Rational design of heptaprenyl diphosphate synthase and metabolic optimization drive biosynthesis of menaquinone-7 by Escherichia coli.

Menaquinone-7 (MK-7) is a fat-soluble vitamin with important nutritional and pharmaceutical applications. However, microbial biosynthesis of MK-7 is limited by low catalytic efficiency of key enzymes, insufficient precursor supply, and imbalanced availability of nicotinamide adenine dinucleotide phosphate (NADPH). In this study, Escherichia coli was engineered for de novo MK-7 biosynthesis through pathway reconstruction, enhanced supply of 1,4-dihydroxy-2-naphthoic acid and farnesyl pyrophosphate, and screening of isopentenyl diphosphate isomerase enzymes. Subsequently, deep learning models and molecular docking were used to identify an efficient heptaprenyl diphosphate synthase combination, SolHepS/HepT, which was further engineered by targeting active-site residues, subunit interfaces, and distal residues. Fusion protein construction, dynamic expression regulation, attenuation of competing branches, and enhanced NADPH regeneration synergistically balanced cell growth and metabolic flux toward MK-7 biosynthesis. Finally, the engineered strain yielded 2.51 g/L of MK-7 in a 5-L bioreactor. The multi-level synergistic strategy developed in this study provided a useful reference for the efficient biomanufacturing of MK-7 and other natural complex isoprenoid products.