Abstract: The starting strain，Escherichia coliMG1655 (M0)， was selected for this study, and the CRISPR/Cas9 genome editing technology was utilized to optimize theL-arginine synthesis gene cluster. Initially, the ornithine decarboxylase genesspeCandspeFwere knocked out to enhance the ornithine supply, a precursor of arginine. Subsequently, integration of theargCJBDFgene cluster fromCorynebacterium glutamatewas used to deactivate the arginine decarboxylase geneadiA, blockingL-arginine degradation while enhancing its synthesis pathway. The resulting recombinant strain M1 (MG1655ΔspeCΔspeFΔadiA::argCJBDF) achieved an arginine yield of 1.35 g/L, representing a 0.88 g/L increase compared to strain M0. Untargeted metabolomics analysis revealed significant alterations in the arginine metabolic pathway and related metabolite concentrations in recombinant strain M1, impacting amino acid synthesis such as arginine and lysine, organic acids, and other metabolites. This study offers insights into potential strategies for regulating these alternate metabolic pathways and developing high-yieldL-arginine engineering strains.

Key words: E. coliMG1655;L-arginine, gene editing, optimization engineering, metabolomics