Inhibition of paclitaxel and baccatin III accumulation by mevinolin and fosmidomycin in suspension cultures of Taxus baccata

To achieve a better understanding of the metabolism and accumulation of paclitaxel and baccatin III in cell cultures of Taxus, inhibitors of the early steps in the terpenoid pathway were applied to a cell suspension culture of Taxus baccata: fosmidomycin as an inhibitor of the non-mevalonate branch of the pathway, and mevinolin as an inhibitor of the mevalonate branch. Synthesis of both taxanes in the cell suspension was first increased when cultured in the product formation medium supplemented with methyljasmonate (100 microM). The product formation medium was selected after assaying 24 different culture media. When fosmidomycin (200 microM) was added to the product formation medium together with the elicitor, the accumulation of paclitaxel and baccatin III was reduced by up to 3.0 and 1.5 times, respectively, whereas the inhibitory effect of mevinolin (1 microM) was only clearly exerted in the case of paclitaxel. Under the conditions of our experiment, we conclude that in the synthesis of both taxanes, the non-mevalonate pathway is the main source of the universal terpenoid precursor isopentenyl diphosphate (IPP).     

Effects of immobilization by entrapment in alginate and scale-up on paclitaxel and baccatin III production in cell suspension cultures of Taxus baccata

Paclitaxel and baccatin III-producing cells of Taxus baccata were immobilized within Ca(2+)-alginate beads. Under established optimum conditions for the biosynthesis of both taxanes, the yields of paclitaxel and baccatin III in shake-flask cultures of free cells increased by factors of up to 3 and 2, respectively, in the corresponding cultures of immobilized cells. Although the scale-up from shake-flask to bioreactor culture usually results in reduced productivities when both free and immobilized cells were grown in the same optimum conditions in three different bioreactor types (Stirred, Airlift, and Wave) running for 24 days in a batch mode and with the system optimized in each case, there was a considerable increase in the yields of paclitaxel and baccatin III. Among the reactors, the Stirred bioreactor was the most efficient in promoting immobilized cell production of paclitaxel, giving a content of 43.43 mg.L(-1) at 16 days of culture, equivalent to a rate of 2.71 mg.L(-1).day(-1). To our knowledge, the paclitaxel productivity obtained in this study is one of the highest reported so far by academic laboratories for Taxus species cultures in bioreactors.     

The kinetics of taxoid accumulation in cell suspension cultures of Taxus following elicitation with methyl jasmonate

Cell suspension cultures of Taxus canadensis and Taxus cuspidata rapidly produced paclitaxel (Taxol) and other taxoids in response to elicitation with methyl jasmonate. By optimizing the concentration of the elicitor, and the timing of elicitation, we have achieved the most rapid accumulation of paclitaxel in a plant cell culture, yet reported. The greatest accumulation of paclitaxel occurred when methyl jasmonate was added to cultures at a final concentration of 200 microM on day 7 of the culture cycle. The concentration of paclitaxel increased in the extracellular (cell-free) medium to 117 mg/day within 5 days following elicitation, equivalent to a rate of 23.4 mg/L per day. Paclitaxel was only one of many taxoids whose concentrations increased significantly in response to elicitation. Despite the rapid accumulation and high concentration of paclitaxel, its concentration never exceeded 20% of the total taxoids produced in the elicited culture. Two other taxoids, 13-acetyl-9-dihydrobaccatin III and baccatin VI, accounted for 39% to 62% of the total taxoids in elicited cultures. The accumulation of baccatin III did not parallel the pattern of accumulation for paclitaxel. Baccatin III continued to accumulate until the end of the culture cycle, at which point most of the cells in the culture were dead, implying a possible role as a degradation product of taxoid biosynthesis, rather than as a precursor.     

Effect of the culture medium and biotic stimulation on taxane production in Taxus globosa Schltdl in vitro cultures

