谷氨酸棒杆菌SYPS-062 L-丝氨酸脱水酶活性分析及其基因敲除对L-丝氨酸积累的影响  

Activity Analysis of L-serine Dehydratase and Effect of the Gene Knockout on L-serine Accumulation in Corynebacterium glutamicum SYPS-062

作　　者：金雪霞[1,2] 张晓梅 窦文芳 许泓瑜 许正宏[1,2] 

机构地区：[1]江南大学医药学院制药工程研究室,无锡214122 [2]江南大学教育部工业生物技术重点实验室,无锡214122

出　　处：《中国生物工程杂志》2011年第1期29-34,共6页China Biotechnology

基　　金：国家"863"计划(2006AA020104);国家"973"计划(2007CB707804);教育部新世纪优秀人才支持计划(NCET-07-0380)资助项目

摘　　要：以谷氨酸棒杆菌(Corynebacterium glutamicum)SYPS-062基因组DNA为模板,扩增得到L-丝氨酸脱水酶(L-SerDH)的编码基因sdaA。将其克隆到表达载体pET-28a(+),并在E.coli BL21(DE3)中诱导表达,对纯化的L-SerDH进行了酶活测定,并与来自C.glutamicum ATCC13032的重组L-SerDH进行了比较,结果显示,两种不同菌株来源的重组L-SerDH降解L-丝氨酸的酶比活力差异并不显著。在此基础上敲除菌株SYPS-062的sdaA基因,探讨该基因对C.glutamicum SYPS-062生长及产酸的影响。通过构建自杀型重组质粒pK18mobsacB-△sdaA,电击转入C.glutamicum SYPS-062中,以同源重组的方式获得了sdaA基因缺失突变株,并用PCR方法对突变株C.glutamicum SYPS-062△sdaA进行了验证。与出发菌株相比,突变菌株生长缓慢,单位菌体L-丝氨酸的产量(YP/X)提高了15.13%。Corynebacterium glutamicum SYPS-062,a strain isolated from soil sample can directly product L-serine from sugar substances.The sdaA gene encoding L-serine dehydratase（EC 4.3.1.17,L-SerDH） from C.glutamicum SYPS-062 was amplified and sequenced.The plasmid pET-28a-sdaA was constructed and transfromed into E.coli BL21（DE3）.SDS-PAGE showed that the gene sdaA was expressed successfully in recombinant E.coli BL21.Then L-SerDH was purified by Ni-NTA affinity chmmatography.The results showed a single band about 46 kDa on SDS-PAGE gel,and the specified activity was about 0.57 U/mg.However,compared the sdaA genes between C.glutamicum SYPS-062 and ATCC13032,it was appeared that the two sdaA cannot lead to the differences of activity of L-SerDH.To further investigate the mechanism of L-serine accumulation by SYPS-062.A sdaA deleted mutant of C.glutamicum SYPS-062 was successfully constructed.The resulting plasmid,pK18mobsacB-△sdaA was constructed and introduced into C.glutamicum SYPS-062 by electroporation.The Recombinant bacteria C.glutamicum SYPS-062△sdaA was conformed by PCR.The deletion of gene sdaA in C.glutamicum SYPS-062 yielded 15.13% increase of L-serine production with slow growth.

关 键 词：L-丝氨酸 谷氨酸棒杆菌 sdaA L-丝氨酸脱水酶 

分 类 号：Q786[生物学—分子生物学]