and taxane accumulation by Taxus chinensis cells were investigated in detail under various initial sucrose concentrations. Furthermore, based on our newly obtained information, an efficient feeding strategy was formulated to enhance taxane productivity in the cell suspension cultures.
0032-9592/99/$ - see front matter © 1999 Elsevier Science Ltd. All rights reserved. PII: S 0 0 3 2 - 9 5 9 2 ( 9 9 ) 0 0 0 9 4 - 1
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H.Q. Wang et al. / Process Biochemistry 35 (1999) 479–483
2. Materials and methods
2.1. Plant materials and cell cultures Suspension cells of T. chinensis were cultured in Murashige and Skoog medium supplemented with 0.5 mg 6-benzyladenine/l, 0.2 mg 2,4-dichlorophenoxyacetic acid/l, 0.5 mg naphthaleneacetic acid/l, 100 mg ascorbic acid/l, and 30 g sucrose/l. The cells were subcultured at an interval of 2 weeks in a 250-ml Erlenmeyer flask containing 50 ml medium on a rotary shaker in darkness, 110 rpm and 25°C [10]. In this work, the initial sucrose concentrations were set at 20, 30, 40 and 50 g/l. Furthermore, other cultures with 20 g/l of initial sucrose concentration, were also fed with a dense sucrose solution to make the total amount of sucrose supply identical to 30, 40 and 50 g/l. Before inoculation, the subcultured cells from a number of flasks were thoroughly mixed to provide homogeneous cell inocula to each flask. In all cases, the inoculation density was controlled at 7.0 g DW/l.
2.2. Determination of cell weight The cells from a sampled shake flask were washed with a large amount of distilled water, and filtrated under vacuum. The cells were then weighed as fresh weight. The fresh cells were dried at 50°C to a constant weight, when the dry cell weight was determined. Finally, the cell concentrations based on fresh and dry cell weights were calculated.
[10]. After 2 years of subcultures, taxol accumulation was found to be unstable and finally became undetectable. On the other hand, in HPLC analysis, an unknown elutant with a large peak was detected and isolated and identified by IR, MR and NMR. Its structure was determined to be 2a,5a,10b,14b-tetra-acetoxy-4(20),11-taxadiene (taxuyunnanine C), which was the same as that from the roots of Taxus yunnanensis as reported in 1994 by Zhang et al. [13].
3. Results and discussion
3.1. Effect of initial sucrose concentration on cell growth and sugar consumption As shown in Fig. 1A and B, in the presence of 20 and 30 g/l initial sucrose concentration, the cells entered their growth phase from day 3 with a specific growth rate of 0.12 per day. At high initial sucrose concentrations of 40 and 50 g/l, extended lag phases of 6 and 9 days were observed, respectively. After that, the cells grew very rapidly. The growth rate [(max. cell mass− initial cell mass)/(initial cell mass)/(time)] was 0.10 per day, similar to that under 20 and 30 g/l initial sucrose concentration (Table 1). The final dry cell weight accumulated in the cultures was significantly affected by the initial sucrose level in the medium. The maximum dry biomass obtained was 13.9 (on day 9), 17.0 (on day 14),
2.3. Measurement of medium nutrients Residual sugar concentration was determined as reported by Hodge and Hofreiter [11]. Inorganic phosphate concentration in media was analysed according to the ascorbic acid method [12].
