Structure-Guided Stepwise Engineering of a Microbial Glycosyltransferase for Highly Regioselective and Efficient Biosynthesis of Salidroside

Abstract Uridine diphosphate glycosyltransferases (UGTs) are among the key rate-limiting enzymes in the biosynthesis of salidroside. Plant-derived UGTs often exhibit poor solubility and low catalytic activity, whereas microbial UGTs typically show insufficient regioselectivity for salidroside production. In this study, we performed stepwise engineering of the UGT from Paenibacillus durus (PdUGT) to generate a highly regioselective biocatalyst for salidroside production. Through stepwise reshaping of the active site and the access tunnel, we progressively enhanced the regioselectivity for tyrosol glycosylation from 81.2 to 99.9%, reaching a level comparable to that of natural plant enzymes. Mutations prioritized by SaProt and ΔΔG calculations further increased the melting temperature by 11.2 °C. The final variant, M4, exhibited a 20-fold increase in catalytic efficiency, with a specific activity of 58.5 U·mg−1. In a UDP-glucose recycling cascade, M4 enabled the production of 193.1 mM salidroside with 99.0% conversion and a space-time yield of 5.7 g·L−1·h−1. Molecular dynamics simulations and substrate docking suggested that the improved performance of M4 is associated with tighter binding of tyrosol, suppression of unproductive tyrosol reorientation, and enhanced protein compactness under thermal stress. Substrate profiling confirmed that PdUGT exhibits broad substrate promiscuity, while M4 displays high specificity toward tyrosol and related aromatic alcohols. This work establishes PdUGT as a valuable microbial UGT scaffold and provides experimentally supported design principles for engineering glycosyltransferases. Moreover, this study lays a solid foundation for the industrial bioproduction of salidroside and other glycosides.