Backgroundl-Methionine is the only bulk amino acid that has not been industrially produced by the fermentation method. Due to highly complex and strictly regulated biosynthesis, the development of microbial strains for high-levell-methionine production has remained challenging in recent years.ResultsBy strengthening thel-methionine terminal synthetic module via site-directed mutation ofl-homoserine O-succinyltransferase (MetA) and overexpression ofmetAfbr,metC, andyjeH,l-methionine production was increased to 1.93 g/L in shake flask fermentation. Deletion of thepykAandpykFgenes further improvedl-methionine production to 2.51 g/L in shake flask fermentation. Computer simulation and auxotrophic experiments verified that during the synthesis ofl-methionine, equimolar amounts ofl-isoleucine were accumulated via the elimination reaction of cystathionine γ-synthetase MetB due to the insufficient supply ofl-cysteine. To increase the supply ofl-cysteine, thel-cysteine synthetic module was strengthened by overexpression ofcysEfbr,serAfbr, andcysDN, which further increased the production ofl-methionine by 52.9% and significantly reduced the accumulation of the byproductl-isoleucine by 29.1%. After optimizing the addition of ammonium thiosulfate, the final metabolically engineered strain MET17 produced 21.28 g/Ll-methionine in 64 h with glucose as the carbon source in a 5 L fermenter, representing the highestl-methionine titer reported to date.ConclusionsIn this study, a high-efficiency strain forl-methionine production was derived from wild-typeEscherichia coliW3110 by rational metabolic engineering strategies, providing an efficient platform for the industrial production ofl-methionine.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13068-023-02347-7.