Enhanced paclitaxel production induced by the combination of elicitors in cell suspension cultures of Taxus chinensis

Taxus chinensis suspension cells were cultured in the modified Gamborg's B5 medium. Addition of 50 mg chitosan l^–1, 60 M methyl jasmonate and 30 M Ag⁺ resulted in the greatest paclitaxel production, at 25 mg l^–1 in the cultures, being almost 40 times higher than that of the control culture, 10 times higher than that of the culture exposed to Ag⁺, 6 times higher than that of the culture elicited by chitosan and almost double that of the culture elicited by methyl jasmonate.     

Enhancement of paclitaxel production by abscisic acid in cell suspension cultures of Taxus chinensis

Addition of 5 mg abscisic acid l^–1 after 12 days' growth of Taxus chinensis suspension culture gave the greatest paclitaxel accumulation at 11 mg l^–1, which was almost 5 times that of the control culture. The highest paclitaxel production, 18 mg l^–1, was obtained using 5 mg abscisic acid l^–1 and 20 mg methyl jasmonate l^–1.     

Apoptotic cell death in suspension cultures of Taxus chinensis var. mairei

Apoptotic cell death was observed in suspension cultures of Taxus chinensis var. mairei under normal cultivation conditions by using microscopy, total DNA agarose gel electrophoresis and in situ end-labeling of fragmented DNA. The morphological and biochemical changes of cells occurred mainly in the non-dividing cell clusters, indicating that the T. chinensis cells died mainly by apoptosis. There exists a close relationship between cell apoptosis and Taxol formation. Taxol concentration increased with the increase in content of apoptotic cells and reached a maximum (14.2 mg l^–1) after 23 days of culture, corresponding to a maximum ratio of apoptotic to total cells of about 13%.     

红豆杉细胞培养生产紫杉醇产量稳定性的探讨

通过磷酸盐双饥饿和秋水仙碱这两种经典的同步化方法处理悬浮培养的红豆杉细胞 ,以实现培养物的均一性 ,并比较了同步化与非同步化细胞及不同同步化方法处理的细胞紫杉醇产量。结果表明 ,不同同步化方法处理的细胞紫杉醇产量有差异 :秋水仙碱同步处理处于中期的细胞紫杉醇产量高于非同步化细胞 ,而磷酸盐双饥饿同步处理处于间期的细胞紫杉醇产量则相反。这表明紫杉醇产量与培养物的均一性有关 ,且与细胞同步的周期时相有关 ,采用同步化方法来选择合适的细胞周期时相有利于紫杉醇产量的稳定 ,通过比较不同同步化方法处理对细胞生物量和 POD活性的影响进一步探讨紫杉醇产量产生差异的原因     

Involvement of NADPH oxidase in hydrogen peroxide accumulation by Aspergillus niger elicitor-induced Taxus chinensis cell cultures

After determining that hydrogen peroxide (H2O2) accumulation induced by a fungal elicitor from Aspergillus niger was from the superoxide dismutase-catalyzed dismutation of superoxide radical, the site of H2O2 generation in cell suspension cultures of Taxus chinensis was studied. The results showed that 90% and 10% of the elicitor-induced H2O2 accumulation respectively appeared in intracellular and extracellular fractions of cells, and that the elicitor-induced H2O2 accumulation in protoplasts and plasma membranes was similar to that in intact cells, indicating that the site of H2O2 accumulation was plasma membranes but not in extracellular fraction of Taxus cells. The H2O2 forming enzyme was also investigated. The elicitor-induced H2O2 accumulation in intact cells was not changed by loss of apoplastic peroxidase (POD) by the washing, and the H2O2 accumulation in plasma membranes was inhibited by the mammalian neutrophil NAD(P)H oxidase inhibitor diphenylene iodonium (DPI), but was slightly affected by exogenous POD and its inhibitor. Furthermore, in plasma membranes, the H2O2 accumulation was more significantly enhanced by NADPH than by NADH, and the former was more obviously decreased by DPI than the latter. The present results show that NADPH oxidase in plasma membranes is involved in H2O2 accumulation in fungal elicitor-induced Taxus chinensis cell cultures.     

