谷氨酰胺合成酶基因的过量表达有效提高谷氨酸棒杆菌中L-谷氨酰胺产量

Significantly improving the productivity of glutamine with over-expression of glnA in Corynebacterium glutamicum

以谷氨酸棒杆菌Corynebacterium glutamicum ATCC13032基因组为模板,通过PCR扩增,得到大小为1434bp的谷氨酰胺合成酶基因glnA.以大肠杆菌/谷氨酸棒杆菌穿梭质粒pXMJ19为载体,将扩增得到的目的基因片段克隆至C.glutamicum Res167,获得重组菌株C.glutamicum Res167/pXMJ19-glnA.在摇瓶发酵水平上,通过IPTG诱导glnA基因的表达,并采用反相高效液相色谱方法测定了发酵液中的L-谷氨酰胺含量.结果显示,与未经诱导的对照菌相比,诱导后的重组菌发酵液中的L-谷氨酰胺产量提高了1.8倍,最高产量达1.54g·L-1.

A 1434bp fragment of Corynebacterium glutamicum glnA gene encoding glutamine synthetase （GS） was proliferated by polymerase chain reaction by using chromasomal DNA of the wild-type strain C. glutamicum ATCC13032 as the template. The obtained glnA fragment was inserted into E. coli/C, glutamicum shuttle vector pXMJ 19 to construct an expression plasmid pXMJ19-glnA. It was then introduced into a restriction-deficient mutant strain C. g lutamicum Res167 via electrotransformation, and a recombinant C. glutarnicum Res167/pXMJ19-glnA was obtained. The glutamate in the broth was detected by HPLC when the culture in the shake flask was induced with 0.5 mmol · L^-1 IPTG. The results showed that after 5-day cultivation, the recombinant stain could produce 1. 54 g · L ^-1 glutamine, about 1.8 folds higher than those with un-induced culture.