Taxol production in bioreactors: Kinetics of biomass accumulation, nutrient uptake, and taxol production by cell suspensions of Taxus baccata

The kinetics of biomass accumulation, nutrient uptake and taxol production of Taxus baccata cell suspensions were examined in three bioreactor configurations, viz. 250-mL Erienmeyerflasks, 1-L working volume pneumatically mixed (PMB), and stirred tank (STB) bioreactors. Qualitatively similar kinetics were observed in all three bioreactor types. Biomass accumulation and specific nutrient uptake rates exhibited biphasic characteristics. Carbohydrate uptake and biomass accumulation substantially ceased when phosphate was depleted from the medium. Phosphate was identified as a possible growth-limiting nutrient. Taxol accumulated exclusively in the second phase of growth. A maximum taxol concentration of 1.5 mg/L was obtained in the PMB which was fivefold greater than that obtained in the Erienmeyer flasks and the STB, but the relative kinetics of taxol production was the same in all three reactor types. Biomass yields were calculated from the kinetic data and a stoichiometry for biomass formation was evaluated. The similarity of kinetics in the three bioreactor configurations suggests that taxol production by T. baccata cell suspensions is amenable to scateup. (c) 1995 John Wiley & Sons, Inc.     

红豆杉细胞悬浮培养结构化数学模型的探讨

用１０Ｌ机械搅拌式生物反应器悬浮培养红豆杉细胞,得到细胞生长、基质消耗和紫杉醇合成动力学曲线。经过代谢动力学分析建立了结构化数学模型。并将模型值与实验值进行比较,结果表明模型预测值与实验值较吻合。     

Monoterpenoid oxindole alkaloid production by Uncaria tomentosa (Willd) D.C. cell suspension cultures in a stirred tank bioreactor

Cell growth, monoterpenoid oxindole alkaloid (MOA) production, and morphological properties of Uncaria tomentosa cell suspension cultures in a 2-L stirred tank bioreactor were investigated. U. tomentosa (cell line green Uth-3) was able to grow in a stirred tank at an impeller tip speed of 95 cm/s (agitation speed of 400 rpm), showing a maximum biomass yield of 11.9 +/- 0.6 g DW/L and a specific growth rate of 0.102 d(-1). U. tomentosa cells growing in a stirred tank achieved maximum volumetric and specific MOA concentration (467.7 +/- 40.0 microg/L, 44.6 +/- 5.2 microg/g DW) at 16 days of culture. MOA chemical profile of cell suspension cultures growing in a stirred tank resembled that of the plant. Depending on culture time, from the total MOA produced, 37-100% was found in the medium in the bioreactor culture. MOA concentration achieved in a stirred tank was up to 10-fold higher than that obtained in Erlenmeyer flasks (agitated at 110 rpm). In a stirred tank, average area of the single cells of U. tomentosa increased up to 4-fold, and elliptical form factor increased from 1.40 to 2.55, indicating enlargement of U. tomentosa single cells. This work presents the first report of U. tomentosa green cell suspension cultures that grow and produce MOA in a stirred tank bioreactor.     

补料培养与溶氧控制联合应用对红豆杉细胞培养的影响

为进一步提高红豆杉 (Taxuschinensis (Pilg.)Rehd .)细胞培养过程中紫杉醇的产量 ,采用细胞悬浮培养方法研究了补料培养与溶氧控制联合应用对紫杉醇产量的影响。 5L反应器中补料培养研究表明 ,培养过程中第 16天添加含 2 0g/L蔗糖的补料培养液有利于细胞的生长及紫杉醇的合成。 2 0L反应器中补料培养的研究结果表明 :2 0 %饱和度培养时紫杉醇含量最高 (0 .98mg/gDW) ,但 4 0 %～ 6 0 %溶氧饱和度能提高紫杉醇的产量。进一步研究表明 ,细胞在 6 0 %溶氧饱和度培养 2 0d后转入 2 0 %溶氧饱和度继续培养 12d ,能显著提高紫杉醇产量。补料培养与溶氧控制联合应用时 ,2 0L反应器中红豆杉细胞培养紫杉醇产量可达 18.7mg/L。     

Large-scale growth and taxane production in cell cultures of Taxus cuspidata (Japanese yew) using a novel bioreactor

