甲醇利用菌MB200丝氨酸乙醛酸氨基转移酶基因(sga)的克隆、突变及对产L-丝氨酸的影响  

Cloning,mutation,and effect on biosynthesis L-serine of serine glyoxylate aminotransferase gene(sga)from M.sp.MB200

作　　者：申佩弘[1] 王亚莉[1] 武波[1] 

机构地区：[1]微生物及植物遗传工程教育部重点实验室

出　　处：《工业微生物》2008年第1期15-19,共5页Industrial Microbiology

基　　金：广西青年科学基金资助项目(9912005);广西科技攻关资助项目(桂科攻033002-5)

摘　　要：sga基因是大多数Methylobacteriumsp.中丝氨酸循环途径里的一个关键酶基因,与单碳化合物的同化及二碳化合物的代谢有着密切的关系。根据已报道的M.extorquensAM1的sga基因序列,合成寡核苷酸引物,用本实验室保存的甲醇利用菌M.sp.MB200的染色体DNA为模板,通过PCR方法扩增出sga基因,测序分析表明该基因与M.extorquensAM1的sga基因在核苷酸水平上有93%的同源性,氨基酸水平上具有85%的同源性。利用自杀质粒pK18mob构建含有sga基因部分片段的重组质粒,通过三亲本结合后导入原始菌株MB200中,经过进一步同源单交换,sga基因被pK18mob插入突变,利用PCR和Southern杂交进行验证,选择发生正向突变的突变株MB200sTB用于进一步研究分析。分析结果表明突变菌株的丝氨酸乙醛酸氨基转移酶(SGAT)的酶活性消失,同时失去了在以甲醇为唯一碳源的培养基上生长的能力,不能再合成L-丝氨酸,但仍能以二碳化合物和多碳化合物进行生长。The gene, sga, encodes a serine glyoxylate aminotransferase（SGAT） in most Methylobacterium. sp with serine cycle, plays a central role in the assimilation of C1 compouds, and also involved in catalyzation the interconverting of glyoxylate to glycine and serine to hydroxypyruvate. Primers were designed according to the sga gene sequence of M. extorquens AM1, with the chromosomal DNA of M. sp. MB200 as a template, a DNA fragment was obtained by PCR. The identity of the cloned fragment was confirmed by sequencing, and the result showed that this DNA fragment is high homologous to M. extorguens AM1 sga at nucleotide and amino acid level. The vector pK18mob-psga was constructed with suicide plasmid pK18mob and a part of sga fragment, This recombinant plasmid was conjugated from E. coli DH5α into MB200 by triparental mating by using the helper plasmid pRK2073, mutants were selected on plates containing Nm and Km, and were screened for disruption of sga gene by PCR and Southem blotting. Mutants in which the sga gene was flanked by genes transcibed in the right orientation were analyzed. The results showed that the mutant had no glyoxylate aminotransferase activity and lost its ability to grow on methanol and synthesize L-serine, but retained the ability to grow on C-2 compounds and multi-carbon compounds.

关 键 词：MB200 L-丝氨酸 丝氨酸乙醛酸氨基转移酶基因 突变菌株 

分 类 号：TQ920.1[轻工技术与工程—发酵工程]