High-level production of L-methionine through collaborative modification of multiple modules in Escherichia coli

L-Methionine is the only sulfur-containing essential amino acid, and it plays a pivotal role in various industries. Due to the unique sulfur-containing structure and complex synthetic regulation, the biological production of L-methionine is still difficult to rival with the chemical synthesis method. In this study, L-cysteine synthesis and one-carbon unit supply modules were successively modified based on a non-auxotrophic L-methionine producing strain. Strengthening the genes in sulfate pathway effectively boosted L-methionine production to 2.85 g/L, and the reduction of SO₄²⁻ to SO₃²⁻ and the supply of cofactors for sulfite reductase were key limitations for sulfur assimilation in this pathway. And the modification of the thiosulfate, new thiosulfate, and the one-carbon unit yielded no significant results. However, improving the cell membrane permeability could enhance the L-methionine production to 3.45 g/L. Finally, the L-methionine production reached 36.06 g/L for fed-batch fermentation in a 5-L bioreactor, which was the highest reported L-methionine titer to date. The study provides a well research foundation for L-methionine production by microbial fermentation with the capacity for industrial application.