希瓦氏菌(Shewanella marinintestina MCCC1A01703)二十碳五烯酸生物合成基因簇的钓取及鉴定

摘要

希瓦氏菌(Shewanella marinintestina MCCC 1 A01703)是从海洋动物肠道分离得到的1株产二十碳五烯酸(Ecicosapentaenoic acid,EPA)的海洋细菌.利用PCR方法、Overlap PCR及Gibson Assemble技术克隆该菌中包含加A、pfaB、pfaC和pfaD的EPA生物合成基因簇,全长18.4 kb.序列分析表明所钓取的合成基因簇与来自希瓦氏菌SCRC-2738的EPA合成基因簇有88％的相似度,均编码聚酮合酶.以构建的低拷贝表达载体pACYC-Trc为骨架,通过Gibson Assemble技术构建EPA基因簇表达质粒pLYSCY03.钓取大肠埃希菌(Escherichia coli DH5α)细胞中的entD基因,克隆至表达载体pTrc99a中,构建成为重组质粒pLYSCY01.将两个表达质粒同时导入大肠埃希菌(Escherichia coli DH5c)中,获得产EPA的工程菌株.结果表明,希瓦氏菌中含有EPA聚酮生物合成基因簇,大肠埃希菌(Escherichia coli DH5α)中的entD基因可以替代pfaE基因与钓取的EPA合成基因协同合成EPA.%Shewanella marinintestina MCCC 1A01703,a marine bacterium producing EPA (eicosapentaenoic acid),was isolated from the intestine of marine animal.A total length of 18.4 kb of the EPA biosynthetic gene cluster,including pfaA,pfaB,pfaC and pfaD,were cloned by PCR,Overlap-PCR and Gibson Assemble methods.The similarity of this gene cluster.with the EPA gene cluster from S.pneunatophri SCRC-2738 was 88％.And sequences analysis revealed that all encoded polyketide synthases.The complete gene cluster was inserted into the low copy number expression vector pACYC-Trc as a framework by Gibson Assemble method,to construct plasmid pLYSCY03.The entD gene,which performs the same function as pfaE,was fished from E.coli DH5o,cloned into expression vector pTrc99a,to construct recombinant plasmid pLYSCY01.The two expression plasmids were co-transformed into E.coli DH5α,and obtained an EPA engineered strain.The results showed that the EPA polyketide biosynthesis gene cluster contained in marine animal intestine S.marinintestina,and entD gene in E.coli DH5α could replace pfaE gene to synthesize EPA cooperate with the fished EPA synthase.