Hydroxysalidroside is an important natural phenylethanoid glycoside with broad application prospects in the food and pharmaceutical fields. However, its low concentration in plants and complex extraction hinder its production. Despite being a promising way to synthesize hydroxysalidroside in Escherichia coli , glycosylation remains the limiting factor for its production. A de novo biosynthetic pathway for hydroxysalidroside was successfully constructed in E. coli via the screening of glycosyltransferase, overexpressing phosphoglucomutase ( pgm ) and UDP-glucose pyrophosphorylase ( galU ) to ensure a sufficient supply of UDP-glucose (UDPG). Additionally, a semi-rational design of UGT85A1 was conducted to expand the acceptor-binding pocket to eliminate steric hindrance interfering with the binding of hydroxytyrosol. The endogenous genes ushA and otsA were knocked out to further reduce the consumption of UDPG. Finally, a titer of 5837.2 mg/L was achieved in a 5 L fermenter by optimizing the feeding times of carbon sources. This laid the foundation for the subsequent biosynthesis of phenylethanoid glycosides.
He De-Jing,Zhang Han-Lin,Liu Wen-Kai,Yu Shi-Yi,Zhang Yan-Zu,Xu Lian,Lin Juan
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.
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