2.4. Taxane extraction and analysis For taxane extraction, 200 mg of powdered dry cells was soaked in 3 ml methanol for 2 days, then ultrasonicated twice for 40 min. The extracts were dried at 25°C. The residue was dissolved by 2 ml dichloromethane and 2 ml distilled water. After sufficient mixing, the mixture was centrifuged at 4000 rpm for 5 min. The organic phase was collected and dried at 25°C, then dissolved in 0.5 ml HPLC grade methanol for HPLC analysis. A reverse-phase column (Whatman Pentafluorophenyl, 5 mm, 250 × 4.6 mm) was used. The mobile phase consisted of acetonitrile and water (40:60, v/v), and the flow rate was 1 ml/min. Taxanes were monitored at a wavelength of 227 nm [10]. In our previous cultivation of T. chinensis cells, taxol was detected although with a low content in the cells
Fig. 1. Dynamic profiles of fresh and dry mass accumulation and sugar consumption in suspension cultures of T. chinensis. A, B, C: cells cultured at four different initial sucrose concentrations, 20 g/l,
30 g/l, 40 g/l, 50 g/l; D, E, F: cultures started with 20 g/l sucrose concentration, and fed sucrose at day 7, + 10 g/l, +20 g/l, +30 g/l. Each value is an average of two to three samples, and the bar in the figure represents the standard error.
H.Q. Wang et al. / Process Biochemistry 35 (1999) 479–483
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Table 1 Effect of initial sucrose concentration and sucrose feeding on cell growth rate (GR), cell yield (Yx/s), maximum taxane production (Pr) and productivity (Pv) in suspension cultures of T. chinensis a Initial sucrose concentration (g/l)
GR (d−1) Yx/s (g g−1) Pr (mg/l) Pv (mg l−1 d−1) a
Sucrose feed (g/l) at day 7
20
30
40
50
10
20
30
0.11 0.36 168.3 7.7
0.11 0.35 206.7 7.3
0.10 0.38 147.4 4.8
0.10 0.41 209.0 5.1
0.10 0.35 192.6 9.4
0.11 0.39 274.4 9.3
0.13 0.41 263.1 7.8
GR is calculated as: (max. cell mass−initial cell mass)/(initial cell mass)/(time).
20.8 (on day 20) and 26.3 g/l (on day 26) at an initial sucrose concentration of 20, 30, 40 and 50 g/l, respectively (Fig. 1B). Compared to the case of 30 g initial sucrose/l, the apparent cell yield over sugar (Yx/ s) was also relatively high at a high initial sucrose concentration of 50 g/l (i.e. 0.41 vs. 0.35 g g − 1) (Table 1). In the case of Vitis 6inifera cell cultures, both a lag phase and a reduced cell concentration were observed under a relatively high sucrose concentration of 50 g/l [14]. For cell cultures of Coleus blumei, a high initial sucrose concentration of 60 g/l led to a high biomass accumulation without an obvious lag phase [15]. With suspension cultures of Perilla frutescens, the growth rate increased with an increase of initial sucrose level up to 60 g/l in the medium [16]. It is clear that initial sucrose concentration is important to the growth of plant cells and its effect is dependent on a specific cell line. The time profile of sugar consumption is shown in Fig. 1C. In all cases, almost all the medium sugar was exhausted during the monitored cultivation period. Sugar consumption under 40 and 50 g/l of initial sucrose level was characterised with two phases as separated on day 12 (for the former case) and 15 (for the later case). This time corresponded to the onset of rapid cell growth in each case. In both cases, the sugar consumption rate in the second phase (days 12–21 and days 15 – 26) was much faster than that in the first phase (days 0 – 12 and days 0–15), i.e. 2.6 versus 1.2 g sugar/(l day). The cell yield (Yx/s) in the second phase was still higher than that in the first phase. For example, at an initial sucrose concentration of 50 g/l, the cell yield was 0.14 and 0.62 g g − 1 in the first and second phase, respectively. Medium phosphate was taken up by the cells at a very fast rate (data not shown). For the cases of low initial sucrose concentrations (20 and 30 g/l), phosphate was depleted by day 5, but its exhaustion was delayed to day 9 and 12 in the cultures with high initial sucrose concentrations of 40 and 50 g/l, respectively.
