Engineering Corynebacterium glutamicum for efficient l-homoserine production

l-homoserine is an important precursor in synthesizing the essential amino acids derived from l-aspartate and other valuable bio-based products, and is widely used in cosmetics, food, and pharmaceutical industries. However, the yield of l-homoserine in Corynebacterium glutamicum is limited. In this study, we successfully engineered a C. glutamicum to efficiently produce l-homoserine. First, a l-threonine-producing strain TWZ023 was optimized by knocking out thrB, resulting in the strain HWZ006 which could produce 20.8 g/L l-homoserine with a yield of 0.231 g/g glucose. Then, the 240th Arg residue of homoserine kinase (HK) in TWZ023 was identified as an important site for l-homoserine binding. Subsequently, the optimal mutant strain R240I was screened through site-directed saturation mutagenesis of HK in TWZ023, it could produce 26.8 g/L l-homoserine with a yield of 0.298 g/g glucose. The HK-homoserine binding mechanism was analyzed by using molecular docking, further providing some potential mutation sites. The final strain R240I/pXTuf-Cgl2344 was obtained by optimizing the efflux of l-homoserine, it could produce 29.9 g/L l-homoserine with a yield of 0.332 g/g glucose in shake flasks. In a 15 L fermenter, the final strain produced 78.3 g/L with a yield of 0.28 g/g glucose. These metabolic engineering strategies used in this study have fundamentally enhanced the ability of C. glutamicum to produce l-homoserine.