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^E266M 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^E160L/E193Q with enzyme activity 1.35 times higher than AdSHMT was identified. Additionally, the target protein AdSHMT^{E160L/E193Q/E266M} was efficiently overexpressed by improving its mRNA stability. Finally, combining the substrate addition strategy and system optimization, the optimized strain BL21/pET28a-AdSHMT^{E160L/E193Q/E266M}-5'UTR-REP3^S16 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.