Productions of Taxol and Related Taxanes by Cell Suspension Cultures of Taxus yunnanensis

A high taxol yield cell line of Taxus yunnanensis Cheng et L. K. Fu keeps a high taxol_producing level after successive subcultures for more than eight years. In this study, eight taxanes were isolated from the suspension cell cultures of this cell line. Based on NMR and MS analyses, and comparison with literature data and standards, their structures were determined to be 2α,5α,10β_triacetoxy_14β_propionyloxy_4(20),11_taxadiene (1), 2α,5α,10β_triacetoxy_14β_(2′_methyl)_butyryloxy_4(20),11_taxadiene (2), 2α,5α,10β_14β_tetra_acetoxy_4 (20),11_taxadiene (3, taxuyunnanine C), 2α,5α,10β_triacetoxy_14β_(2′_methyl_3′_hydroxy)_butyryloxy_4(20),11_taxadiene (4, yunnanxane) and its 3′_epimer (5), baccatin Ⅳ (6), baccatin Ⅲ (7) and taxol (8), respectively. Among those compounds, 3, 5, 6 and 7 were reported to be isolated from the suspension cell cultures of T. yunnanensis for the first time. TLC and HPLC analyses indicated that the chemical constituents of the culture solution were similar to those of cultured cells. Moreover, the highest taxol content of this cell line reached 0.3% and the cell line could be applied for a large_scale culture.

Dynamic Effects of Cerium on Syntheses of Soluble Protein and Taxol in Suspension Culture of Taxus Chinensis Var. Mairei Cells

The dynamic effects of Ce4+ on the syntheses of soluble protein and taxol in suspension cultures of Taxus chinensis var. mairei cells were studied. The phenomena of 'partition' and 'bifurcation' were observed in studying the dynamic effect of Ce4+ on soluble protein synthesis and cell activity. That is, Ce4+ of low concentration improves the soluble protein synthetic strength and cell activity, while Ce4+ of high concentration is harmful to protein synthesis and cell activity. In addition, Ce4+ of appropriate concentration enhances taxol synthesis.

APOPTOSIS AND TAXOL PRODUCTION IN SUSPENSION CULTURES OF Taxus spp.CELLS

The hallmark of apoptosis, in suspension cultures of Taxus spp. cells induced by fungal extractive or by abiotic means, was studied by total DNA agarose gel electrophoresis and in situ end-labeling. The cleavage of nuclear DNA (nDNA) into oligonucleosomal fragments(DNA laddering) was a characteristic of apoptosis, which involved cell shrinkage, condensation of cytoplasm and tracheary elements differentiation. Terminal deoxynucleotidy transferase-mediated dUTP nick end in situ labeling (TUNEL) assay of Taxus spp. cells showed that fungal extractive or abiotic elicltors (Ce4+, Taxol, H2O2) induced TUNEL positive. Also, the increase of the apoptotic cell ratio was accompanied by the increase of secondary metabolites (especially Taxol). These results suggest that apoptosis may have some coincidence with biosynthesis of Taxol. The implication of apoptosis for the production of secondary metabolites in plant cell cultures is discussed.

Effect of La,Ce on Taxus Cuspidata Cell Growth,Biosynthesis and Release of Taxol

The effect of La, Ce was firstly tested on growth of the Taxus(T.) cuspidata cell suspensions, biosynthesis and release of taxol. The results indicate that the growth pattern of T.cuspidata cells is altered significantly by adding high concentration of rare earth in the medium. The lag and exponential phases of cell growth are shortened, the stationary phase disappears and the biomass fluctuates periodically during the decline phase. The rare earth compounds added in the exponential phase obviously increase the taxol biosyntheis and release yields of T.cuspidata cells, and the supplement of carbon source in the medium containing rare earth is also favorable to taxol biosynthesis.

Effect of elicitors, precursors and metabolic inhibitors on paclitaxel production by Taxus cuspidata cell culture

In this study, using Taxus cuspidata as a raw material, we obtained stable high-yielding cell lines by subculturing and quantified paclitaxel content using ultra- sonic extraction combined with TLC-UV spectrophotom- etry. In single factor and multiple factors tests to optimize design and study the effects of elicitors, precursors, and metabolic inhibitors on paclitaxel production by Taxus cuspidata cells, paclitaxel production reached 4.32 mg/L when 100 μmol/L methyl jasmonate, 20 mg/L salicylic acid, 400 mg/L phenylalanine and 2 mg/L gibberellin （GA3） were added to the culture medium of suspension cells. When adding metabolic adjustment factors on the 7th day of culture, extra- and intracellular paclitaxel production was the highest at 4.855 mg/L, paclitaxel release rate was 10.48 %, fresh mass and paclitaxel production of cell increased, respectively, by 6.08 and 11.57 %. By controlling the anabolism of paclitaxel, paclitaxel yield was significantly improved.

