Discovery and engineering of a uridine diphosphate glycosyltransferase for efficient gastrodin biosynthesis in yeast

Gastrodin is a bioactive phenolic glucoside naturally found in Gastrodia elata and is widely recognized for its health-promoting properties. However, its limited natural supply necessitates sustainable microbial production strategies. Here, we report the first experimental characterization of a uridine diphosphate glycosyltransferase (UGT) from Triticum aestivum , termed HGT, for the biosynthesis of gastrodin from p -hydroxybenzyl alcohol ( p HBA) in Saccharomyces cerevisiae . While wild-type HGT demonstrated robust activity with 86.7% conversion at 5 mmol/L p HBA, its efficiency decreased significantly at elevated substrate concentrations, limiting its applicability in high-titer bioprocesses. To address this limitation, we employed a directed evolution strategy combined with fluorescence-based screening. This approach identified two mutants, A133T and T256I, with significantly enhanced performance. In fermentations with 40 mmol/L p HBA, the A133T and T256I mutants achieved maximum gastrodin titers of 5.37 g/L and 5.45 g/L, corresponding to 42.5% and 44.4% improvements in conversion efficiency compared to the wild-type enzyme. In vitro kinetic assays further validated their enhanced performance, with A133T and T256I exhibiting 1.93-fold and 1.73-fold increases in catalytic efficiency, respectively. Finally, computational modeling provided insights into potential structural determinants of the improved activity. This work identifies and engineers HGT as an efficient uridine diphosphate glycosyltransferase for gastrodin biosynthesis, providing a useful enzymatic module for improving yeast whole-cell biocatalytic production of this bioactive glycoside.