Metabolic engineering of plasmid-free Escherichia coli for enhanced glutarate biosynthesis from glucose

Glutarate is a crucial synthetic precursor for the production of polyesters and polyamides. However, recent studies have predominantly focused on plasmid-based systems for high-level glutarate production, which are impractical for industrial applications. In this study, we aimed to construct a plasmid-free recombinant E. coli strain to enhance glutarate biosynthesis from glucose. First, to increase the supply of the precursor L-lysine, ARTP mutagenesis was applied to generate a lysine-overproducing mutant strain. Moreover, various gene integration sites were characterized to facilitate the chromosomal integration of the glutarate biosynthetic pathway in this mutant strain. Subsequently, a glutarate biosensor was developed to optimize the rate-limiting DavTD module in the glutarate synthetic pathway through copy number optimization. Additionally, transcriptome analysis identified a potential glutarate transporter, YidE, which was co-expressed with the lysine transporter LysP to further enhance glutarate production. Ultimately, in a 5-L fed-batch fermentation, the optimal strain RY29 achieved a glutarate titer of 44.8 g/L, with a yield of 0.28 g/g and a productivity of 0.62 g/L/h. These findings provide valuable insights into the stable production of glutarate in microbial cell factories.