Function-Guided Tunnel Remodeling Enables Regioselective Synthesis of Hydroxysalidroside via Engineered Microbial UDP-Glycosyltransferase with Record Productivity

Abstract

Regioselective glycosylation of polyhydroxylated natural products remains a major bottleneck for deploying glycosyltransferases in sustainable biomanufacturing. Herein, we developed a function-guided substrate tunnel remodeling strategy to redesign a microbial UDP-glycosyltransferase from Bacillus paralicheniformis (BparUGT), enabling the highly regioselective biosynthesis of hydroxysalidroside (2a), a high-value phenylethanoid glucoside with antioxidant and antiaging bioactivities. The poor intrinsic regioselectivity and low catalytic activity of wild-type BparUGT toward hydroxytyrosol (1a) can be ascribed to three coexisting parallel substrate-access tunnels (T1, T2, and T3), which enable unregulated substrate entry and subsequent off-target glycosylation. To address this issue, we adopted an iterative engineering workflow to sterically occlude nonproductive T1/T2 tunnels while structurally reshaping the functional T3 tunnel. The best mutant BparUGT-Mu11 afforded >99% regioselectivity and a 41-fold improvement in catalytic efficiency. Molecular dynamics simulations revealed that tunnel remodeling restricts 1a binding orientation, exclusively directing the aliphatic hydroxyl group (2′-OH) for targeted glycosylation. By coexpressing BparUGT-Mu11 and sucrose synthase GmSUS from Glycine max, we constructed a robust whole-cell biocatalyst BL21(HA05). This biocatalytic system achieved gram-scale 2a production with a titer of 48.5 mM and an exceptional space−time yield of 1.3 g/L/h, setting a new benchmark among all reported biosynthetic routes. Moreover, BL21(HA05) exhibited broad substrate compatibility toward diverse phenylethanoid scaffolds, enabling efficient synthesis of salidroside and phenylethyl glucosides. Collectively, this work not only provides a high-efficiency biocatalytic platform for green industrial production of phenylethanoid glucosides but also establishes a generalizable tunnel engineering framework for the rational design and regiocontrol of multitunnel enzymes.