Structure-Guided Engineering of Glycosyltransferase UGT73-327-2 Coupled with UDP-Glucose Regeneration Enables Highly Efficient Biosynthesis of Mogroside VI.
Aug 2026· Journal of Agricultural and Food Chemistry· Vol 74 32, pp.
25369-25379
· 0 citations· 50 references
Medicine
Abstract
Mogroside VI (Mog VI) is a rare triterpene glycoside from Siraitia grosvenorii with promising bioactivities. However, its biosynthesis is limited by a single rate-limiting glycosylation step converting mogroside V, catalyzed by the inherently low-activity plant glycosyltransferase UGT73-327-2. In this study, we applied a structure-guided engineering strategy to overcome this catalytic bottleneck. By combining substrate-channel expansion with catalytic pocket remodeling, the double mutant W192F/K206E was generated, showing a 22.2-fold increase in catalytic activity. Molecular dynamics simulations and kinetic analyses indicated that the enhanced performance results from an enlarged substrate-access channel, improved substrate-binding stability, and a more favorable active-site geometry that reduces key catalytic distances. Furthermore, coupling the engineered UGT with Arabidopsis thaliana sucrose synthase enabled an in situ UDP-glucose regeneration system, achieving a Mog VI titer of 5.6 g·L-1 with a 76.8% molar conversion. This work establishes an efficient biocatalytic route for Mog VI production and highlights the potential of structure-based glycosyltransferase engineering for the synthesis of rare natural glycosides.
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.
Mu-Yang Li, Yi-Wei Meng, Ji-Shan Li et al.· ACS Catalysis· 0 citations
The biocatalytic synthesis of UDP-glucuronic acid (UDPGA), a key precursor for glucuronides and high-value carbohydrates, is limited by an unstable UDP-glucose (UDPG) supply in coupled systems. In this study, through an extensive screening of 10 sucrose synthases derived from both eukaryotic and prokaryotic microorganisms, we identified a broad-spectrum, highly efficient enzymatic scaffold suitable for the synthesis of UDP-sugars. A rational design integrating PROSS and ThermoMPNN algorithms with molecular dynamics (MD) analysis yielded the V353L mutant, which retained 97% of wild-type activity and extended the half-life at 55°C from 0.4 to 5.5 h. Tetrameric MD simulations indicated that V353L strengthens hydrophobic stacking in the GT-B linker region, reducing conformational fluctuations and driving the enzyme toward a stable low-energy state. In a dual-enzyme cascade synthesizing UDPG and UDPGA, the mutant sustained UDP consumption and increased the accumulated UDPGA concentration from 0.60 to 2.09 mM after 8 h. Engineering sucrose synthase thermostability thus improves UDPG supply in cascade biocatalysis and offers a viable enzymatic strategy for UDPGA-centered synthesis of high-value carbohydrate derivatives.
Qian Zhao, Ya-Min Chen, Zhi-Min Li et al.· Biotechnology Journal· 0 citations
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.
De He, Han-Lin Zhang, Wen-Kai Liu et al.· ACS Sustainable Chemistry &a...· 0 citations
Cyanidin-3-O-glucoside (C3G) is a water-soluble, value-added natural flavonoid with extensive applications in the nutraceutical and cosmetic industries. However, its efficient bioproduction is hampered by intermediate instability, metabolic imbalance and enzyme kinetic constraints. Here, we successfully constructed an efficient biosynthesis pathway from the dihydroquercetin (DHQ) to C3G in Escherichia coli through a multilevel engineering strategy. Initially, the integration of glutathione S-transferase (GST) redirected the metabolic flux towards target cyanidin formation. To minimize the dissipation of labile intermediates, pathway enzymes were spatially organized within a protein cage to enhance cascade efficiency. Furthermore, genomic integration of Glycine max sucrose synthase established an in-situ UDP-glucose regeneration module to ensure a continuous precursor supply for the final glycosylation step. After these pathway-level optimizations, the key enzyme dihydroflavonol 4-reductase (FaDFR) emerged as a new rate-limiting bottleneck due to substrate inhibition under increased DHQ loading. Structure-guided and evolution-informed engineering generated FaDFR variants with improved high-substrate tolerance, as supported by in vitro activity profiling and molecular dynamics simulations. Through combined pathway and enzyme engineering, the G130C-containing strain achieved a C3G titer of 1.34 g/L, representing a 23-fold improvement over the GST-assisted baseline strain. Our platform enables efficient, value-added C3G production and provides a promising framework for constructing downstream pathways toward structurally diverse anthocyanin derivatives.
Benzylisoquinoline alkaloids (BIAs) hold broad pharmaceutical potential, yet poor water solubility and low bioavailability limit their application. Glycosylation improves these properties, but glycosyltransferases (GTs) efficiently catalyzing diverse BIAs remain scarce. Here, we identify UGT74AN1 from Asclepias curassavica, capable of glycosylating various BIAs. Structure-guided semirational engineering yielded the double mutant UGT74AN1M2, exhibiting a 341-fold increase in catalytic efficiency. