Engineering serine hydroxymethyltransferases for efficient synthesis of L-serine in Escherichia coli.

L-serine is a high-value amino acid widely used in the food, medicine, and cosmetic industries. However, the low yield of L-serine has limited its industrial production. In this study, a cellular factory for efficient synthesis of L-serine was obtained by engineering the serine hydroxymethyltransferases (SHMT). Firstly, after screening the SHMT from Alcanivorax dieselolei by genome mining, a mutant AdSHMT with high thermal stability was identified through rational design. Subsequently, an iterative saturating mutant library was constructed by using coevolutionary analysis, and a mutant AdSHMT with enzyme activity 1.35 times higher than AdSHMT was identified. Additionally, the target protein AdSHMT was efficiently overexpressed by improving its mRNA stability. Finally, combining the substrate addition strategy and system optimization, the optimized strain BL21/pET28a-AdSHMT-5'UTR-REP3 produced 106.06 g/L L-serine, which is the highest production to date. This study provides new ideas and insights for the engineering design of SHMT and the industrial production of L-serine.