水杨酸对真菌诱导子诱导红豆杉悬浮细胞膜脂过氧化和紫杉醇生物合成的影响

研究了 5 0 mg· L- 1真菌诱导子 (F5) ,5 0 mg· L- 1水杨酸 (SA) ,5 0 mg· L- 1F5+5 0 mg· L- 1SA3种处理 ,对红豆杉悬浮细胞膜脂过氧化和紫杉醇合成的影响。结果表明 :F5和 SA单独处理红豆杉细胞均引起细胞膜脂过氧化。SA+F5联合处理可以减轻 F5单独处理细胞所引起的膜脂过氧化程度 ,SA+F5联合处理与真菌诱导子处理相比 ,较大地提高了过氧化物酶的活性 ,得到较多的生物量。3种处理方法均可提高红豆杉细胞紫杉醇产量 ,特别以 F5+SA处理得到产量最高 ,达到 1 1 .5 mg· L- 1,分别为 F5,SA和对照组的 1 .5倍、2 .0倍和7.5倍。结果显示 :在真菌诱导子诱导与水杨酸的联合作用下提高紫杉醇产量 ,可能与水杨酸减轻真菌诱导子所引起的细胞膜脂过氧化程度有关     

Taxol-induced apoptotic cell death in suspension cultures of Taxus cuspidata

Taxol caused apoptotic cell death of Taxus cuspidata in suspension cultures. Typical morphological and biochemical changes of apoptosis were observed by microscopy and total DNA agarose gel electrophoresis. Taxus cuspidata responded to the added Taxol by increasing the biosynthesis of Taxol. The percentage of apoptotic cells in total cells increased with the concentration of added Taxol. With Taxol added at 10 mg l^–1, the maximum concentration of Taxol produced was 23 mg l^–1, 3 times higher than that of the control culture.     

Repeated elicitation enhances taxane production in suspension cultures of Taxus chinensis in bioreactors

Methyl jasmonate added at 100 M on days 8, 14 and 20 to cultures of Taxus chinensis growing in flasks and 1 l airlift bioreactors improved taxuyunnanine C (TC) production by 50–60% in flasks and by about 80% in airlift bioreactors compared with single addition of 100 M methyl jasmonate on day 8. The final TC production in the airlift bioreactors reached 565±47 mg l^–1, which is the highest reported for taxoid production in a bioreactor.     

脂氧合酶在诱导红豆杉细胞产紫杉醇中的作用

对红豆杉悬浮培养细胞中脂氧合酶(LOX)在诱导子诱导紫杉醇合成中的作用进行了探讨。结果表明真菌诱导子处理可提高细胞内LOX的活性和紫杉醇的产量,而诱导前用LOX抑制剂菲尼酮处理,可完全抑制诱导子对LOX活性和紫杉醇合成的诱导作用。说明LOX途径可能参与了紫杉醇的合成过程。外加茉莉酸甲酯也可激活LOX活性和紫杉醇合成,诱导前用菲尼酮处理可抑制诱导子诱导的LOX活性和紫杉醇合成,说明外源茉莉酸甲酯可能是通过激活细胞内LOX途径而启动下游紫杉醇的合成。为了进一步研究脂氧合酶在紫杉醇合成中的作用。我们还对红豆杉细胞脂氧合酶的分布和分子量等性质进行了研究。     

Paclitaxel production in suspension cell cultures of Taxus

Five separate cell lines, three of Taxus canadensis Marsh. and two of Taxus cuspidata Sieb. et Zucc., were used to test the effect of carbohydrates and plant growth regulators on the growth of cells and production of paclitaxel in culture. There was no significant correlation between growth of cells and paclitaxel production. While no single medium was developed that was optimal for all cell lines, it was possible to develop a medium for each species that represented a superior combination of growth and paclitaxel production. A combination of NAA and thidiazuron produced the best combination of growth and paclitaxel production in cell lines of T. canadensis, while IAA and BA produced the best results in cell lines of T. cuspidata. A mixture of sucrose and fructose gave the best combination of growth and paclitaxel production. The addition of carbohydrates midway through the growth cycle increased the rate at which paclitaxel accumulated in the culture medium. The highest paclitaxel concentration obtained was 14.78±0.86 mg 1^–1 (n=3).Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 2ip 6-(,-dimethylamino)-purine - BA 6-benzyladenine - IAA indole-3-acetic acid - IBA indole-3-butyric acid - kinetin 6-furfurylaminopurine - NAA -napthaleneacetic acid - picloram 4-amino-3,5,6-trichloropicolinic acid - thidiazuron 1-phenyl-3 (1,2,3-thiadiazol-5-yl)urea     

