High-level production of vitamin K2 in Escherichia coli via modular molecular engineering

Menaquinone-7 (MK-7), a key form of vitamin K2 with wide-ranging nutritional and pharmaceutical applications, has attracted increasing interest for microbial production. Here, we developed an integrated modular metabolic engineering strategy in <i>Escherichia coli</i> to enhance MK-7 biosynthesis. Cellular membrane capacity and acetate metabolism were rewired to improve precursor supply for the mevalonate (MVA) pathway, while arabinose induction was applied to overexpress three critical enzymes, including BsHepPPS (<i>Bacillus subtilis</i>), EcMenA (<i>E. coli</i>), and BsUbiE (<i>B. subtilis</i>). Among them, EcMenA was identified as a major bottleneck. Rational protein engineering based on folding free energy analysis and consensus design yielded the EcMenA mutant G110W, which produced 102.55 mg/L MK-7 in shake-flask fermentation, a 57.2 % increase compared with the wild-type (WT) enzyme. Further active-site hotspot random mutagenesis generated a G110W-Q57T double mutant, raising MK-7 production to 176.38 mg/L, a 72 % increase compared to the single mutant. Optimization of EcMenA expression cassette by ribosome binding site redesign using a generative network further improved MK-7 titer to 227.53 mg/L in shake flasks. Finally, scale-up fermentation in a 50-L bioreactor, combined with optimized fermentation strategies, achieved a maximum MK-7 titer of 2.18 g/L. This study establishes a systematic framework integrating metabolic rewiring, enzyme engineering, and expression optimization, providing a robust platform for industrial-scale MK-7 production in microbial hosts.