代谢工程改造谷氨酸棒状杆菌合成及分泌途径生产L-缬氨酸  

Metabolic engineering of L-valine synthesis and secretory pathways in Corynebacterium glutamicum for higher production

作　　者：张海灵 李颜颜[1,2] 王小元[1,2] Hailing Zhang1,2, Yanyan Li1,2, and Xiaoyuan Wang1,2(1 State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu, China ;2 School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China)

机构地区：[1]江南大学食品科学与技术国家重点实验室,江苏无锡214122 [2]江南大学生物工程学院,江苏无锡214122

出　　处：《生物工程学报》2018年第10期1606-1619,共14页Chinese Journal of Biotechnology

基　　金：国家自然科学基金面上项目(No.31370131);江苏省普通高校研究生科研创新计划(No.CXZZ12-0755)资助~~

摘　　要：谷氨酸棒状杆菌是目前微生物发酵生产L-缬氨酸的主要工业菌株。文中首先在谷氨酸棒状杆菌VWB-1中敲除了alaT(丙氨酸氨基转移酶),获得突变菌株VWB-2,作为出发菌株。进而对L-缬氨酸合成途径关键酶——乙酰羟酸合酶(ilvBN)的调节亚基进行定点突变(ilvBN_1^(M13)),解除L-缬氨酸对该酶的反馈抑制。然后辅助过量表达L-缬氨酸合成途径关键基因ilvBN_1^(M13)、乙酰羟酸异构酶(ilvC)、二羟酸脱水酶(ilvD)、支链氨基酸氨基转移酶(ilvE),加强通往L-缬氨酸的碳代谢流,提高菌株的L-缬氨酸水平。最后,基于过量表达L-缬氨酸转运蛋白编码基因brnFE及其调控蛋白编码基因lrp_1,提高细胞的L-缬氨酸转运能力。最终获得工程菌株VWB-2/pEC-XK99E-ilvBN_1^(M13)CE-lrp_1-brnFE在5 L发酵罐中的L-缬氨酸产量达到461.4 mmol/L,糖酸转化率达到0.312 g/g葡萄糖。Corynebacterium glutamicum is the main industrial strain to produce L-valine by microbial fermentation.In this study,a low L-alanine producing C.glutamicum strain VWB-2 was constructed by knocking out the alanine aminotransferase encoding gene alaT in a high L-valine producing strain VWB-1.Meanwhile,a site-directed mutagenesis（ilvBN_1^（M13））was done on the regulatory subunit of acetohydroxyacid synthase（ilvBN）,a key enzyme in the L-valine synthesis pathway.Furthermore,the overexpression of the genes involved in the biosynthesis of L-valine,the mutated ilvBN_1^（M13）,the acetohydroxy acid isomerase coding genes ilvC,the dihydroxy-acid dehydratase coding gene ilvD and branched-chain amino acid aminotransferase coding gene ilvE,could all promote the L-valine production of VWB-1 by strengthening the carbon flow towards L-valine.With the overexpression of the branched chain amino acid transporter coding gene brn FE and its regulator lrp_1,the L-valine producing capability of VWB-1 was further enhanced.The finally obtained engineered strain VWB-2/pEC-XK99E-ilvBN_1^（M13）CE-lrp_1-brnFE could produce 461.4 mmol/L L-valine in a 5 L fermentor with a sugar acid conversion rate of 0.312 g/g glucose.

关 键 词：L-缬氨酸 代谢工程 发酵 谷氨酸棒状杆菌 

分 类 号：Q936[生物学—微生物学]