Taxus globosa is the only species of the Taxus genus that grows in Mexico. In this study, callus cultures from leaves and young shoots of T. globosa were established in Gamborg’s B5 medium supplemented with 2,4-dichlorophenoxiacetic acid (2 mg/L), kinetin (0.5 mg/L) and gibberellic acid (0.25 mg/L). Callus growth and taxane production were evaluated using two culture media: Woody Plant Medium and Gamborg’s B5 supplemented with picloram (2 mg/L), kinetin (0.1 mg/L) and gibberellic acid (0.5 mg/L). The effect of the inoculum size (50, 100 and 150 g FW/L) and culture media (Woody Plant Medium and Gamborg’s B5) with and without the presence of methyl jasmonate (100 μM) on T. globosa cell suspensions was assessed. Taxane analysis revealed that the calli in Gamborg’s B5 produced taxol (50 μg/g DW), baccatin III, 10-deacetyl baccatin III and 10-deacetyl taxol. Woody Plant Medium also induced the production of taxol, although to a lesser extent. The optimum inoculum size was 50 g FW/L. In cell suspension cultures, both media had a significant effect on taxane production when supplemented with methyl jasmonate. In Woody Plant Medium, at day 14, a total concentration of 197.999 μg/L of taxol, 160.622 μg/L of baccatin III, 633.724 μg/L of 10-deacetyl baccatin III and 229.611 μg/L 10-deacetyl taxol were obtained, with total excretion of baccatin III and 10-deacetyl taxol to the culture medium. In Gamborg’s B5, cephalomanine was obtained at a concentration of 91.428 μg/L without elicitation, and all taxanes were excreted to the medium to a variable extent.     

The configuration of methyl jasmonate affects paclitaxel and baccatin III production in Taxus cells

All stereoisomers of methyl jasmonate (MJA) were prepared, and their effects on cell yield and promotion of paclitaxel (Taxol) and baccatin III production investigated in cell suspension cultures of Taxus media. (3R,7S)-MJA showed the strongest cell growth inhibition, followed by (3R,7R)-MJA. In contrast, (3S,7R)- and (3S,7S)-MJA had very low inhibitory effects, indicating that this inhibition depends largely on the (3R)-configuration. In terms of the promotion of paclitaxel and baccatin III production, (3R,7R)-MJA had the highest activity. Although it showed considerable activity at low concentration, at higher concentrations the activity was decreased due to strong inhibition of cell growth. Interestingly, paclitaxel and baccatin III contents increased even at a high (3S,7R)-MJA concentration, whereas the other isomers had the opposite effects. These findings are interpreted to suggest that the optimum configuration is (3R,7R), the (3R)-configuration not being indispensable, and that the (7R)-configuration is suitable for the promotion of paclitaxel and baccatin III production.     

Cell cycle analysis of Taxus suspension cultures at the single cell level as an indicator of culture heterogeneity

Single cell growth and division was measured via flow cytometry in order to characterize the metabolic variability of Taxus cuspidata suspension cultures, which produce the valuable secondary metabolite Taxol. Good agreement was observed between the cell cycle distribution and biomass accumulation over the batch culture period. Specific growth rates of 0.13 days(-1) by fresh weight and 0.15 days(-1) by dry weight were measured. Elicitation with methyl jasmonate (MJ) significantly decreased both cell cycle progression and biomass accumulation, as the specific growth rate decreased to 0.027 days(-1) by fresh and dry weight. Despite the decrease in biomass accumulation for MJ elicited cultures, sucrose utilization was not significantly different from control cultures. MJ elicitation also increased the accumulation of paclitaxel and other taxanes. The accumulation of upstream taxanes (baccatin III and 10-deactylbaccatin III) increased during exponential growth, reached a maximum around day 12, and then declined throughout the stationary phase. The paclitaxel concentration increased during both exponential growth and stationary phase, reaching a maximum around days 20-25. Throughout the culture period, greater than 70% of the cells were in G(0)/G(1) phase of the cell cycle. Studies using bromodeoxyuridine (BrdU) incorporation showed that approximately 65% of the Taxus cells are noncycling, even during exponential growth. Although the role of these cells is currently unknown, the presence of a large, noncycling subpopulation can have a significant impact on the utilization of plant cell culture technology for the large-scale production of paclitaxel. These results demonstrate that there is a high degree of metabolic heterogeneity in Taxus cuspidata suspension cultures. Understanding this heterogeneity is important for the optimization of plant cell cultures, particularly the reduction of production variability.     

Paclitaxel and baccatin III production induced by methyl jasmonate in free and immobilized cells of Taxus baccata

The effects of 100 and 200 μM methyl jasmonate (MJA) on cell proliferation and paclitaxel and baccatin III production were investigated in free and alginate immobilized cells of Taxus baccata growing in a selected product formation culture medium. The greatest accumulation of paclitaxel (13.20 mg dm⁻³) and baccatin III (4.62 mg dm⁻³) occurred when 100 μM MJA was added to the culture medium of cells entrapped using a 1.5 and 2.5 % alginate solution. The effects of different treatments on the viability of cultured cells and their capacity to excrete both taxanes into the surrounding medium were considered.     

