Rationally Engineered Novel Glycosyltransferase UGT74DD1 from Siraitia grosvenorii Catalyzes the Generation of the Sweetener Mogroside III

that catalyzes the conversion of mogrol to mogroside IIE. Site-directed mutagenesis yielded the UGT74DD1-W351A mutant, which exhibited the new capability to transform mogroside IIE into the valuable sweetener mogroside III, but with low catalytic activity. Subsequently, using structure-guided directed evolution with combinatorial active-site saturation testing, the superior mutant M6 (W351A/Q373 K/E49H/Q335W/S278C/D17F) were obtained, which showed a 46.1-fold increase in catalytic activity compared to UGT74DD1-W351A. Molecular dynamics simulations suggested that the enhanced activity and extended substrate profiles of M6 are due to its enlarged substrate-binding pocket and strengthened enzyme-substrate hydrogen bonding interactions. Overall, we redesigned UGT74DD1, yielding mutants that catalyze the conversion of mogrol into mogroside III. This study thus broadens the toolbox of UGTs capable of catalyzing the formation of valuable polyglycoside compounds.