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

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(Y_P/X)提高了15.13%。

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

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.