Oxidative burst, jasmonic acid biosynthesis, and taxol production induced by low-energy ultrasound in Taxus chinensis cell suspension cultures

This work aims to detect the two signal events in the elicitation of plant defense responses and secondary metabolism in plant cell cultures by low-energy ultrasound (US), transient production of reactive oxygen species (ROS) or the oxidative burst and jasmonic acid (JA) biosynthesis, and examine their influence on secondary metabolism. Experiments were carried out in Taxus chinensis cell suspension culture which produces the anticancer diterpenoid Taxol (paclitaxel). The culture was exposed to low-frequency US for a short period of time (2 min). At sufficiently high US power levels the US exposure significantly enhanced the Taxol production and slightly depressed cell growth and viability. The US exposure induced transient production of O(2)*- and H(2)O(2) and an increase in the intracellular JA level as well as the activities of enzymes for JA synthesis, lipoxygenase (LOX), and allene oxide synthase (AOS). Inhibition of the ROS production by putative ROS scavengers or the JA accumulation by LOX inhibitors effectively suppressed the US-stimulated Taxol production. Inhibition of the ROS production also suppressed the US-induced JA accumulation. These results suggest that oxidative burst is an upstream event to JA accumulation, and both ROS from the oxidative burst and JA from the LOX pathway are key signal elements in the elicitation of Taxol production of T. chinensis cells by low-energy US.     

Translocation of isopentenyl pyrophosphate for Taxol biosynthesis in suspension cultures of Taxus chinensis var. mairei

Two inhibitors of metabolite translocators, sodium pyrophosphate (NaPP) and D,L-glyceraldehyde (DLG), respectively, were added into suspension cultures of Taxus chinensis var. mairei at the early and late growth phases to investigate the translocation of isopentenyl pyrophosphate (IPP) from cytoplasm to plastids for Taxol biosynthesis. NaPP and DLG hardly affected cell biomass and viability regardless of the phases of their introduction. The Taxol content varied less when NaPP or DLG was added at day 7 but decreased obviously when NaPP or DLG was introduced at day 14. It is thus inferred that NaPP and DLG inhibited Taxol biosynthesis by blocking IPP translocation at the late growth phase, suggesting that the translocation of IPP be involved only at the late growth phase of Taxus cells.     

Intermittent maltose feeding enhances paclitaxel production in suspension culture of Taxus chinensis cells

High paclitaxel production (26 mg l^–1) was achieved through the intermittent feeding of 3, 1, and 2% (w/v) sucrose at days 0, 7, and 21, respectively, to a suspension culture of Taxus chinensis cells. Intermittent feedings of 1 and 2% (w/v) maltose at days 7 and 21, respectively, to the culture increased the paclitaxel production up to 67 mg l^–1.     

Salicylic acid-induced taxol production and isopentenyl pyrophosphate biosynthesis in suspension cultures of Taxus chinensis var. mairei

The influences of salicylic acid (SA) on taxol production and isopentenyl pyrophosphate (IPP) biosynthesis pathways in suspension cultures of Taxus chinensis var. mairei were investigated by adding SA and mevastatin (MVS), a highly specific inhibitor of 3-hydroxy-3-methylglutaryl-CoA reductase in the mevalonate pathway for IPP biosynthesis, into the culture systems. The cell death and taxol production were induced upon the introduction of SA, and 20mg/l was proved to be the optimal SA concentration in terms of the less damage to Taxus cells and marked activation of phenylalanine ammonia lyase (PAL). In the coexistence of SA (20mg/l) and MVS (100 nmol/l), the taxol content (1.626 mg/g dry wt) was higher than that (0.252 mg/g dry wt) of the MVS-treated system but almost equal to that (1.581 mg/g dry wt) of the SA-treated system. It is thus inferred that the activated non-mevalonate pathway should be responsible for the formation of IPP in taxol biosynthesis in the presence of SA.     

