Glycosyltransferase engineering and multi-glycosylation routes development facilitating synthesis of high-intensity sweetener mogrosides

Mogrosides are widely served as natural zero-calorie sweeteners. To date, the biosynthesis of high-intensity sweetness mogrosides V from mogrol has not been achieved because of inefficient and uncontrollable multi-glycosylation process. To address this challenge, we reported three UDP-glycosyltransferases (UGTs) catalyzing the primary and branched glycosylation of mogrosides and increased the catalytic efficiency by 74-400-folds toward branched glycosylation using an activity-based sequence conservative analysis engineering strategy. The computational studies provided insights into the origin of improved catalytic activity. By virtue of UGT mutants, we provided regio- and bond-controllable multi-glycosylation routes, successfully facilitating sequential glycosylation of mogrol to three kinds of mogroside V in excellent yield of 91-99%. Meanwhile, the feasibility of the routes was confirmed in engineered yeasts. It suggested that the multi-glycosylation routes would be combined with mogrol synthetic pathway to