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

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5 L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110 g/L、0.51 g/g和2.29 g/(L·h)。

Abstract:

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.