摘要
以谷氨酸棒杆菌SPT9(Phe-、Tyr-、4-FPr、6-FTr)为出发菌株,采用PCR方法扩增色氨酸合成途径中解除了反馈抑制的关键酶aroⅡ和trpEGD基因,应用大肠杆菌-谷氨酸棒杆菌穿梭表达载体pEC-XK99E在谷氨酸棒杆菌SPT9中对其进行了分别过量表达和串联过量表达。首先对aroⅡ和trpEGD基因分别进行过量表达,得到菌株SPT9-aroⅡ、SPT9-trpEGD,摇瓶发酵试验表明,菌株SPT9-aroⅡ、SPT9-trpEGD的色氨酸产量相对于出发菌株SPT9分别提高了30%、23%;进一步对aroⅡ和trpEGD进行了串联过量表达,得到菌株SPT9-trpEGD-TP-aroⅡ,摇瓶发酵试验表明,该菌株色氨酸产量相对于出发菌株SPT9提高了84%。荧光定量PCR检测表明,菌株SPT9-trpEGD-TP-aroⅡ中aroⅡ、trpEGD基因的表达量分别为出发菌株SPT9的4.0倍和1.3倍。
The host strain in this paper was C.glutamicum SPT9（Phe-,Tyr-,4-FPr,6-FTr）.In order to improve bio-production of tryptophan,feedback inhibition resistant aroⅡand trpEGD genes were cloned by PCR and over-expressed in C.glutamicum SPT9 respectively using Escherichia coli-C.glutamicum shuttle expression vector pEC-XK99E.Over-expressions were effective,and the amounts of tryptophan biosynthesis in the resulted strains SPT9-aroⅡ,SPT9-trpEGD were increased by 30% and 23% respectively,compared with that of the host strains SPT9.Then,aroⅡ and trpEGD genes were co-expressed using the same shuttle expression vector pEC-XK99E,and the amount of tryptophan biosynthesis in the resulted strain SPT9-trpEGD-TP-aroⅡ was increased by 84% compared with that of the host strains SPT9.Fluorescence quantitative PCR showed that the relative expressions of aroⅡ and trpEGD genes in strain SPT9-trpEGD-TP-aroⅡ were increased to 4.0 folds and 1.3 folds,respectively.
出处
《生物技术通报》
CAS
CSCD
北大核心
2011年第5期151-156,171,共7页
Biotechnology Bulletin
基金
中央高校基本科研业务费资助项目(2009ZM0289)
国家科技支撑计划项目(2007BAK36B03)
关键词
谷氨酸棒杆菌
L-色氨酸
关键酶
过量表达
串联表达
Corynebacterium glutamicum L-tryptophan Key enzymes Over-expression Co-expression
