Zhang Hailing, Li Yanyan, Wang Xiaoyuan
State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, Jiangsu, China.
School of Biotechnology, Jiangnan University, Wuxi 214122, Jiangsu, China.
Sheng Wu Gong Cheng Xue Bao. 2018 Oct 25;34(10):1606-1619. doi: 10.13345/j.cjb.180112.
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 (ilvBN₁ (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 ilvBN₁ (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 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-ilvBN₁ (M13)CE-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.
谷氨酸棒杆菌是通过微生物发酵生产L-缬氨酸的主要工业菌株。在本研究中，通过敲除高产L-缬氨酸菌株VWB-1中编码丙氨酸转氨酶的基因alaT，构建了低产L-丙氨酸的谷氨酸棒杆菌菌株VWB-2。同时，对L-缬氨酸合成途径中的关键酶乙酰羟酸合酶（ilvBN）的调节亚基进行了定点诱变（ilvBN₁ (M13)）。此外，L-缬氨酸生物合成相关基因、突变的ilvBN₁ (M13)、乙酰羟酸异构酶编码基因ilvC、二羟基酸脱水酶编码基因ilvD和支链氨基酸转氨酶编码基因ilvE的过表达，均能通过增强碳流向L-缬氨酸的流动来促进VWB-1的L-缬氨酸产量。随着支链氨基酸转运蛋白编码基因brnFE及其调节因子lrp₁的过表达，VWB-1的L-缬氨酸生产能力进一步提高。最终获得的工程菌株VWB-2/pEC-XK99E-ilvBN₁ (M13)CE-lrp₁-brnFE在5 L发酵罐中可产生461.4 mmol/L的L-缬氨酸，糖酸转化率为0.312 g/g葡萄糖。
