Ancestral Sequence Reconstruction and SemirationalEngineering of Glycosyltransferase for Efficient Synthesis of RareGinsenoside Rh1

Rare ginsenoside Rh1, exhibiting great potential in the food industry, is limited by its natural scarcity. This constraint has driven the development of biocatalytic synthesis approaches, yet robust enzymes capable of efficient production remain elusive. Here, we employed the ancestral sequence reconstruction (ASR) approach to create a thermostable UDP-dependent glycosyltransferase (UGT227) for Rh1 synthesis from 20(<i>S</i>)-protopanaxatriol (PPT). UGT227 exhibited enhanced thermostability (<i>t</i>_1/2 = 44.2 h at 60 °C) but initially yielded only 15% Rh1. Semirational engineering generated the I83A/F285 M variant, increasing the yield to 92%. For economic viability, the I83A/F285 M variant was coexpressed with Arabidopsis thaliana sucrose synthase (AtSUS1), enabling the use of cost-effective sucrose for UDP-glucose regeneration. This integration achieved a 99.9% yield at a 1 mM PPT. Molecular dynamics simulations revealed that the enlarged binding pocket entrance of I83A/F285 M contributed to the enhanced Rh1 yield. Our findings offer strategies for efficient biosynthesis of Rh1 and pave the way for economically feasible production.