3.2. Effect of initial sucrose concentration on taxane production The dynamic profile of taxane content of the cells was dependent on a different initial sucrose concentration in medium (Fig. 2A). At a relatively high initial sucrose concentration of 50 g/l, a significant increase of taxane accumulation in the cells was observed during the lag phase; after that, the taxane content showed a decrease. However, at the end of cultivation, it was increased again from 5 mg/g DW on day 23 to 8.3 mg/g DW on day 29. A similar taxane accumulation pattern was observed in cells in media containing 40 g/l initial sucrose. Although the increase of taxane content in cells during the lag phase was less significant, its final value (on day 26) was comparable to that under 50 g/l of initial sucrose. At the low initial sucrose concentrations of 20 and 30 g/l, the taxane content showed a decreasing trend during the exponential growth phase (days 3–7 and days 3–12, respectively). When growth approached stationary phase, taxane accumulation in-
Fig. 2. Time courses of content and production of taxuyunnanine C in suspension cultures of T. chinensis. A, B: cells cultured at four different initial sucrose concentrations, 20 g/l, 30 g/l, 40 g/l, 50 g/l; C, D: cultures started with 20 g/l sucrose concentration, and fed sucrose at day 7, +10 g/l, +20 g/l, +30 g/l. Each value is an average of two to three samples, and the bar in the figure represents the standard error.
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creased sharply to a level of 14 mg/g DW within 5 days. Fig. 2B showed the time courses of total taxane production in cell cultures under the various initial sucrose concentrations. In all cases, the total taxane amount showed a significant increase in the later part of cultivation due to its higher content and/or much higher biomass level. Under a high initial sucrose concentration of 50 g/l, although the taxane content was relatively low, its highest volumetric production (209.0 mg/l) was achieved on day 29 due to a relatively high biomass concentration in this case. It is clear that raising the cell culture density was effective in the improving taxane production by T. chinensis cells. The significance of biomass accumulation was also claimed in other Taxus cell cultures [2]. On the other hand, for taxane productivity, with an increase in initial sucrose concentration from 20 to 50 g/l, this was decreased from 7.7 to 5.1 mg/(l day) due to the long time of cultivation for the later case (Table 1). At low initial sucrose concentrations, the taxane accumulation pattern seemed to be antagonistic with cell growth. Taxane content decreased in the exponential growth phase, while a large amount of taxane accumulated when the growth was approaching or had entered its stationary phase. In cell cultures of T. baccata, Srinivasan et al. also reported that taxol was produced exclusively during a period of declining growth rate [7]. At high initial sucrose concentrations, the increase of taxane content during the lag phase may be due to the effects of high osmotic pressure, as claimed by Zhang et al. in suspension cultures of Panax notoginseng for production of ginseng saponin [17]. In cell cultures with a relatively high initial sucrose concentration, when the cell growth entered stationary phase, taxane was not synthesised as much as under a low initial sucrose concentration. The above information indicates that the effect of initial sucrose level on the kinetics of taxane formation is quite complicated. In cell cultures of T. bre6ifolia, more than 60 g/l of sucrose in a production medium was claimed to be desirable for production of taxol and related taxane (cephalomannine) [9]. Since only a static result was presented in that study, it was not clear how an initial sucrose concentration affected the kinetics of both cell growth and taxol synthesis. The positive effects of a relatively high initial sucrose concentration on metabolite production were also reported in a number of other plant cell cultures [15 – 18].
