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Methionine is the only one of the essential amino acids that contain sulfur, widely used as a feed additive in agriculture. In this study, the availability of 5-methyl-tetrahydrofolate was confirmed as the main limitation in the complex multibranched biosynthetic pathway of L-methionine. The cycle of one-carbon units was thoroughly investigated and modified to supply 5-methyl-tetrahydrofolate for L-methionine production, such as enhancing the supply of precursor, expediting the conversion rate of the cycle, introducing exogenous serine hydroxymethyltransferase and increasing pool size of one-carbon units carrier. The final strain MYA/pAmFA-4 was able to produce 20.89 g/L L-methionine by fed-batch fermentation, which was the highest titer reported in the literatures. This study is instructive for other metabolites biosynthesized needing one-carbon units or having a complex multibranched biosynthetic pathway., Supplementary Information: The online version contains supplementary material available at 10.1007/s13205-023-03625-9., Competing Interests: Conflict of interestThe authors declare that they have no conflict of interest in the publication., (© King Abdulaziz City for Science and Technology 2023, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.)

O -succinyl-l-homoserine (OSH) is a promising platform chemical for the production of C4 chemicals with huge market potential which can be produced by fermentation from glucose. To construct a strain capable of producing OSH with high yield, the metJ (encodes transcriptional repressor) and metI (encodes a subunit of dl-methionine transporter) were deleted in Escherichia coli W3110 to obtain a strain E. coli ∆JI. Then, overexpression of metL (encodes bifunctional aspartate kinase/homoserine dehydrogenase II) and inactivation of metB (encodes cystathionine γ-synthase) were implemented in one step, and the OSH titer of the resulting strain E. coli ∆JIB * Trc metL was dramatically increased to 7.30 g/L. The feedback regulation was further relieved by progressively overexpressing metA fbr (encodes homoserine O -succinyltransferase), yjeH (encodes l-methionine exporter), and thrA fbr (encodes bifunctional aspartate kinase/homoserine dehydrogenase I) to increase the metabolic flux from aspartate to OSH. The 100% rationally designed strain E. coli ∆JIB * Trc metL /pTrc- metA fbr -Trc- thrA fbr - yjeH produced 9.31 g/L OSH from 20 g/L glucose (0.466 g/g glucose) in batch fermentation, which represents the highest OSH yield from glucose reported to date. The culture profiles of the newly constructed strains were recorded to investigate their productive properties. The effects of l-methionine addition on the fermentation process of the optimal strain were also studied. Our results demonstrate that tuning the expression level of metL , inactivation of metB , and attenuation of feedback resistance of the crucial enzymes in the biosynthetic pathway are the key factors that impact the OSH production in E. coli ., Competing Interests: Compliance with ethical standardsThe authors declared that there is no conflict of interest.