 A novel type of bioreactor was successfully developed for the production of taxol and its precursors by culturing cells of Taxus cuspidata (Japanese yew) on a pilot-scale. Rapidly growing cell lines were selected from callus cultures derived from immature embryos of yew. The cells were inoculated in 20-l capacity bioreactors of different types to test the growth performance. The models of small-scale bioreactors incorporated in this study included a balloon-type bubble bioreactor (BTBB), a bubble-column bioreactor (BCB), a BCB with a split-plate internal loop, a BCB with a concentric draught-tube internal loop, a BCB with a fluidized bed bioreactor, and two different models of stirred tank reactors. Among the reactors, BTBB appeared to be the most efficient in promoting cell growth. The doubling time of cell growth in BTBB was 12 days with a 30% inoculation cell density. The optimum time for medium replacement or feeding was 12–15 days after inoculation as determined by monitoring both the levels of sugars and medium conductivity. When yew tree cells were grown in different sizes (100–500-l) of BTBBs, more than 70% cell viability was recorded at the time of harvest. The growth pattern of the cells in the pilot-scale BTBB appeared to be the same as that of cells in the 20-l bioreactors. Approximately 3 mg/l of taxol and 74 mg/l total taxanes were obtained after 27 days of culture. Received: 6 April 1999 / Revision received: 23 August 1999 / Accepted: 31 August 1999     

超声波对红豆杉悬浮细胞生长及紫杉醇释放的研究

分析了超声波对中国红豆杉悬浮细胞培养的生长,紫杉醇合成及释放的影响,细胞对不同强度及作用时间的超声波反应不同,用38kHz,120s的超声强度处理悬浮细胞,紫杉醇胞外释放率由对照的10％左右提高到40－50％,总产量提高了47％,超声波处理植物细胞,提供了在保持细胞生长的前提下有效刺激胞内次生代谢物的简易操作方法。     

Model of dissolved organic carbon distribution for substrate-sufficient continuous culture.

The growth yields (Yobs) are greater under substrate-limited conditions than those under substrate-sufficient conditions in continuous cultures. This indicates that the excess substrate should cause uncoupling between anabolism and catabolism. It appears that the excess substrate could determine metabolic pathways of microorganisms, which further control dissolved organic carbon (DOC) distribution under substrate-sufficient conditions. However, how to quantitatively describe the DOC distribution remains unclear in substrate-sufficient continuous culture. Based on a balanced DOC reaction, a DOC distribution model was developed in relation to residual substrate concentration for substrate-sufficient continuous cultures. Results showed that a considerable portion of the DOC consumed was directly oxidized to carbon dioxide through energy spilling under substrate-sufficient conditions. The proposed model for the first time quantified the DOC distribution between nongrowth-associated and growth-associated metabolisms of cells. The proposed model was verified with literature data very well.     

Importance of glucose-6-phosphate dehydrogenase in taxol biosynthesis in <Emphasis Type="Italic">Taxus chinensis</Emphasis> cultures

The roles of glucose-6-phosphate dehydrogenase (G6PDH) in paclitaxel production were investigated in cell suspension cultures of Taxus chinensis. In the normal cultures, the trend of G6PDH activity was similar to that of cell growth. Addition of glutamate increased G6PDH activity, while dehydroepiandrosterone (DHEA) decreased G6PDH activity. In elicitor-treated cultures, cell growth was depressed, while G6PDH activity and taxol production were enhanced compared with the control. Glutamate recovered the depression of cell growth, and resulted in further increase in G6PDH activity and taxol production. Contrarily, DHEA exacerbated the depression of cell growth, and decreased G6PDH activity and taxol production induced by fungal elicítor. The results indicated that G6PDH played a critic role of taxol production by affecting cell viability.     

红豆杉悬浮细胞放大培养的细胞生长与紫杉醇合成动力学

研究了在Murashige&skoog s(MS)和 6 2号两种不同的培养基中 ,红豆杉细胞悬浮细胞从摇瓶到 1 0L机械通气搅拌式反应器放大培养过程中细胞生长与紫杉醇合成动力学 .结果表明 :尽管在不同的培养条件下 ,细胞生长曲线均呈现“S”型 .紫杉醇在延迟期与指数生长期中基本上没有积累 ,而且随着培养规模的增大 ,紫杉醇的含量逐渐降低 .进一步对各级放大培养的细胞生长 ,比生长率与胞内外紫杉醇合成量进行分析 ,发现MS利于细胞生长但不利于紫杉醇合成 ,而 6 2号则相反 .根据此文的结果 ,提出了红豆杉细胞培养条件的优化和大规模细胞培养生产紫杉醇应采取的策略     

