A metabolic optimization strategy based on modular genomic integration of the MVA pathway to enhance lycopene production in Escherichia coli

Lycopene, a bioactive tetraterpenoid antioxidant valued in food, pharmaceutical, and cosmetic sectors, remains constrained by low plant extractability and expensive chemical synthesis; microbial engineering now presents a cost-effective alternative. In this study, key genes from the methylerythritol phosphate (MEP) pathway—dxs, idi, and ispDF—were integrated into the genome of the chassis strain Escherichia coli MG1655 using a CRISPR-Cpf1-based system, resulting in MEP pathway-overexpressed strains. Additionally, the downstream module (MBot) of the mevalonate (MVA) pathway was optimized by introducing T7 RNA polymerase, mvaE, and mvaS from different species, including Saccharomyces cerevisiae, Streptococcus pneumoniae, and Staphylococcus aureus. Integration of the MEP genes improved lycopene production by 2 mg/L compared with the initial strain. Notably, fermentation performance varied significantly depending on the source of the downstream MBot module. The optimal combination—erg12 from Saccharomyces cerevisiae, mvaK and mvd1 from Streptococcus pneumoniae, and idi from Escherichia coli—achieved a lycopene titer of 86 mg/L in shake-flask cultures, representing a 21-fold increase compared to the parental strain. Paradoxically, dxr deletion to eliminate endogenous MEP flux precipitated a 7-fold drop in lycopene titre, whereas MEP overexpression failed to enhance production—revealing that MEP–MVA pathway synergy, rather than simple precursor supply, governs efficient carotenoid biosynthesis.