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

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 (ilvBN1M13)，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ilvBN1M13、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lrp1，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-ilvBN1M13CE-lrp1-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。

Abstract:

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 (ilvBN1M13) 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 ilvBN1M13, 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 brnFE and its regulator lrp1, the L-valine producing capability of VWB-1 was further enhanced. The finally obtained engineered strain VWB-2/pEC-XK99E-ilvBN1M13CE-lrp1-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.