【作者】 谢鸿观；

【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 sp.139能够产生一种新型胞外多糖依博素。研究表明其具有明显的抗类风湿性关节炎作用,已申报临床研究,有望发展成为新药。本室已确定依博素的生物合成基因簇(ste)由27个ORFs组成(GeneBankAccession Number:AY131229)。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衍生物,已对不同开放阅读框的功能进行深入研究。本文以ste16基因为研究对象。ste16大小为1,236bp,编码一个412-aa的蛋白。经同源性分析,ste16编码的蛋白与不同微生物来源的NDP-己糖甲基转移酶具有较高同源性,因此推测ste16编码NDP-己糖甲基转移酶,该酶能催化NDP-己糖的甲基化,反应需S-腺苷甲硫氨酸(S-adenosylmethionine,AdoMet)作为甲基供体。为了确定ste16编码蛋白的性质,以pET-30a为载体将ste16克隆至E.coliBL21(DE3),在IPTG诱导下表达,SDS-PAGE分析表明,在47kD处出现了一条新的蛋白条带,与预计的分子量相符。采用镍亲合柱层析对表达产物进行纯化,纯度达90%。重组蛋白Ste16经酶学分析其具有催化甲基从供体分子AdoMet转移至受体分子dTDP-4-keto-6-deoxy-D-glucose的活性,因此确定Ste16是NDP-己糖甲基转移酶,Km值为0.43±0.02mM。为了研究ste16在依博素生物合成中的功能,对ste16进行了同源重组双交换阻断实验。经Southern杂交确证获得了基因缺失株Streptomyces sp.139(ste16~-),采用基因互补获得了该基因互补株Streptomyces sp.139(ste16c)。与依博素相比,基因缺失株产生的多糖EPS-16m单糖比略有变化;基因互补株产生的多糖EPS-16c单糖组成类似于EPS-16m。分子量测定显示,EPS-16m和EPS-16c分子量均显著低于依博素。IL-1R拮抗活性分析结果显示,与依博素比较,EPS-16m活性降低,经基因互补后EPS-16c活性可恢复。据此推测依博素分子中的6-脱氧己糖(6DOHs)鼠李糖或岩藻糖可能侧链被甲基化,ste16作为一种修饰基因参与依博素的生物合成。以往研究显示ste15和ste22分别编码葡萄糖糖基转移酶和鼠李糖糖基转移酶,本研究通过基因同源重组双交换,在ste15基因缺失突变株Streptomyces sp.139(ste15~-)基础上,再进行ste22基因阻断,经Southern杂交验证,得到了ste15和ste22双基因缺失突变株Streptomyces sp.139(ste15~-ste22~-)。与依博素相比,双基因缺失株产生的多糖EPS-15m22m的分子量降低了50%;IL-1R拮抗活性分析结果显示,EPS-15m22m的活性显著降低。该多糖的单糖组分分析正在进行中,本研究表明葡萄糖和鼠李糖与依博素活性密切相关,与以往结果相符。ste15-ste22双敲除对依博素重复单元聚合度有突出影响。为证明依博素生物合成基因簇(ste)的完整性,我们将该基因簇克隆至变铅青链霉菌TK24中,获得了克隆菌株Streptomyces lividans TK24(ste)。其胞外多糖EPS-ste与Streptomyces lividans TK24原株和Streptomyces lividans TK24(pOJ446)分别产生的EPS-TK及EPS-pOJ比较,三种EPS均没有岩藻糖和木糖,其它六种单糖组分含量基本相似,但与依博素相比差异显著。三种EPS的分子量相近,但明显低于依博素。三种EPS均无IL-1R拮抗活性。这些结果表明尽管ste基因簇已克隆至变铅青链霉菌TK24,但未成功表达依博素或新结构EPS。综上所述,本研究确定了ste16在依博素生物合成中的修饰基因作用,进一步证明了葡萄糖和鼠李糖与依博素活性的关系,为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基础。更多还原

【Abstract】 Streptomyces sp. 139 was identified to produce a novel exopolysaccharide (EPS) designated Ebosin. It has obvious anti-rheumatic arthritis activity in vivo and may be developed to be a new drug in the future.The biosynthesis gene cluster consisting of 27 ORFs for Ebosin was confirmed in our laboratory. The individual roles of the putative genes in the cluster have been characterized for studying of the relationship between structure and activity of Ebosin and obtaining new derivatives. In this research, the objective of present investigation was to functionally assess ste16.ste16 (1,236bp) was predicted to specify a protein with homology to known NDP-hexose methyltransferases from different sources of microbes, which has the ability catalyzing transfer of the methyl group from S-adenosylmethionine (AdoMet) to dTDP-4-keto-6-deoxy-D-glucose.For characterization of the protein encoded by ste16, the gene was expressed in E. coli BL21 and the recombinant protein was purified to ~ 90% with a Mw 47kD in agreement with the expected size. Enzymatic reaction showed that Ste16 protein possesses the methyltransferase activity which catalyzes transfer of the methyl group from S-adenosylmethionine (AdoMet) to dTDP-4-keto-6-deoxy-D-glucose and then production of S-adenosylhomocysteine (AdoHcy). So that Ste16 was identified as a NDP-hexose methyltransferase with Km 0.43±0.02mM.For understanding the function of ste16 in the biosynthesis of Ebosin, the gene was disrupted with a double crossover via homologous recombination. The mutant strain Streptomyces sp.139 (ste16~-) was confirmed with Southern blot. The monosaccharide composition of EPS-16m produced by the mutant strain was found varied from that of Ebosin. The monosaccharide analysis of EPS-16c produced by the ste16 complemented mutant strain showed no remarkable changes compared with EPS-16m. The Mw for both of EPS-16m and EPS-16c were remarkably smaller than Ebosin. The antagonist activity of EPS-16m for IL-1R was significantly lower than Ebosin, but the activity of EPS-16c recovered partially compared with that of EPS-16m. According to these results, we assume Ste16 transfers methyl groups to rhamnose or fucose residues (6DOHs) of Ebosin in vivo, although the direct evidence has yet to be provided. Ste16 may be as a modificator gene during the biosynthesis of Ebosin. The products encoded by ste15 and ste22 were identified as the glucosyltranferase and the rhamnose-glycosyltranferase respectively. The ste22 was disrupted with a double crossover via homologous recombination in Streptomyces sp.139 (ste15~-). Southern blot demonstrated the mutant strain Streptomyces sp.139 (ste15~- ste22~-). The Mw of EPS-15m22m produced by the mutant strain was smaller more than 50% comparing with Ebosin. The antagonist activity of EPS-15m22m for IL-1R was significantly lower than the original levels of Ebosin. The monosaccharide analysis of EPS-15m22m has been carried out. From these research data, it can be concluded that both of stel5 and ste22 played essential roles in relationship between structure and activities in Ebosin and affected tremendously the polymerization of the repeating units of Ebosin also.The biosynthesis gene cluster (ste) of Ebosin was cloned into Streptomyces lividans TK24 with cosmid pOJ446 as vector. The monosaccharide analysis of EPS-TK, EPS-pOJ and EPS-ste produced by Streptomyces lividans TK24, Streptomyces lividans TK24 (pOJ446) and Streptomyces lividans TK24 (ste) separately showed that them were similar for the EPSs. The Mw of EPSs were remarkably lower than Ebosin and all of them had not the antagonist activities for IL-1R. From the results mentioned above, the biosynthesis gene cluster (ste) of Ebosin has not been expressed in Streptomyces lividans TK24. More detail research works should be performed further.In a word, the presenting studies identified the function of ste16 in the biosynthesis of Ebosin as a modificator gene and the essential roles of glucose, rhamnose in the bioactivity of Ebosin. These studies have laid the better groundwork for studying the relationship between structure and bioactivity of EPSs and obtaining more novel derivatives of Ebosin with new activities.更多还原