关键词: 谷氨酸棒杆菌；菌株构建；过表达；筛选钝化酶；代谢流分析

Abstract: Objective: To a new strain for improved microbial production of methionine. Methods: Aspartate kinase (AK), homoserine dehydrogenase (HSD) and homoserine acetyltransferase (HAT), the key enzymes of methionine synthesis pathway, were modified and overexpressed in vitro. In addition, site-directed mutagenesis and characterization of the substrate binding site of the key enzyme, homoserine kinase (HSK) A20, were carried out to reduce the enzymatic activity and to prevent carbon flow from entering the threonine pathway. Results: Compared with the original strain, the expression levels of lysC, hom and metX in strain WTg1/PEC-lysCm-SD-homm-SD-metX increased by 6.896, 2.378 and 1.659 times, respectively. The liquid chromatographic analysis showed that the production of methionine was 4.14 g/L, 2.26 times higher than that of the original strain. The activities of HSK mutants thrB-A20Y, thrB-A20H and thrB-A20L were 39%, 43% and 49% as compared to HSK. Conclusion: In this experiment, the downstream metabolism of aspartic acid in Corynebacterium glutamicum was regulated by overexpression of the key enzymes in the trunk and weakening of the key enzymes in the branch. As a result, the methionine carbon transport system was strengthened, the accumulation of carbon flow was enhanced, and the production of methionine was effectively increased. This study provides theoretical reference for the construction of engineered bacteria for methionine production.

Key words: Crynebacterium glutamicum; strain construction; overexpression; screening of passivating enzymes; metabolic flux analysis