Antibiotic-Free High-Level <scp>l</scp>-Methionine Production in Engineered <i>Escherichia coli</i>

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 <i>hok</i>/<i>sok</i> system was incorporated into the plasmid. The resulting plasmid pAm10 enabled strain M2 <i>thrB</i>^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.