The selection and stability analysis of stable and high Taxol-producing cell lines from Taxus cuspidata

In this study to screen for stable, high Taxolproducing cell lines（CL5, CL12, and CL21） of Taxus cuspidata, stem tissues were used to induce calli, which were then subcultured nine times to establish suspension cell cultures. From 97 cell lines obtained from conditioned cultures, 10 cell lines with high Taxol content were selected. Stability analyses on solid and liquid B5 media were then used to obtain lines that stably produced high levels of Taxol. Fresh biomass and Taxol production of the ninth generation became stable. Taxol content of selected CL5, CL12, and CL21 samples was 0.0448, 0.0477, and0.0428% of dry mass（DW）, respectively. Proliferation of CL5, CL12 and CL21 was 346.3, 382.5, and 409.2%,respectively. From work over about 2 years, the three cell lines appear suitable for mass production of Taxol,promoting the industrialisation and commercial-scale production of Taxol using cell culture.

Study on the Browning in Cell Suspension Culture of Taxus cuspidata

[Objective] This study aimed to investigate the browning of T. cuspidata cells in suspension culture and provide the guidance for the cell suspension culture of T. cuspidata. [Method] T. cuspidata callus was used as experimental materials, to explore the effect of different medium, N/P ratio, pH, shaking speed, illumination time and light intensity and other factors on browning of T. cuspidata cells in suspension culture. [Result] Non-browning callus was transferred to 2MB5 medium （pH 7.0） for illumination culture at 22℃ under light intensity of 1 500 lx with shaking speed of 90 r/min for 24 h. Results showed that the cell browning was significantly inhibited. [Conclusion] This study laid the foundation for cell suspension culture of T. cuspidata and had important significance to the large-scale industrial production of paclitaxel.

Ce^(4+)-Induced Apoptosis of Taxus cuspidata Cells in Suspension Culture

The standard detection hallmarks of apoptosis of Taxus cuspidata cells in suspension culture with Ce 4+ were studied. The condensation and margination of chromatin were observed under the electron microscopy. DNA fragmentation ranged 'DNA ladder' on agarose gel electrophoresis. TdT mediated dUTP nick end labeling (TUNEL) analysis of the cells reveals that the nuclear DNA strand breaks can be identified by labeling free 3′ OH termini. These results suggest that Ce 4+ can induce apoptosis of Taxus cuspidata cells and also indicate that there is a certain relationship between apoptosis and secondary metabolite product Taxol.

Neural networks modeling signal responses and taxol production of cultured Taxus chinensis cells induced by bio-elicitor

Quantitatively describing the signal transduction process is important for understanding the mechanism of signal regulation in cells,and thus,poses both a challenge and an opportunity for chemical and biochemical engineers.An artificial neural network(ANN),in which we took the signal molecules as neural nodes,was constructed to simulate the generation of active oxygen species(AOS)in Taxus chinensis cells induced by a bio-elicitor.The relative contents of AOS in cells predicted by the ANN model agreed well with the experimental data and three notable stages of AOS increase were observed from the 3D figure of AOS generation.The robustness of AOS trajectories indicated that signal regula-tion in vivo was an integral feedback control model that ensured the adaptation of Taxus chinensis to environmental stress.The artificial neural network was able to predict taxol production as well as determine the optimal concentration of oligosaccharides needed for it.

Optimization of Cell Line Building for Taxus chinensis var. mairei

[Objective] The paper aims to optimize the abduction and domestication conditions of Taxus chinensis var.Mairei callus.[Method] We compared the efficiencies of callus between different explants and investigated the multiplication conditions of callus and suspended cell culture conditions with the buds,young stems and young leaves from T.chinensis var as the explants.[Results] The effect was the best with the bud as the explants; the best way for sterilizing the explants of T.chinensis var mairei was：streptomycin detergent for 2 h ＋ suds for 3 h ＋ 75% alcohol disinfection for 30 s ＋ 10% sodium hypochlorite solution for 25 min ＋ 1‰ mercury chloride for 10 min; the optimum formula of callus subculture was：B5 ＋ 4.0 mg/L NAA ＋ 0.5 mg/L 2,4-D ＋ 0.2 mg/L GA ＋ 0.5 mg/L 6-BA ＋ 2 g/L AC.[Conclusion] This research built the high efficient regeneration system of T.chinensis var.