3.3. Effect of sucrose feeding on cell growth and sugar consumption From the above experiments, it is clear that a shorter lag phase, higher taxane content, and higher taxane productivity were achieved at a low initial sucrose concentration (e.g. 20 g/l), while the final biomass level and total taxane production titre were high under a high
initial sucrose concentration (e.g. 50 g/l). Thus, it is logical for us to consider a series of new cell cultures with low initial sucrose concentration (20 g/l) combined with sucrose feeding at a later stage of cultivation in order to enhance both the specific production (content) and productivity of taxanes while maintaining a high final cell density in the cell cultures. As shown in Fig. 1D and E, cells grown in 20 g/l initial sucrose concentration with sucrose feeding on day 7 displayed a similar growth pattern to those of batch cultures under low initial sucrose concentrations. In the case of feeding 30 g sucrose/l on day 7, the cell growth seemed to be repressed again during the period of days 7–12. This was assumed to be due to the osmotic shock caused by a relatively high medium sucrose concentration (about 42 g/l) immediately after sucrose feeding. Compared to that in batch cultures with equal amounts of total sugar (40 and 50 g/l), the growth rates in the corresponding two fed-batch cultures increased by 10– 30% (Table 1). The reason was that a shorter time was required to reach a relatively high biomass level in the later case. For example, at day 15, 19.2 and 16.5 g DW/l were reached by feeding 20 and 30 g/l of sucrose, respectively, while only 15.6 and 9.84 g/l were reached for initial sucrose concentrations of 40 and 50 g/l, respectively. The maximum dry weights were comparable to those of batch cultures. They were 16.9 (at day 14), 22.3 (at day 20) and 27.2 g/l (at day 23) for sucrose feed of 10, 20 and 30 g/l, respectively. The medium sucrose fed at day 7 was also consumed rapidly in a similar way to that of batch cultures at low initial sucrose concentrations (Fig. 1F). In all cases, the time of maximum growth reached corresponded to medium sugar depletion.
3.4. Effect of sucrose feeding on taxane production Fig. 2C and D show the dynamic profiles of the specific production (i.e. content) and total production of taxuyunnanine C under sucrose feeding. The patterns of taxane formation were similar for all three cases of feeding experiments (Fig. 2C). The taxane content was relatively low and stable (around 6 mg/g DW) during the exponential growth phase. After that, a significant increase in taxane formation was observed after the beginning of the slowing-down phase of the cell growth in the cases of feeding 10 (at day 12), 20 (at day 21) and 30 g/l of sucrose (at day 24). At the end of cultivation, a high taxane content of 14 mg/g DW was reached in all cases. Compared to that in batch cultures under high initial sucrose concentrations (40 and 50 g/l), the increase of taxane content in the later stage of cultivation was much more significant in the experiments with a sucrose feed of 20 and 30 g/l. The final amount of total taxane produced was 192.6 (at day 14), 274.4 (at day 23) and 263.1 mg/l (at day 26) for the cases with sucrose feeding of 10, 20 and 30 g/l, respectively (Fig. 2D and
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Table 1). For taxane productivity, it was higher in feeding experiments than in their corresponding batch cultures. The highest productivity of 9.3 mg/(l day) was successfully achieved in the both cases of feeding 10 and 20 g sucrose/l (Table 1). Compared with that at high initial sucrose concentrations, it is evident that sucrose feeding improved both taxane production (Fig. 2D vs. Fig. 2B) and productivity (Table 1). The manipulation of medium sucrose level based on the characteristics of cell growth and metabolite production was very important and effective for optimal cell growth and metabolite production. For example, Zhang et al. reported a successful medium sucrose manipulation for hyper-production of saponin and polysaccharide in suspension cultures of Panax notoginseng [18]. In the present case, both the initial sucrose concentration and the total amount of medium sucrose were important for the enhancement of both production and productivity of taxuyunnanine C. In conclusion, a relatively high initial sucrose concentration above 30 g/l could increase the cell density, but decreased taxane productivity in suspension cultures of T. chinensis. Taxane accumulation was significantly increased when growth approached its stationary phase. The operation of a cell culture at a low initial sucrose concentration (20 g/l) in combination with sucrose feeding at a later stage could effectively improve cell growth and both the production and productivity of taxane. The above results are useful for the understanding of the sucrose effect in Taxus cell cultures and for largescale cultivation of plant cells for production of anticancer taxane diterpenes.
Acknowledgements This work was supported by National Natural Science Foundation of China (No. 29606003). We also thank Z.W. Pan and Y.Z. Zhang for their helpful assistance during the experiments.
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