【作者】 王岚；

【导师】 李元；

【作者基本信息】 中国协和医科大学 ， 微生物与生化药学， 2008， 博士

【摘要】 链霉菌(Streptomyces)是工业重要微生物之一,能产生大量的次级代谢物,主要有抗生素等,然而有关链霉菌胞外多糖的研究除本实验室外国内外尚未见报道。本研究室自行构建了以基因重组白细胞介素-1(interleukin-1,IL-1)可溶性受体为靶位的受体拮抗剂筛选模型,获得了一种由链霉菌139产生的新型胞外多糖(exopolysaccharide,EPS)依博素,经药效学研究证明依博素对类风湿性关节炎有明显疗效,并且毒性较低,已申报临床研究,有可能发展成为临床治疗类风湿性关节炎的新药。我室已确定了由27个开放阅读框架(ORF)构成的依博素生物合成基因簇(ste)。为了阐明依博素生物合成途径,研究依博素结构与生物活性关系并获得新型依博素衍生物,已对不同基因功能进行了深入研究,本文的研究对象为ste5和ste22基因。根据基因同源性分析,ste5基因编码的蛋白可能是引导糖基转移酶,在依博素生物合成中启动第一步反应。为了解ste5在依博素生物合成中的功能,通过同源重组双交换对ste5进行了基因阻断研究,获得了基因缺失突变株Streptomyces sp.139(ste5),其产生的多糖EPS-5m单糖组分中半乳糖含量明显下降,全部丧失对IL-1R的拮抗活性,同时EPS-5m的分子量较依博素明显降低。为了确定ste5编码产物性质,在大肠杆菌成功进行了基因克隆表达,并纯化了重组蛋白,其分子量为54KD。酶学活性研究证明Ste5为半乳糖糖基转移酶,能够催化1-磷酸半乳糖由UDP-半乳糖转移到Streptomyces sp.139细胞膜脂质载体上,形成磷酸糖脂,从而作为引导糖基转移酶启动了依博素的生物合成,因此ste5是依博素的关键的生物合成基因。为了证实ste22基因编码产物性质,以pBV220为载体,在大肠杆菌成功进行了基因克隆表达,并纯化了重组蛋白Ste22,其分子量为35KD。酶学活性研究证明Ste22为鼠李糖糖基转移酶,能够催化鼠李糖基由TDP-L-rhamnose转移到多糖重复单元增长链,形成糖苷键,在依博素生物合成中具有重要作用。本文有效确定了ste5和ste22基因在依博素生物合成中的作用,为通过组合生物学:途径研究多糖结构与生物活性关系,获得具有生物活性的新结构多糖奠定了较好的基础。上述研究均未见国内外报道。更多还原

【Abstract】 It is well known that members of the genus Streptomyces produce a great many antibiotics and other classes of biologically active secondary metabolites,but little is known of the EPSs produced by these valuable microbes.Recently,a novel EPS named Ebosin was isolated and identified in our laboratory from the culture of Streptomyces sp. 139,which has antagonist activity for IL-1R in vitro and remarkable anti-rheumatic arthritis activity in vivo.The application of clinical trial for Ebosin has been made. The biosynthesis gene cluster(ste) of Ebosin consisting of 27 ORFs was identified in our lab.We have endeavored to elucidate the functions of the ste genes present in this cluster.Here we presented our study on ste5 and ste22.Based on the database analysis,the deduced product of ste5 showed high homology with glycosyltransferases,which may take responsibility in Ebosin biosynthesis at the first step.For understanding the function of ste5 in the biosynthesis of Ebosin,the gene was disrupted with a double crossover via homologous recombination.The proportions of monosaccharides in EPS-5m produced by the gene deficient strain Streptomyces sp. 139(ste5) were changeable especially in reducing galactose compared with Ebosin, while its antagonist activity for IL-1R was lost and Mw was apparently lower than Ebosin.The gene ste5 was cloned and expressed in E.coli.The recombinant protein with Mw～54 KD was purified,which was identified as a galactosyltransferase catalyzing the transfer of galactose-1-phosphate from UDP-galactose to the lipid carrier in the cytoplasmic membrane of Streptomyces sp.139.As a priming glycosyltransferases,Ste5 is functional at the first step during biosynthesis of Ebosin.Gene ste22 was also cloned and expressed in E.coli with pBV220 as vector.The recombinant protein Ste22 with Mw～35 KD was purified.The result of enzymatic study showed that the protein of ste22 was rhamnosyltransferase catalyzing the transference of rhamnose from TDP-rhamnose to a rhamnose-acceptor,a lipophilic carrier in the cytoplasmic membrane of Streptomyces sp.139.Gene ste22 plays essential role undoubtedly in biosynthesis of Ebosin.In short,the functions of ste5 and ste22 were identified during biosynthesis of Ebosin.The foundation has been laid on explaining the relationship between structure and biological activities in Ebosin and producing the novel derivates of Ebosin with activities.更多还原