Enhanced Menaquinone Biosynthesis by Engineering 2-Succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate Synthase MenD to Alleviate Feedback Inhibition in <i>Bacillus subtilis</i>

Vitamin K₂ (menaquinones, MKs), a group of fat-soluble vitamins with a 2-methyl-1,4-naphthoquinone core, plays vital roles in bone metabolism and cardiovascular health. Although microbial biosynthesis has become the primary method for MKs production, regulatory constraints and metabolic bottlenecks in the MK pathway limit its production efficiency. In this study, we identified that 1,4-dihydroxy-2-naphthoic acid (DHNA), an intermediate in the MK pathway of <i>Bacillus subtilis</i>, exerts feedback inhibition on the key enzyme 2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate (SEPHCHC) synthase MenD. Through structure-guided evolution, we obtained a feedback-resistant variant (MenD^W322I/R323F) with 43% higher activity than wild-type, maintaining full function at 1 μM DHNA. This mutant boosted Menaquinone-7 (MK-7) production by 22.3% to 186.5 mg/L. Molecular dynamics (MD) simulation indicates that the attenuation of the feedback inhibition results from weakened interaction forces between the mutant and DHNA. This work advances the understanding of MKs pathway regulation and paves the way for high-efficiency microbial production of MKs.