Enhancing one-step d-tagatose production from d-fructose through combinatorial structure-guided and evolution-based engineering of tagatose 4-epimerase.

) of the wild-type, respectively. Using 500 g/L d-fructose, 28% d-tagatose yield was achieved with a relatively low enzyme loading of 1 mg/mL within 12 h, substantially outperforming all currently known T4Es. Molecular dynamics simulations revealed that reduced flexibility of a key loop in the substrate-binding channel stabilized substrate binding and facilitated catalysis. The industrial applicability of M6 was further demonstrated, suggesting its potential for promoting the low-cost and high-efficiency large-scale bio-manufacturing of d-tagatose and offering insights for engineering of epimerases lacking in nature.