Antibiotic-Free High-Level l-Methionine Production in Engineered Escherichia coli

l -Methionine, a valuable sulfur-containing amino acid, holds great significance as a feed additive, nutraceutical, pharmaceutical, or even in the cosmetic industry. However, achieving efficient microbial production of l -methionine remains challenging due to its complex biosynthetic pathway and plasmid loss during fermentation. Herein, l -methionine biosynthesis was improved by enhancing succinyl-CoA supply, introducing a direct-sulfurylation pathway, and weakening the l -threonine branched pathway. The engineered strain produced 21.55 g/L l -methionine with a yield of 0.14 g/g glucose in a 5 L bioreactor. To eliminate the need for antibiotics and minimize plasmid loss, the hok / sok system was incorporated into the plasmid. The resulting plasmid pAm10 enabled strain M2 thrB A1G to produce 20.39 g/L of l -methionine without antibiotics in 5 L of fed-batch cultivation, a 42.58% increase compared to the control. This study highlights the potential of plasmid-based antibiotic-free fermentation for efficient and cost-effective production of l -methionine, as well as other amino acids or chemicals.