Callus initiation and subculture of Taxus chinensis

Conditions have been established for the callus initiation and subculture of T chinensis. The calliwere induced by the explants cultured first on the medium MS supplemented with 1 .0 mg/L 2,4-D, 2g/L CH, and 25g/L sucrose, then on The medium f MS+1 .0 mg/L NAA+0.5 mg/L BA+2 g/L CH+25 g/L sucrose. When the callus was Subcultured and tamed several times, it could grow fast and stable on the medium : MS+0.2mg/L 2,4-D+0.5mg/L NAA+0.5 mg/L BA+2 g/LCH+25 g/L sucrose. The contamination of explants was a result of endophytic microbes of T Chinensis. This could be avoided by adopting the tender shoots 3-5 cm long collected in early spring as the source of explants. The browning of the Cultures could be prevented and controlled by means of the selection of a suitable explants, hormonal regime in the medium, culture methods and the use of antioxidants.

Involvement of NO in fungal elicitor-induced activation of PAL and stimulation of taxol synthesis in Taxus chinensis suspension cells

Elicitor prepared from the cell walls of Peni-cillium citrinum induces multiple responses of Taxuschinensis cells, including nitric oxide (NO) generation, se-quentially followed by the activation of PAL and synthesis oftaxol. NO scavenger cPITO and nitric oxide synthase (NOS)inhibitor PBITU prevent the latter two reactions, all of whichare triggered in the absence of elicitor by NO donor sodiumnitroprusside (SNP). The elicitor-induced NO release ofTaxus chinensis suspension cells is strongly inhibited byPBITU. These results demonstrate a causal relationship be-tween NO generation and the latter two reactions of Taxus chinensis cells to the elicitor, and also indicate that NO, pro-duced via NOS in Taxus chinensis cells treated with fungalelicitor, might act as an essential signaling molecule for trig-gering the activation of PAL and synthesis of taxol.

Nitric oxide mediates the fungal elicitor-induced Taxol biosynthesis of Taxus chinensis suspension cells through the reactive oxygen species-dependent and -independent signal pathways

Nitric oxide and reactive oxygen species are two important signal molecules that play key roles in plant defense responses. Nitric oxide generation and oxidative burst and accumulation of reactive oxygen species are the early reactions of Taxus chinensis suspension cells to fungal elicitor prepared from the cell walls of Penicillium citrinum. In order to investigate the relationship and/or interactions of ni- tric oxide and reactive oxygen species in the elici- tor-induced Taxol biosynthesis of T. chinensis sus- pension cells, we treated the cells with nitric oxide specific scavenger 2-4-carboxyphenyl-4,4,5,5-tetra- methylimidazoline-1-oxyl-3-oxide (cPITO), nitric ox- ide synthase inhibitor S,S′-1,3-phenylene-bis(1,2-eth- anediyl)-bis-isothiourea (PBITU), membrane NAD(P) H oxidase inhibitor diphenylene iodonium (DPI), su- peroxide dismutases (SOD) and catalase. The results show that pretreatment of T. chinensis cells with cPITO and DPI inhibited not only the elicitor-induced nitric oxide biosynthesis and oxidative burst, but also the elicitor-induced Taxol production, suggesting that both nitric oxide and reactive oxygen species are involved in elicitor-induced Taxol biosynthesis. Fur- thermore, pretreatment of the cells with cPITO and PBITU suppressed the elicitor-induced oxidative burst, indicating that the oxidative burst might be dependent on NO. Application of nitric oxide via its donor sodium nitroprusside (SNP) triggered Taxol biosynthesis of T. chinensis cells. The nitric ox-ide-induced Taxol production was suppressed by DPI, showing that the oxidative burst is involved in NO-triggered Taxol biosynthesis. However, nitric ox- ide and the fungal elicitor induced Taxol biosynthesis even though the accumulation of reactive oxygen species wass completely abolished in T. chinensis cells. Our data show that nitric oxide may mediate the elicitor-induced Taxol biosynthesis of T. chinensis suspension cells through both reactive oxygen spe- cies-dependent and -independent signal pathways. Moreover, the results of our work show that the elici- tor- and nitric oxide-induced Taxol biosynthesis is inhibited by catalase, indicating that H2O2 from the oxidative burst might be the signal molecule involved in induced Taxol production of T. chinensis cells.

