Tailoring Regioselectivity‐Controlled UDP‐Glycosyltransferase for Bidirectional Glycosylation of Tyrosol via Free Energy‐Driven Pocket Reshaping and Tunnel Engineering

Abstract UDP‐glycosyltransferases (UGTs) are pivotal biocatalysts for synthesizing pharmaceutically valuable active components; however, their application is frequently constrained by poor regioselectivity and suboptimal catalytic efficiency. In this study, a tailored, free energy‐driven, substrate‐binding pocket reshaping strategy is implemented to pinpoint the specific residues in UGT _BL 1 that control bidirectional regioselective glycosylation of tyrosol, enabling the synthesis of salidroside and icariside D2 without the need for large‐scale screening. Additionally, modifications in the tunnel lead to two strictly regioselective mutants with improved catalytic efficiency due to the faster release of the products. Remarkably, while wild‐type UGT _BL 1 exhibits poor regioselectivity toward the alcoholic and phenolic hydroxyl groups of tyrosol, generating an almost equal mixture of products (1:1 ratio), mutant M2 achieved 99.2% regioselectivity toward the alcoholic hydroxyl group of tyrosol, coupled with a 14.8‐fold enhancement in catalytic efficiency for salidroside production. Similarly, mutant M2‐1 displays 99.1% regioselectivity toward the phenolic hydroxyl group, along with a 3.6‐fold improvement in catalytic efficiency for icariside D2 synthesis. Molecular dynamics simulations reveal details about the mechanism for improved regioselectivity and catalytic efficiency. This work provides important insights for protein engineering of UDP‐glycosyltransferase with the spacious active pocket in constructing small but smart mutant libraries.