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.

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.

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.

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.

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.

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．

代谢调节剂对紫杉醇和Taxuyunnanine C生物合成的调控作用

研究了诱导子、前体和抑制剂对东北红豆杉生产紫杉醇和taxuyunnanineC的影响。结果表明,诱导子在第12d添加,前体和抑制剂在第15d添加能有效地提高紫杉醇和taxuyunnanineC的含量。水杨酸与氯化氯胆碱的交互作用对紫杉醇的合成有很大影响,水杨酸与赤霉酸的交互作用对taxuyunnanineC的合成有很大影响。

灵芝诱导子对南方红豆杉悬浮培养细胞产紫杉醇的影响

为提高红豆杉悬浮细胞紫杉醇产量,采用制备的灵芝(Ganoderma lucidum)诱导子处理南方红豆杉(Taxus wallichiana var.mairei)悬浮细胞,通过分析细胞生长量、紫杉醇总产量,研究灵芝诱导子对红豆杉悬浮细胞的影响。结果表明,灵芝诱导子添加质量浓度为100µg/mL时,红豆杉悬浮细胞生长指数和紫杉醇总产量显著高于其他质量浓度(P<0.05),该质量浓度为灵芝诱导子的最佳添加质量浓度;灵芝诱导子在细胞生长的第8天加入时,第21天的细胞生长指数和紫杉醇总产量显著高于其他添加时间(P<0.05),细胞生长的第8天为灵芝诱导子的最佳添加时间;灵芝诱导子持续作用6 d时,细胞生长指数和紫杉醇总产量显著高于其他持续作用时间(P<0.05),该持续作用时间为灵芝诱导子的最佳持续作用时间。在悬浮细胞培养的第8天加入100µg/mL灵芝诱导子培养至第21天时,细胞干质量和紫杉醇总产量达到峰值,分别为对照(等量蒸馏水处理)的221.13%、569.69%;细胞膜表面黏膜和球状物质增加;苯丙氨酸解氨酶活性和总酚含量提高,细胞活力和多酚氧化酶活性降低。

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。

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。

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。

促进紫杉醇合成真菌诱导物制备方法的选择及组分的分离

Four methods were used to prepare four crude elicitors containing different ingredients from mycelia of fungus F 5: (a) Polysaccharides A containing lipids and proteins; (b) Micromolecular polysaccharides B without lipids and proteins; (c) Macromolecular polysaccharides F without lipids and proteins; (d) Polysaccharides H containing proteins but no lipids. The crude preparation B had the highest ability to induce taxol biosynthesis among other preparations did. The crude preparation B was through Sephadex G15 column to separate two compositions: BⅠ whose molecular weight was over 1500 D and BⅡ whose molecular weight between 700～ 1500 D. The BⅡ activity to induce taxol biosynthesis was 2.3 times higher than that of the crude preparation B, while BⅠ hadn’t the activity to induce taxol biosynthesis. By analysis of PAL activity induced by elicitors, we conclude that the changes of PAL activity could be regarded as a useful physiological indicator to select the effective elicitor.

前体物对红豆杉培养细胞中紫杉醇生物合成的影响(英文)

本文报道了添加７ 种紫杉醇前体物／调节物后，红豆杉（ Ｔ．ｃｈｉｎｅｎｓｉｓ（Ｐｉｌｇｅｒ） Ｒｅｈｄ）ＴＣ１５８ 细胞系的反应。在红豆杉细胞悬浮培养２５ 天时，分别加入不同浓度乙酸钠、苯甲酸钠、Ｌ－ 苯丙氨酸、甘氨酸、丝氨酸、α－ 蒎烯、松节油。试验结果表明，各前体物对红豆杉细胞生长无明显影响。

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。

铈对悬浮培养南方红豆杉细胞可溶性蛋白和紫杉醇合成动态的影响

研究Ce4+ 对悬浮培养南方红豆杉细胞可溶性蛋白和紫杉醇合成的动态影响。Ce4+ 对可溶性蛋白合成和细胞活力的影响存在“分区”和“分叉”现象 ;低浓度Ce4+ (0 10mmol·L- 1 )可显著提高可溶性蛋白的合成强度和细胞活力 ,高浓度Ce4+ (5 0 0mmol·L- 1 )对细胞表现为强伤害作用。适宜浓度Ce4+ (1 0 0mmol·L- 1 )在短期内可大幅度提高紫杉醇的合成速度 。

几种真菌诱导子对云南红豆杉细胞产生紫杉醇的影响

The effects of the cultures of Penicilliun citrinum,Absidia glauca,Mucor rouxianus and Botrytis cinerea on the growth and taxol production in suspension cells of Taxus yunnanensis were investigated.The results showed that the addition of the cultures of Penicilliun citrinum or Botrytis cinerea into the suspension cell cultures of T.yunnanensis at the final stage of fast growing phase(the 12th day of culture) did not inhibited the cell growth,but dramatically increased taxol yield;The best result was obtained in the elicitation with Penicilliun citrinum (3.1 g/L dry mycelium) for 3 days,in which the taxol yield was increased by 1～2 times.