东北红豆杉细胞两液相培养中紫杉醇释放行为研究

在东北红豆杉细胞悬浮培养中,分别研究了稀土化合物(硫酸铈铵)、有机溶剂(油酸和邻苯二甲酸二丁脂)和稀土化合物与有机溶剂的协同作用对紫杉醇释放的影响。在此基础上深入研究了在东北红豆杉细胞两液相培养中,紫杉醇释放率随不同的有机溶剂(烷烃、有机酸、醇和脂)、有机溶剂的体积分数、有机溶剂的加入时间和有机溶剂相毒性的变化规律。结果表明分别加入稀土化合物和有机溶剂都明显促进紫杉醇的释放,特别是有机溶剂更显著促进紫杉醇的释放。但在东北红豆杉细胞两液相培养中,稀土化合物加入不能进一步促进紫杉醇的释放。因此两液相培养中有机溶剂本身就是很好的产物释放剂。紫杉醇的释放率由对照组的40%提高到75%以上。     

Azadirachtin production in stirred tank reactors by Azadirachta indica suspension culture

Successful scale-up of Azadirachta indica suspension culture for azadirachtin production was done in stirred tank bioreactor with two different impellers. The kinetics of biomass accumulation, nutrient consumption and azadirachtin production of A. indica cell suspension culture were studied in a stirred tank bioreactor equipped with centrifugal impeller and compared with similar bioreactor with a setric impeller to investigate the role of O₂ transfer efficiency of centrifugal impeller bioreactor on overall culture metabolism. The maximum cell mass for centrifugal impeller bioreactor and stirred tank bioreactor (with setric impeller) were 18.7 and 15.5 g/L (by dry cell weight) and corresponding azadirachtin concentrations were 0.071 and 0.05 g/L, respectively. Glucose and phosphate were identified as the major growth-limiting nutrients during the bioreactor cultivation. The centrifugal impeller bioreactor demonstrated less shearing and improved O₂ transfer than the stirred tank bioreactor equipped with setric impeller with respect to biomass and azadirachtin production.     

Stimulation of taxol production and excretion in Taxus spp cell cultures by rare earth chemical lanthanum

The trivalent ion of a rare earth element, lanthanum, was tested for elicitor-like effects on taxol production in suspension cultures of four different Taxus spp cells. In T. yunnanensis cell cultures, the lanthanum ion at concentrations from 1.15 to 23.0 microM stimulated taxol production. The lanthanum ion also promoted taxol excretion by the T. yunnanensis cells considerably. The maximum stimulation of taxol production was achieved by the addition of 5.8 microM La3+ to the culture during mid-log growth phase, increasing the volumetric taxol yield by nearly threefold, from 2.61+/-0.37 to 9.89+/-1.92 mg l(-1) over a 28 day culture period. At higher concentrations, i.e. 23.1 and 46.2 microM, however, the lanthanum ion caused significant growth inhibition. For the other three Taxus cell lines, namely an embryo and a leave cell of T. chinensis and a stem cell of T. chinensis marv, the addition of lanthanum ion to the culture only had a significant effect on taxol production by the T. chinensis marv stem cells, increasing the volumetric yield by about threefold to 4.69+/-0.76 mg l(-1). These results suggest that lanthanum has elicitor-like effects on secondary metabolite synthesis of plant cell cultures.     

Biotransformation of hyoscyamine into scopolamine in transgenic tobacco cell cultures

Hyoscyamine-6beta-hydroxylase (H6H) catalyses the conversion of hyoscyamine into its epoxide scopolamine, a compound with a higher added value in the pharmaceutical market than hyoscyamine. We report the establishment of tobacco cell cultures carrying the Hyoscyamus muticus h6h gene under the control of the promoter CAMV 35S. The cell cultures were derived from hairy roots obtained via genetically modified Agrobacterium rhizogenes carrying the pRi and pLAL21 plasmids. The cultures were fed with hyoscyamine, and 4 weeks later the amount of scopolamine produced was quantified by HPLC. The transgenic cell suspension cultures showed a considerable capacity for the bioconversion of hyoscyamine into scopolamine, and released it to the culture medium. Although the scale-up from shake-flask to bioreactor culture usually results in reduced productivities, our transgenic cells grown in a 5-L turbine stirred tank reactor in a batch mode significantly increased the scopolamine accumulation.     

