L-serine is one of the 30 most important skeleton compounds in chemical and materials industries. In order to explore the mechanism of L-serine production by Corynebacterium glutamicum, the whole-genome sequencing and comparative genomics analysis were performed, and the relationship between genotype and phenotype was studied through reverse mutation, gene deletion and overexpression. The pK18mobsacB plasmid was used for reverse mutation of four genes (faS, folC, rhtA, and tyrP) related to transport or metabolism in the starting strain ΔSSAAI, and corresponding mutants Kf, Ko, Kr, and Kt were obtained. The fermentation of the mutants showed that, compared with the starting strain ΔSSAAI, the biomass of Kf, Ko, Kr, and Kt did not change significantly, and the L-serine titer of Kf and Ko did not change significantly, while that of Kr and Kt were 31.1 g/L and 31.04 g/L, increased by 18.5% and 18.3%, respectively. The deletion strains ΔrhtA and ΔtyrP were further constructed, and the fermentation results showed that there was no significant change in the L-serine titer. Then rhtA and tyrP were overexpressed in ΔSSAAI, L-serine titer improved slightly. rhtA and tyrP were overexpressed in themutantstrainA36,andtherecombinantstrainspD-RandpD-Twere obtained, resulting in L-serine titer of 32.55 g/L and 33.7 g/L, which was 6.5% and 10.3% higher than that of A36. At the same time, rhtA and tyrP overexpression resulted in a decrease of the biomass by 13.88% and 24.5%, respectively. The results indicated that rhtA and tyrP were related to the cell growth and L-serine production.
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