东北红豆杉(Taxus cuspidata)细胞悬浮培养研究

用东北红豆杉的愈伤组织建立细胞悬浮系。培养研究发现，最适宜细胞生长的培养基仍为Ｂ５，最佳接种量应控制在２ｇ鲜重／瓶左右。在培养基中添加酪蛋白水解物比水解乳蛋白更利于悬浮细胞生物量的增加。培养基的ｐＨ值随细胞的培养周期而有相应的变化。

Chitosan对红豆杉PAL及Taxol合成的影响

研究了Ｃｈｉｔｏｓａｎ（聚氨基葡糖）对红豆杉悬浮培养细胞ＰＡＬ（苯丙氨酸解氨酶）活性及Ｔａｘｏｌ（紫杉醇）生物合成的影响．种胚来源的红豆杉细胞培养在改良ＭＳ液体培养基中，于培养的第１８ｄ添加不同浓度的Ｃｈｉｔｏｓａｎ．Ｃｈｉｔｏｓａｎ浓度为１００～１０００ｍｇ·Ｌ－１时对红豆杉细胞ＰＡＬ活性有明显的诱导作用，且诱导作用随浓度的增加而增强，在诱导作用下ＰＡＬ活性在１６ｈ达到峰值；Ｃｈｉｔｏｓａｎ浓度在１００ｍｇ·Ｌ－１时对红豆杉细胞的生长基本无抑制作用，增产效果最显著（约为对照组的１０倍），Ｃｈｉｔｏｓａｎ可作为Ｔａｘｏｌ合成的诱导子．

云南红豆杉愈伤组织培养及其生产紫杉醇的研究

云南红豆杉愈伤组织培养及其生产紫杉醇的研究甘烦远彭丽萍郑光植（中国科学院昆明植物研究所昆明６５０２０４）从红豆杉科红豆杉属植物如短叶红豆杉（Ｔａｘｕｓｂｒｅｖｉｆｏｌｉａ）等的树皮或枝叶提取到的紫杉醇（Ｔａｘｏｌ）是一种具有强抗癌活性的二萜烯类化合...

镧、铈对红豆杉细胞生长及紫杉醇合成与释放的影响

研究镧、铈对东北红豆杉悬浮细胞生长及紫杉醇合成与释放的影响。结果表明，当发酵液中稀土浓度较高时，均能明显改变东北红豆杉细胞的生长模式，使细胞生长的延迟期与对数期缩短，平稳期消失，并于衰亡期出现生物量的周期性振荡；在细胞生长的对数期加入稀土化合物能显著提高东北红豆杉细胞合成与释放紫杉醇的量；在加入稀土化合物的同时，补加碳源，有利于细胞合成紫杉醇。

Cu^(2+)对红豆杉培养细胞中紫杉醇形成的影响

用Ｃｕ２＋对红豆杉（Ｔａｘｕｓｃｈｉｎｅｎｓｉｓ）培养细胞中紫杉醇形成的影响进行了研究。在红豆杉细胞悬浮培养２０ｄ即指数生长期末，每１Ｌ细胞悬浮培养物中加入３０μｍｏｌＣｕＣｌ２，Ｃｕ２＋促进紫杉醇形成的作用最大。添加ＣｕＣｌ２对红豆杉细胞生长没有明显影响，但引起培养细胞中可溶性蛋白质含量、苯丙氨酸解氨酶活性及培养基ｐＨ值的变化。

植物细胞工程技术生产紫杉醇研究进展

紫杉醇是一种二萜类生物碱,主要从红豆杉树皮中分离提取。目前采用植物细胞工程技术是提高紫杉醇产率、保护稀缺资源红豆杉、解决紫杉醇药源紧缺的一种最有效方法。该文对近十年来国内外有关采用植物细胞工程技术生产紫杉醇的研究进展,包括红豆杉细胞系的建立、悬浮培养条件的研究、生物反应器培养以及紫杉醇合成代谢调控方面的最新研究进展进行综述。并重点论述了前体、诱导子和代谢支路抑制剂对红豆杉细胞悬浮培养生产紫杉醇的影响,为植物细胞工程技术生产紫杉醇提供借鉴和参考。

桔青霉诱导子对红豆杉培养细胞中紫杉醇生物合成的影响

在红豆杉培养红胞中，桔青霉诱导子促进紫杉醇的合成。将培养６—７ｄ的桔青霉菌丝体的粗提物，以５０μｇ碳水化合物／ｍｌ培养液的浓度加入到处于指数生长期末期的红豆杉培养细胞中，诱导子促进紫杉醇合成的作用最大。高压灭菌处理２０—９０ｍｉｎ，不影响诱导子的活性。