Self-immobilized aggregate culture of Taxus cuspidata for improved taxol production

The suspension culture of self-immobilized aggregates of Taxus cuspidata, consisting of spherical, compact aggregates displaying some level of cellular/tissue differentiation was established. Total taxol yield of the self-immobilized aggregate culture was 4.9 mg/l, 5 times as much as that of the dispersed cell cultures. © Rapid Science Ltd. 1998     

Effects of amino acids, nitrate, and ammonium on the growth and taxol production in cell cultures of Taxus yunnanensis

The effects of concentration of amino acids, nitrate, and ammonium on the growth and taxol production in cultures of cell line TY-21 of Taxus yunnanensis were investigated. Addition of 20 different amino acids each at 15–20 mg l^–1 to B5 medium significantly improved callus growth but inhibited taxol formation in the cultures. The optimum nitrate concentration was 20–30 mM for both growth and taxol production. Ammonium greatly suppressed growth but strongly promoted taxol formation in the cells when it was the sole inorganic nitrogen in the medium. Culturing the suspension cells in nitrate-containing medium for 15 days and then in a medium in which ammonium was the sole inorganic nitrogen for 7 days increased taxol yield by 104%, reaching up to 28.1 mg l^–1.     

Anthraquinones production in Rubia tinctorum cell suspension cultures: Down scale of shear effects

The effect of turbulence on suspended cells is one of the most complex problems in the scale-up of cell cultures. In the present paper, a direct comparison of the effects of turbulence on suspension cultures of Rubia tinctorum in a standard bioreactor and in shake flask cultures was done. A procedure derived from the well known global method proposed by Nishikawa et al. (1977) [39] was applied. Standard flasks and four-baffled shake flasks were used. The effect of turbulence and light irradiation on cell viability, biomass, and anthraquinones (AQs) production was evaluated. The biomass concentration and AQs production obtained using baffled shake flasks agitated at 360 rpm were similar to that achieved in R. tinctorum suspension cultures growing in a stirred tank bioreactor operating at 450 rpm, previously published (Busto et al., 2008 [17]). The effect of light on AQs production was found to be very significant, and a difference of up to 48% was found in cells with and without illumination after 7 days of culture. It is concluded that this down-scaled and simple flask culture system is a suitable and valid small scale instrument for the study of intracellular mechanisms of turbulence-induced AQs production in R. tinctorum suspension cultures.     

Dynamic change of metabolites and nutrients in suspension cells of <Emphasis Type="Italic">Panax Quinquefolium</Emphasis> L. in bioreactor

In order to understand how the nutrient elements were taken up during the cell growth as well as the production of metabolites, it was quite necessary to identify the dynamic change of metabolites and nutrients in suspension cells of Panax quinquefolium in bioreactor. In this study, dynamic accumulation of biomass and ginsenosides Re, Rb1 and polysaccharide as well as major nutrients consumption in cell suspension culture of P. quinquefolium in a 5-L stirred tank bioreactor were investigated. The dry cell weight and the contents of ginsenosides Re, Rb1 and polysaccharide reached the maximum peak simultaneously on about 21 days and the results showed that cell growth and metabolites synthesis related to nutrients consumption. For this reason, we supposed that the contents of metabolites can be increased through added nutrient at the right moment. These results provided theory reference for two-stage or continuous perfusion culture in suspension cells of P. quinquefolium in bioreactor.     

Model of energy uncoupling for substrate-sufficient culture

The growth yields (Y(obs)) are greater under substrate-limited conditions than those under substrate-sufficient conditions in continuous cultures. This indicates that the excess substrate should cause uncoupling between anabolism and catabolism, which leads to energy spilling. Although the uncoupling between anabolism and catabolism has already been recognized in the microbiology literature, how to quantitatively describe such uncoupling remains unclear. Based on a balance on substrate reaction, a growth yield model was developed in relation to residual substrate concentration for substrate-sufficient continuous cultures. On the basis of that yield model, the concept of an uncoupling coefficient between anabolism and catabolism is defined in this work. A model describing the effect of the residual substrate concentration on the uncoupling coefficient of anabolism to catabolism is proposed. This model agrees very well with literature data. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 571-576, 1997.     

红豆杉细胞培养的研究

从红豆杉（Ｔａｘｕｓｃｈｉｎｅｎｓｉｓ（Ｐｉｌｇ）Ｒｅｈｄ）的嫩茎及针叶诱导的出愈伤组织,对愈伤组织培养及细胞悬浮培养进行了研究,利用ＨＰＬＣ方法测定它们合成紫杉醇的能力,发现了能够提高培养细胞生长速率及紫杉醇含量的一些因子,红豆杉愈伤组织及悬浮培养细胞的生长速率已分别达到０．２５ｇ／Ｌ．ｄ和０．２８ｇ／Ｌ．ｄ。而他们的紫杉醇含量分别是０．００２６％和０．０１２％。