Structure-Guided Engineering of a Multifunctional Glycosidase for Exclusive High-Yield Production of Ginsenoside F1 from Panax Notoginseng

Ginsenoside F1 (G-F1), a therapeutically valuable compound from Panax notoginseng, faces production challenges due to its low natural abundance. Herein, we engineered the glycoside hydrolase BgDU via structure-based design for efficient bioconversion of Notoginsenoside R1 (NG-R1) to G-F1. Through a semirational design approach, we generated the triple mutant I73L/G138H/W509Y (DUase). This engineered variant demonstrated significant catalytic improvements, showing an 85-fold reduction in K_M for NG-R1 along with 16-fold and 5-fold increases in k_cat/K_M values for NG-R1 and ginsenoside Rg1, respectively. DUase achieved a 95.02% molar yield of G-F1 (12.82 g/L) within 10 h, while reducing the byproduct Rh1 to below 0.7 g/L. DUase’s catalytic improvement resulted from a remodeled substrate channel with expanded proximal and contracted distal regions, overcoming the classic activity–selectivity trade-off and enabling scalable production of rare ginsenosides.