中国红豆杉悬浮培养细胞的超低温保存

对中国红豆杉悬浮细胞超低温保存中几个主要因素进行多方面对比研究。结果表明,取培养16d的细胞进行超低温保存效果最好,10％DMSO＋8％葡萄糖作为冰冻保护剂对冷冻细胞起到最佳的保护效果;较好的降温程序是在0℃中预处理30min后移入－20℃中停留180min,然后转入－70℃中停留30min,最后投入－196℃液氮中保存。该实验还对保存后细胞的恢复性生长进行了验证。     

Abscisic acid plays critical role in ozone‐induced taxol production of Taxus chinensis suspension cell cultures

Exposure to ozone induced a rapid increase in the levels of the phytohormone abscisic acid (ABA) and sequentially followed by the enhancement of Taxol production in suspension cell cultures of Taxus chinensis. The observed increases in ABA and Taxol were dependent on the concentration of ozone applied to T. chinensis cell cultures. To examine the role of ABA in ozone‐induced Taxol production, we pretreated the cells with ABA biosynthesis inhibitor fluridone to abolish ozone‐triggered ABA generation and assayed the effect of fluridone on ozone‐induced Taxol production. The results showed that pretreatment of the cells with fluridone not only suppressed the ozone‐triggered ABA generation but also blocked the ozone‐induced Taxol production. Moreover, our data indicate that the effect of ABA on Taxol production of T. chinensis cell cultures is dose‐dependent. Interestingly, the suppression of fluridone on ozone‐induced Taxol production was reversed by exogenous application of low dose of ABA, although treatment of low dose ABA alone had no effect on Taxol production of the cells. Together, the data indicated that ozone was an efficient elicitor for improving Taxol production of plant cell cultures. Furthermore, we demonstrated that ABA played critical roles in ozone‐induced Taxol production of T. chinensis suspension cell cultures. © 2011 American Institute of Chemical Engineers Biotechnol. Prog., 2011     

A model for signal transduction in suspension cultures of Taxus chinensis var. mairei induced by an oligosaccharide from Fusarium oxysporum

A non-linear cascade model is proposed to describe the signal transduction pathway in suspension cultures of Taxus chinensis var. mairei induced by an oligosaccharide from Fusarium oxysporum. The oxidative burst intensity, which was defined as the amount of the free radicals including superoxide anion (O² ), H₂O₂ and OH^– and measured by ESR spectrometry, was used as the signal characteristic and the theory of electronic signal transduction cascade was then applied to derive the initial model. The model shows that the signal transduction cascade process is composed of three cascade nets, represented by the phosphorylation of a G-protein, activation of an ion channel and phospholipase C, and phosphorylation of protein kinase C. The three reactions were calculated as beginning at 30.5, 48.8 and 141.4 min after elicitation, and the maximum variation rates of the signal intensity in the three nets occurred at 43.2, 80.6 and 192.6 min, respectively. The validity of the model predictions was verified by experimental data.     

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.     

Improved paclitaxel accumulation in cell suspension cultures of Taxus chinensis by brassinolide

When brassinolide was added at 17 ng l^–1 to Taxus chinensis cell suspension cultures, the paclitaxel content was increased by over 100% to 580 g g^–1 dry weight. Brassinolide may therefore be a novel regulator of paclitaxel biosynthesis.     

Improved Taxol production in suspension cultures of Taxus chinensis var. mairei by in situ extraction combined with precursor feeding and additional carbon source introduction in an airlift loop reactor

Taxus chinensis var. mairei was grown in two-liquid-phase culture in a 2.5 l airlift external loop reactor for production of Taxol. A mixture of oleic acid and dibutyl phthalate (1:1, v/v) in a volume ratio of 8:92 to the culture medium gave higher production of Taxol (up to 12 mg l^–1) with an aeration rate of 1 vvm. By combination of the in situ extraction, precursor feeding and additional sugar introduction, Taxol production (16.7 mg l^–1) was 5 times higher than that in the case without any additives (3.4 mg l^–1) and 1.4 times of that in the case with only thein situ extraction (12 mg l^–1). Taxol was extracted from the organic solvents with water and methanol (1:1:1.4, by vol.) and lyophilized giving a total yield of ca. 90%.