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

d-tagatose is a highly valuable rare sugar with significant application potential. However, its widespread use is severely limited by high production costs. One-step conversion from d-fructose represents an economically attractive bioprocess; nevertheless, the low catalytic efficiency of natural tagatose 4-epimerases (T4Es) toward d-fructose remains a major bottleneck for industrial application. Therefore, developing highly efficient T4Es is essential in this field. In this study, a structure-guided and evolution-assisted engineering strategy was employed to enhance the catalytic performance of a tagatose 4-epimerase (KoT4E) from Kosmotoga sp. The superior mutant (M6) exhibited 5.6-fold the specific activity and 9.6-fold the catalytic efficiency (k_cat/K_m) 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.