紫杉醇的研究

,Analysis of heterologous taxadiene production in K- and B-derived Escherichia coli,紫杉醇(Taxol)是从红豆杉科一些植物的树皮或枝叶中提取到的一种二萜类化合物,其分子式为C17H51NO14,分子量为853.92.,紫杉醇简介,紫杉醇在植物体内含量普遍偏低,即使在目前公认含量最高的短叶红豆杉树皮中也仅有0.060%0.069%. 经临床试验证明,它对肺癌、卵巢癌、结肠癌、白血病等多种癌症有特效.,微管是真核细胞的一种组成成分，它是由两条类似的多肽（a和p)亚单位构成的微管二聚体形成的。在正常情况下，微管和微管蛋白二聚体之间存在动态平衡。 紫杉醇可使二者之间失去这种动态平衡，诱导和促进微管蛋白聚合，防止解聚，稳定微管。这些作用导致细胞在进行有丝分裂时不能形成纺锤体和纺锤丝，抑制了细胞分裂和增殖，从而发挥抗肿瘤作用。,紫杉醇的药理作用,目前国际市场上的紫杉醇仍依靠从红豆杉树皮提取及半合成，迄今为止尚未见有第三种形式形成大规模工业化生产的报道。,为了解决红豆杉资源短缺与紫杉醇需求量的日益增加的矛盾，人们对生产紫杉醇进行了多方面的研究，包括化学合成、红豆杉细胞培养及微生物发酵。,紫杉醇获得的方法,细胞培养不仅能够连续均匀生产，生产条件可控，细胞的外部对其的影响不大，而且可以在生物反应器中大规模培养，易于提高产量和进行产物的分离提取纯化，是进行工业化生产的首选方法。,红豆杉细胞的悬浮培养着方法目前来说可行性还是相对较高的,1、利用红豆杉细胞培养生产紫杉醇,2.直接从红豆杉科植物中分离提取,自紫杉醇药效发现以来，红豆杉资源遭到毁灭性开发，有些种系濒临灭绝。 因此，利用人工种植的红豆杉代替野生红豆杉成为必然这要求人工栽培的红豆杉品种紫杉醇含量高、生长迅速因此，人工种植该品种生产紫杉醇的成本很高。,3、直接从紫杉醇产生菌中提取,紫杉醇产生菌主要是一些红豆杉的内生真菌真菌中最高紫杉醇含量为1854毫克每升。 由此可见，当前利用微生物发酵方法产生紫杉醇的的得率是相当低，难以达到工业化生产的要求。,对微生物途径工程产紫杉醇的研究相对进展较慢，原因是目前还存在生物合成途径的多样性、代谢网络的复杂性、基因表达调控的未知性等问题 。,4、化学合成法,紫杉醇结构复杂，母核有9个手性中心，因此应有2048个非对映异构体,紫杉醇的半合成是以天然来源的baccatin或10一去乙酰baccatin为原料，通过13位酯化得到紫杉醇或其它半合成衍生物,TEXT 03,TEXT 06,inducible T7, Trc, and T5 promoters were tested with the plasmid-borne geranylgeranyl diphosphate synthase and taxadiene synthase genes responsible for the downstream pathway.,In this study,the heterologous production of taxadiene was established in and analyzed between K- and B-derived Escherichia coli strains.,E. coli isoprenoid metabolism with taxadiene in the context of Taxol biosynthesis,Materials and methods,crtE gene (coding for a geranylgeranyl diphosphate synthase (GGPPS) from Taxus canadensis,txs gene (coding for a taxadiene synthase(TXS) from T. brevifolia,E. coli strains JM109(DE3) and BL21(DE3) were used as the original hosts for YW22 and YW23,Materials and methods,Small-scale cultures,Transcript preparation and analysis,Taxadiene analysis,Organic acid analysis,Microplate growth assay,Results,Recombinant parameter optimization,T7(pACYC), T5 (pQE), and Trc (pTrc)promoters,taxadiene production in YW22 isroughly 2.5-fold that of YW23,SDS-PAGE analysis of heterologous protein levels for the three promoter systems tested in YW22 and YW23,A transcriptomics study (global transcript profiling) was undertaken to better understand ：why the K strain produces more than twice as much taxadiene with qualitatively comparable or lower expression of the heterologous genes (particularly for the pTrc plasmid).,Of the 11 genes, four (dxs, ispE, ispF,and ispA) were expressed higher (p0.05) in YW23 and one (ispU) was expressed higher in YW22 (p2) at a statistically significant level (p value0.01). Of these 348 genes, 243 were upregulated in YW22 while 105 were upregulated in YW23.,We observed that YW23 (the B derivative) grew to higher cell densities than YW22 (the K derivative),The cell-density differences achieved were not statisticallysignificant at 22C, 27C, and 32C,Temperature modulation study for cell density,Specific uptake rate of glycerol,The specific uptake rate of glycerol increased roughly linearly with temperature. YW23 showed lower specific uptake rates of glycerol than YW22 but grew to a higher cell density, lending itself to better utilization of the initial carbon source.,Specific production rate of acetate (by-product),The specific production rate of acetate was higher for YW22 than YW23 at all temperatures except 22C also indicating that YW23 better utilizes glycerol.,specific taxadiene titer are plotted,For each strain, the specific taxadiene titers did not vary significantlybetween 12C and 27C, with a slight decrease observed at 32C.,As a function of exogenous indole concentration, the a exponential-phase specific growth rate,The growth was not inhibited for both strains at levels of 10, 25, or 50 mg l 1. At levels of 100, 200, and 300 mg l 1 ,the growth rates steadily declined for both strains.,Exponential-phase specific growth rate normalized to the untreated culture,Indole inhibited the growth of YW22 greater than that of YW23. At 300 mg l 1 indole; the specific growth rate of YW22 was roughly 65.3% of the control, while that of YW23 was 78.5% of that respective control.,Discussion,In YW23, both pyruvate kinase I (pykF) and phosphoenolpyruvate carboxykinase (pck) were upregulated, decreasing pyruvate flux from this pathway by upregulation of these two enzymes is a likely explanation for why the taxadiene production titer in YW23 is less than half that of YW22.,YW22 significantly upregulated phosphofructokinase I(pfkA),YW23 significantly upregulated fructose-1,6-bisphosphatase (fbp),Slightly increased expression of certain genes in the MEP pathway by YW23 does n