【作者】 常明；

【导师】 李元； 石莲英； 洪斌；

【作者基本信息】 中国协和医科大学 ， 微生物与生化药学， 2009， 博士

【摘要】 链霉菌(Streptomyces)是在工业上常用的微生物之一,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,有可能发展成为临床治疗类风湿性关节炎的新药。依博素生物合成基因簇(ste)包含27个完整开放阅读框(ORF),已对不同基因的功能进行深入研究。本文选择了ste7、ste26和ste27基因作为研究对象。根据同源性分析发现,ste7基因编码的蛋白与不同来源的糖基转移酶具有很高的同源性。为了确定ste7编码产物的生化性质,本研究在大肠杆菌中克隆表达了Ste7,对表达产物进行纯化,酶学性质研究证明Ste7为岩藻糖基转移酶,能够催化GDP-岩藻糖中的岩藻糖转移到单糖延长链受体上,在依博素生物合成中起重要作用。该酶的Km值为36.87±3.04μM。为了解ste26在依博素生物合成中的作用,对ste26进行了同源重组双交换基因阻断实验,获得了基因缺失株Streptomyces sp.139(ste26~-)。与依博素相比,基因突变株产生的多糖衍生物EPS-26m的单糖组分不同程度有所变化;对IL-1R拮抗活性分析显示,EPS-26m的活性没有发生明显变化,但EPS-26m的峰位分子量明显低于依博素。据此推测ste26可能在依博素生物合成过程中起修饰作用。同源性分析显示ste26基因编码蛋白与乙酰转移酶具有很高的同源性。为了解ste26编码蛋白的生化性质,我们在大肠杆菌中对ste26进行了克隆和表达,对表达产物进行纯化,酶学性质研究证明Ste26为精胺/精脒乙酰转移酶,能够催化乙酰辅酶A(AcCoA)中的乙酰基转移到精胺、精脒和多聚赖氨酸上,具有一定的底物特异性。其中精胺是最适底物,酶的Km为72.14±7.39μM、最适温度为37℃、最适pH为7.5。为了研究ste27在依博素生物合成中的作用,对ste27进行了基因阻断实验,获得了基因突变株Streptomyces sp.139(ste27~-),其产生的多糖衍生物EPS-27m的单糖组分有所变化;与依博素相比,EPS-27m对IL-1R的拮抗活性没有发生明显变化;此外EPS-27m的峰位分子量明显低于依博素。综上所述,ste27基因的敲除对依博素生物合成有所影响,但未导致其衍生物丧失活性,因此推测ste27可能在依博素生物合成中可能起修饰基因作用。同源性分析显示ste27基因编码蛋白与乙酰转移酶具有很高的同源性。为了解ste27编码蛋白的生化性质,我们在大肠杆菌中对ste27进行了克隆和表达。酶学性质研究证明Ste27为精胺/精脒乙酰转移酶,能够催化AcCoA中的乙酰基转移到精胺、精脒、多聚赖氨酸和6-磷酸葡糖胺上,具有一定的底物特异性。其中精脒是最适底物,酶的Km为58.97±2.47μM、最适温度为28℃、最适pH为7.5。为了获得新型胞外多糖衍生物,更加深入了解ste26和ste27在依博素生物合成中的作用,我们利用同源重组双交换法对ste26和ste27进行了双敲除,经Southernblot验证,获得了双基因缺失株Streptomyces sp.139(ste26~-27~-)。与依博素相比,双基因缺失株Streptomyces sp.139(ste26~--27~-)产生的胞外多糖衍生物EPS-26m27m中的鼠李糖,岩藻糖,木糖和葡萄糖组分都明显下降,对IL-1R的拮抗活性降低,峰位分子量也明显低于依博素。EPS-26m27m作为依博素新衍生物,需对其体内外活性进行深入研究。综上所述,本文通过对ste7、ste26和ste27基因的功能和性质及ste26-ste27基因的双敲除研究,有效地确定了各基因在依博素生物合成中的作用。为依博素生物合成途径的研究和获得具有生物活性的新型胞外多糖衍生物奠定了基础。更多还原

【Abstract】 Streptomyces are well known as important industrial microorganisms for their production of naturally derived antibiotics.However,the research of exopolysaccharide （EPS） in Streptomyces was not reported before.With the screening model for IL-1R antagonist,a novel EPS namely Ebosin produced by Streptomyces was found with remarkable anti-rheumatic arthritis activity in vivo,which may be developed to a new drug.The biosynthesis gene cluster（ste） of Ebosin contains 27 ORFs and the individual roles of these genes were studied.In this thesis,the functions of ste7,ste26 and ste27 have been reported.According to database searches,the protein encoded by ste7 in the cluster is homologous to some known microbial glycosyltransferases.For identification of its biochemical characteristics,ste 7 was expressed in E.coli and the recombinant protein was purified.Ste7 was demonstrated to be a fucosyltransferase with the ability of catalyzing the transfer of fucose specifically from GDP-fucose to a fucose-acceptor,which plays an essential role in the formation of repeating sugars units during Ebosin biosynthesis.Its Km was determined to be 36.87±3.04μM.For the functional study of ste26 gene in Ebosin biosynthesis,it was disrupted with a double crossover via homologous recombination.The monosaccharide composition of EPS-26m produced by the mutant strain Streptomyces sp.139（ste26^-） was found altered compared with Ebosin,but the antagonist activity of EPS-26m for IL-1R showed about similar level with Ebosin.The molecular weight of EPS-26m is remarkably lower than Ebosin.Gene ste26 may act as a modifier gene of Ebosin during its biosynthesis.Searching with the database,Ste26 is homologous to some of the acetyltransferase. For studying its characteristics,ste26 was expressed in E.coli and the recombinant Ste26 was purified,which was identified to be a spermidinse/spermine-N₁-acetyltransferase catalyzing the transfer of an acetyl group from AcCoA to spermine,spennidine and poly-L-lysine respectively.Spermine is the best substrate,and its Km,optimum temperature and pH were determined to be 72.14±7.39μM,37℃and 7.5,respectively.For understanding the function of ste27 in biosynthesis of Ebosin,its Knockout was achieved with a double crossover via homologous recombination.The monosaccharide composition of EPS-27m produced by the mutant strain Streptomyces sp.139（ste27^-） changed and its molecular weight was much lower than Ebosin.The bioactivity of EPS-27m for IL-1R was the same as Ebosin.Gene ste27 may also act as a modifier gene of Ebosin during its biosynthesis.Based on the higher homology with some of the acetyltransferase,ste27 was also expressed in E.coli and the recombinant protein was purified.It was identified to be a spermidinse/spermine-N₁-acetyltransferase catalyzing the transfer of an acetyl group from AcCoA to spermine,spermidine,poly-L-lysine and GlcN-6-P.Substrate specificities of Ste27 showed that spermidine is the best one,and its Km,optimum temperature and pH were identified to be 58.97±2.47μM,28℃and 7.5,respectively.Both ste26 and ste27 were disrupted for producting new derivatives of Ebosin. Compared with Ebosin,the monosacharride composition of EPS-26m27m produced by Streptomyces sp.139（ste26^--27^-） showed that rhamnose,fucose,xylose and glucose decreased remarkably and both of its molecular weight and bioactivity are much lower. As a new derivatives of Ebosin,EPS-26m27m will be studied more detail.In short,ste7,ste26 and ste27 are involved in the biosynthesis of Ebosin,and such study will lay a studying foundation for new EPS drugs.更多还原