Substrate promiscuity enables UDP-glycosyltransferases (UGTs) to glycosylate diverse plant natural products. These broadly acting UGTs could serve as universal glycosylation modules in synthetic biology; however, promiscuity often increases by-product formation, elevating downstream processing demands and overall manufacturing costs. Achieving scalable glycoside production thus requires precise control of regiospecificity without sacrificing high substrate loading. Here, UGT73C1, a highly promiscuous Arabidopsis thaliana UGT, was found to harbor a through-type substrate-binding pocket with an unusually large bottleneck radius and pocket volume. This architecture positions the sugar donor and acceptor at opposite ends of the pocket, providing a structural basis for broad substrate scope and high loading capacity. Guided by computational pocket reshaping, we engineered UGT73C1 variants with defined hydroxyl regiospecificity, enabling the synthesis of functionally improved glycosides at over gram-per-liter titers. Together, these results provide an efficient framework for tuning UGT promiscuity and regiospecificity and yield a broadly applicable glycosylation module for producing high-value glycosides.
Bi et al. (Mon,) studied this question.