Mechanism-guided engineering of inositol dehydrogenase for efficient biocatalytic production of epi-inositol

Epi-inositol is a nutraceutical with broader application potential than myo-inositol, but its supply has long depended on inefficient multi-step chemical synthesis. Here, we identify an inositol dehydrogenase that catalyzes the reduction of 1L-epi-2-inosose to epi-inositol, establishing its first biosynthetic route. Semi-rational engineering—integrating deep-learning turnover prediction (DLkcat), binding energy calculations, and evolutionary-conservation analysis—yielded the variants GtIDH-GHGA, which exhibited about 3.0-fold increase in catalytic efficiency (k_cat/K_m) and improved product yields from 10.2% to 48.7%. Molecular dynamics simulations revealed enhancements due to strengthened substrate binding, optimized active-site geometry, remodeled hydrogen-bond networks facilitate proton and hydride transfers, and reshaped product-release tunnels. Coupling a cofactor regeneration system with reaction optimization achieved 4.92 g/L epi-inositol at 68.3% molar yield (diastereomeric excess >99%)—a 6.7-fold improvement over wild-type. This work establishes the highest reported titer for epi-inositol biosynthesis, providing a practical foundation for its expanded application in food, biology, and medicine.