谷氨酸棒杆菌代谢工程高效生产L-缬氨酸  

Highly efficient production of L-valine by multiplex metabolic engineering of Corynebacterium glutamicum

作　　者：赵阔 程金宇 郭亮 高聪 宋伟 吴静 刘佳[1,2] 柳亚迪 刘立明 陈修来[1,2] ZHAO Kuo;CHENG Jinyu;GUO Liang;GAO Cong;SONG Wei;WU Jing;LIU Jia;LIU Yadi;LIU Liming;CHEN Xiulai(State Key Laboratory of Food Science and Technology,Jiangnan University,Wuxi 214122,Jiangsu,China;International Joint Laboratory on Food Safety,Jiangnan University,Wuxi 214122,Jiangsu,China;School of Life Sciences and Health Engineering,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]江南大学食品安全国际合作联合实验室,江苏无锡214122 [3]江南大学生命科学与健康工程学院,江苏无锡214122

出　　处：《生物工程学报》2023年第8期3253-3272,共20页Chinese Journal of Biotechnology

基　　金：江苏省农业科技自主创新资金项目(CX(22)1012);国家自然科学基金(22122806,22208124,32000037);江苏省自然科学基金(BK20211529,BK20200614)。

摘　　要：L-缬氨酸作为一种支链氨基酸,广泛应用于医药和饲料等领域。本研究借助多种代谢工程策略相结合的方法,构建了生产L-缬氨酸的微生物细胞工厂,实现了L-缬氨酸的高效生产。首先,通过增强糖酵解途径、减弱副产物代谢途径相结合的方式,强化了L-缬氨酸合成前体丙酮酸的供给;其次,针对L-缬氨酸合成路径关键酶—乙酰羟酸合酶进行定点突变,提高了菌株的抗反馈抑制能力,并利用启动子工程策略,优化了路径关键酶的基因表达水平;最后,利用辅因子工程策略,改变了乙酰羟酸还原异构酶和支链氨基酸转氨酶的辅因子偏好性,由偏好NADPH转变为偏好NADH,从而提高了L-缬氨酸的合成能力。在5L发酵罐中,最优谷氨酸棒杆菌工程菌株Corynebacterium glutamicum K020的L-缬氨酸产量、得率和生产强度分别达到了110g/L、0.51g/g和2.29 g/(L·h)。As a branched chain amino acid,L-valine is widely used in the medicine and feed sectors.In this study,a microbial cell factory for efficient production of L-valine was constructed by combining various metabolic engineering strategies.First,precursor supply for L-valine biosynthesis was enhanced by strengthening the glycolysis pathway and weakening the metabolic pathway of by-products.Subsequently,the key enzyme in the L-valine synthesis pathway,acetylhydroxylate synthase,was engineered by site-directed mutation to relieve the feedback inhibition of the engineered strain.Moreover,promoter engineering was used to optimize the gene expression level of key enzymes in L-valine biosynthetic pathway.Furthermore,cofactor engineering was adopted to change the cofactor preference of acetohydroxyacid isomeroreductase and branched-chain amino acid aminotransferase from NADPH to NADH.The engineered strain C.glutamicum K020 showed a significant increase in L-valine titer,yield and productivity in 5 L fed-batch bioreactor,up to 110 g/L,0.51 g/g and 2.29 g/(L‧h),respectively.

关 键 词：谷氨酸棒杆菌 L-缬氨酸 代谢工程 路径优化 

分 类 号：TQ922[轻工技术与工程—发酵工程]