Involvement of nitric oxide in elicitor-induced defense responses and secondary metabolism of Taxus chinensis cells

This work was to characterize the generation of nitric oxide (NO) in Taxus chinensis cells induced by a fungal elicitor extracted from Fusarium oxysporum mycelium and the signal role of NO in the elicitation of plant defense responses and secondary metabolite accumulation. The fungal elicitor at 10-100 microg/ml (carbohydrate equivalent) induced a rapid and dose-dependent NO production in the Taxus cell culture, which exhibited a biphasic time course, reaching the first plateau within 1 h and the second within 12 h of elicitor treatment. The NO donor sodium nitroprusside potentiated elicitor-induced H2O2 production and cell death but had little influence on elicitor-induced membrane K+ efflux and H+ influx (medium alkalinization). NO inhibitors Nomega-nitro-L-arginine and 2-phenyl-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide partially blocked the elicitor-induced H2O2 production and membrane ion fluxes. Moreover, the NO inhibitors suppressed elicitor-induced activation of phenylalanine ammonium-lyase and accumulation of diterpenoid taxanes (paclitaxel and baccatin III). These results suggest that NO plays a signal role in the elicitor-induced responses and secondary metabolism activities in the Taxus cells.     

条件培养液对红豆杉细胞Paclitaxel生产的促进作用

在两步法红豆杉（Taxus chinensis)细胞悬浮培养体系的生产阶段,加入从生长阶段悬浮培养物中制得的条件培养液（conditioned Medium,CM)既能促进细胞的生长,又能提高紫杉醇（paclitaxel)的产率,解决了生产培养时,细胞生长受抑制的问题,特别是,取自生长12天的细胞悬浮培养物的CM按体积分数为25％添加到新鲜生产培养基中时,可使细胞紫杉醇最高产量达28．5mg/L,细胞干重达32．3g/L,分别是对照的2．4倍和2．2倍,对CM中的蔗糖,果糖,NO3－和PO4－3等的含量的进行了分析。     

Enhancement of Taxol production and excretion in Taxus chinensis cell culture by fungal elicitation and medium renewal

An endophytic fungus, Aspergillus niger, isolated from the inner bark of a Taxus chinensis tree, was used as an elicitor to stimulate the Taxol (paclitaxel) production in a Taxus chinensis cell suspension culture. Different elicitor doses and elicitation times were tested in a batch culture; and the highest volumetric Taxol yield was achieved when 40 mg of the fungal elicitor (carbohydrate equivalent) l(-1) was added to the culture during the late exponential-growth phase. The elicitation resulted in a more than two-fold increase in the Taxol yield and about a six-fold increase in total secretion. The Taxol yield was further improved substantially by applying medium renewal and re-elicitation to the culture. In particular, with repeated medium renewal (in a way similar to medium perfusion) and a second elicitation of the culture, the volumetric Taxol yield was increased to 67.1+/-7.5 mg l(-1), which was about seven times the amount obtained in the non-elicited batch culture. The Taxol productivity of the perfusion-like culture with repeated fungal elicitation was 1.5 mg l(-1) day(-1), which was about 40% higher than that of the elicitor-treated batch culture and three times the productivity of the non-elicited batch culture.     

Recovery of extracellular paclitaxel from suspension culture of Taxus chinensis by adding inorganic salts

A new and simple method was developed to recover paclitaxel from the extracellular culture medium of Taxus chinensis. More than 80% of paclitaxel in the medium was obtained by adding 7.8 mM MgSO₄ or greater and then centrifuging. The concentration of baccatin III in the supernatant did not change after MgSO₄ treatment while paclitaxel was precipitated in the pellet. This method was used to recover paclitaxel without baccatin III from the extracellular culture medium of Taxus species.     

Improved β-thujaplicin production in Cupressus lusitanica suspension cultures by fungal elicitor and methyl jasmonate

Production of a novel antimicrobial tropolone, beta-thujaplicin, in Cupressus lusitanica suspension cultures was studied by using a variety of chemicals and fungal elicitors. Sodium alginate, chitin, and methyl jasmonate resulted in 2-, 2.5-, and 3-fold higher beta-thujaplicin production, respectively, than in the control. Significantly improved beta-thujaplicin production (187 mg l(-1)) was obtained using a high cell density (180-200 g l(-1)) and fungal elicitor treatment [10 mg (g fresh cells)(-1)] in a production medium with a high ferrous ion concentration (0.3 mM). This improved volumetric productivity was 3- to 4-fold higher than obtained under standard conditions. A synergistic effect of fungal elicitor and ferrous ion on beta-thujaplicin production was also suggested by our study. Plant cell culture technology is a promising alternative for producing a large variety of secondary metabolites that are widely used as food additives, pharmaceuticals, and dairy products (Verpoorte et al. 1999). Thus, beta-thujaplicin production by plant cell cultures was developed with the goal of commercial application (Berlin and Witte 1988; Itose and Sakai 1997; Ono et al. 1998). However, the production of beta-thujaplicin by plant cell cultures is still not competitive for use in industrial applications. In this study, we assessed the effects of methyl jasmonate, alginate, chitin, and fungal elicitor on beta-thujaplicin production; we obtained a significantly elevated beta-thujaplicin production by using an improved culture strategy.     

