一种谷氨酸棒杆菌基因无痕敲除载体的构建及应用

以谷氨酸棒杆菌（Corynebacteriumglutamicum）23604编码赖氨酸胞内转运蛋白基因lysP为敲除对象，利用重叠PCR技术将 lysP基因的上下游同源臂融合，并构建了由木糖启动子Pxyl和毒素蛋白基因mazF组成的筛选盒，同时无缝克隆连接至带有卡那霉素抗性基因的pTOPO载体中，经电转化及两次同源单交换筛选，实现lysP基因的无痕敲除。 结果表明，经过卡那霉素抗性筛选、木糖二次筛选及基因组PCR鉴定后，成功获得lysP基因缺失菌株C.glutamicum23604ΔlysP；发酵后C.glutamicum23604ΔlysP赖氨酸产量较对照菌株产量提高了10.8%。该实验以大肠杆菌毒素蛋白基因mazF作为反向筛选标记的敲除载体，实现谷氨酸棒杆菌lysP的高效敲除； lysP基因的敲除使得赖氨酸不能进入胞内而在胞外积累，从而达到增产赖氨酸的目的。

Construction and application of a vector for marker-free targeted genetic knockout in Corynebacterium glutamicum

A gene lysPencoding lysine intracellular transporters inCorynebacteriumglutamicum 23604 was knocked out. Two homologous arms of the lysPgene were first fused by overlapping PCR technology, and then ligated to the vector pTOPO which contained a MazF-cassette consisted of a xylose promoter Pxyland a toxin protein genemazF, after electroporation and two single homologous exchanges, the lysPgene was non-trace knocked out successfully. Results showed that one lysPgene deletion strainC.glutamicum23604ΔlysPwas confirmed by kanamycin resistance screening and PCR identification. After fermentation, the yield of lysine by C.glutamicum23604ΔlysPincreased by 10% compared with the control strain. Using the Escherichiacoliprotein toxin knockout geneMazFas reverse filter mark vector, lysPgene ofC.glutamicumwas knocked out effectively.The knockout of gene lysPmade lysine not enter the cell and just accumulate in the extra, thus achieved the aim of increasing lysine production.