Directed Evolution Boosts UGT85A1 Glycosylation for Enhanced Phenylethanoid Glucoside Production

Glycosides are diverse plant metabolites with important biological functions. Salidroside and its derivatives, natural phenylethanoid glycosides, have considerable potential in food and pharmaceutical applications, but their low natural abundance limits large-scale production. Microbial biosynthesis offers a sustainable alternative, where glycosyltransferase efficiency is critical. Here, we engineered UGT85A1 via iterative saturation mutagenesis (ISM), yielding the high-activity mutant DHG (G114D/F217H/C128G) with a 23.6-fold increase in catalytic efficiency. Molecular dynamics analyses indicated that DHG's enhanced activity stems from stabilized substrate binding and optimized catalytic geometry. In tyrosol- and hydroxytyrosol-overproducing yeast strains, DHG enabled salidroside and hydroxysalidroside production of 2.70 g/L (4.6×) and 1.63 g/L (19.4×), respectively─the highest titers reported in <i>S. cerevisiae</i> in shake-flask cultures (50 mL). This work provides a robust platform and general strategy for engineering plant UGTs to expand microbial production of high-value glycosides.