Isolation of an endophytic fungus producing baccatin III from Taxus wallichiana var. mairei

The objective of this study was to isolate endophytic fungi producing baccatin III from yew for the purpose of baccatin III and paclitaxel manufacture. Surface sterilized bark of Taxus wallichiana var. mairei was used as source material with potato dextrose agar culture medium for isolation of endophytic fungi. Fungal cultures were extracted with a mixture of chloroform/methanol (1:1, v/v) and the baccatin III in the extracts was determined and authenticated with LC–MS. An endophytic fungus that produced baccatin III was identified by ITS rDNA and 26S D1/D2 rDNA sequencing. A total of 192 endophytic fungal strains were isolated from T. wallichiana var. mairei. Only one of the 192 strains produced baccatin III and it was identified as Diaporthe phaseolorum. The productivity of this strain cultured in PDA culture medium was 0.219 mg/l. The isolated endophytic fungus produced baccatin III at a relatively high level and shows promise as a producing strain for baccatin III and paclitaxel manufacture after strain improvement.     

Improved Taxol production by combination of inducing factors in suspension cell culture of Taxus baccata

To date enormous attempts have been devoted to improve Taxol production exploiting various methodologies from bioprocess engineering to biotechnological and synthetic approaches. We have developed a 2-stage suspension cell culture of Taxus baccata L. using modified B5 medium in order to improve cell growth as well as productivity. After callus induction and cell line selection, B5 medium was supplemented with vanadyl sulfate (0.1 mg/l), silver nitrate (0.3 mg/l) and cobalt chloride (0.25 mg/l) at the first day of stage I culture to maximize cell growth. This medium was further supplemented with sucrose (1%) and ammonium citrate (50 mg/l) on day 10 and sucrose (1%) and phenylalanine (0.1 mM) on day 20 (i.e., biomass growth medium). At stage II (day 25), two different concentrations of several elicitors such as methyl jasmonate (10 or 20 mg/l), salicylic acid (50 or 100 mg/l) and fungal elicitor (25 or 50 mg/l) were added to the biomass growth medium with the aim of improving cellular productivity. For morphological analysis, microscopic inspection was carried out during cultivation. Cell-associated and extracellular amount of Taxol were detected and measured using HPLC methodology. At stage I, overall Taxol amount of biomass growth medium was 13.75 mg/l (i.e., 5.6-fold higher than that of untreated B5 control). At stage II, treated cells with methyl jasmonate (10 mg/l), salicylic acid (100 mg/l) and fungal elicitor (25 mg/l) produced the highest amount of Taxol (39.5 mg/l), which is 16-fold higher than that of untreated B5 control (2.45 mg/l). Microscopic analyses of Taxus cells in suspension cultures showed various positional auto-fluorescence showing direct correlation with Taxol production. Our studies revealed that intervallic supplementation of B5 medium with combination of biomass growth factors at stage I and mixture of elicitors at stage II could significantly increase Taxol production. Thus, we suggest that the exploitation of this methodology may improve the production of Taxol since demands for Taxol pharmaceuticals are increasingly growing and resource paucities have limited its direct harvesting from Taxus trees.     

杜仲细胞悬浮培养产黄酮及其动力学研究

本文应用正交设计对杜仲细胞悬浮培养的基本培养基和植物生长物质浓度进行了筛选,并对影响杜仲细胞悬浮培养和总黄酮含量的不同因素进行了考察。结果表明,B5培养基+0.5mg/L NAA+0.6mg/L 6-BA、蔗糖30g/L、初始pH 5.0-5.5、接种量20g(FW)/L以及摇床转速110r/min为杜仲细胞悬浮培养的适宜条件。通过对杜仲悬浮细胞生长和代谢动力学的分析表明：杜仲细胞悬浮培养生长符合Logistic生长模型,最大比生长速率( m)为0.417d-1;细胞基于蔗糖的真正比生长得率(YG)与维持系数(m)分别为0.619g/g和0.0206g/(g·d-1);黄酮合成属部分生长耦联型,可用Luedeking-Piret模型进行描述。研究结果为杜仲细胞大规模悬浮培养生产天然活性成分奠定了基础。     

Variation of taxane content in needles of Taxus x media cultivars with different growth characteristics

Needles from 17 different Taxus x media cultivars, belonging to 4 groups showing different growth characteristics, were analyzed using high performance liquid chromatography for their content of 10-deacetylbaccatin III, baccatin III, cephalomannine and paclitaxel (Taxol). The 4 Taxus x media cultivar groups were: 1.) medium to fast growing and upright form; 2.) slow growing and upright form; 3.) fast growing and spreading form; and 4.) slow growing and spreading form. The purpose of this study was to identify yew cultivars of fast growth rate, upright growth and high taxane content in their needles. The highest content of paclitaxel was found in 'Coleana' of group 1 (378 microg/g of the extracted dry weight). Three cultivars in group 1, 'Coleana', 'Stovekenii' and 'Hicksii', make good candidates for taxane extraction because of their high paclitaxel and 10-deacetylbaccatin III content, fast biomass accumulation and upright growing form. They are also good starting materials to develop alternative methods for the production of paclitaxel and its analogous compounds through modern biotechnology approaches.     

One step more towards taxane production through enhanced Taxus propagation

We have developed a high-yielding procedure for the in vitro propagation of juvenile material of Taxus baccata involving a combination of seed handling and culture on WP culture medium supplemented with sucrose (2%), activated charcoal (0.5%) and BAP (22.19 mM) for 30 days, followed by 40 days on hormone-free medium. Shoot apical ends should be decapitated to obtain propagation rates up to 12- to 18-fold per subculture period (70 days). In this way the high genetic variability of the juvenile material can be used in the most productive way. In addition to producing large numbers of yew plants (difficult to get by traditional methods), this procedure allows the fast screening of individuals for their taxane content. A negative correlation between growth and secondary metabolite content was found for paclitaxel. The positive correlation with 10-deacetyl baccatin III accumulation reflects once more the commercial viability of using 10-deacetyl baccatin III extraction as an alternative to taxane production, but this time opening up the possibility of selecting genotypes with both characteristics: fast growth and high productivity. Received: 12 July 1999 / Revision received: 22 November 1999 / Accepted: 22 November 1999     

Enhancement of paclitaxel production by temperature shift in suspension culture of Taxus chinensis

Effect of temperature shift during culture period on cell growth and paclitaxel was investigated to optimize paclitaxel production in suspension culture of Taxus chinensis. Cell growth showed the optimum at 24 degrees C while paclitaxel synthesis showed the maximum at 29 degrees C. To minimize the inhibitory effect of higher temperature on cell growth, temperature was shifted after a certain period of culture time at 24 degrees C. Paclitaxel synthesis in plant cell culture increased dramatically during day 14 to day 21 regardless of treatment, reaching the maximum production of 137.5 mg paclitaxel/L. When the temperature was maintained at 29 degrees C after day 21, the specific productivity of paclitaxel was sustained for prolonged period of 42 days. The possible relationship between temperature and paclitaxel synthetic pathway was also suggested.     

真菌诱导子对悬浮培养西洋参细胞的生理效应

报道了不同真菌诱导子对悬浮培养的西洋参（Ｐａｎａｘｑｕｉｎｑｕｅｆｏｌｉｕｍ）细胞生长、皂甙和多糖合成,以及细胞内和培养液中过氧化物酶活性的生理效应。悬浮培养的西洋参细胞经刺盘孢菌（Ｃｏｌｌｅｔｏｔｒｉｃｈｕｍｎｉｃｏｌｔｉａｎａｅ）丝体诱导子处理后,总皂甙产率可由对照的２９６ｍｇ／Ｌ增加到６７９ｍｇ／Ｌ（约占细胞干重的（１６．３％）,比对照提高约１．３倍,而且总皂甙的８５％排放在培养液中;经黑曲霉（Ａｓｐｅｒｇｉｌｌｕｓｎｉｇｒａｎ）诱导子处理后,细胞多糖含量可达到１１．７９％（细胞干重）,比对照增加１倍多。初步纯化的刺盘孢菌丝体诱导子和尖孢镰刀菌（Ｆｕｓｕｒｉｕｍｏｘｙｓｐｏｒｕｍ）滤液诱导子在诱导处理前期能明显促进西洋参细胞生长,同时细胞内及培养液中过氧化物酶活性显著增加;随时间延长,细胞生长和酶活性逐步受到抑制。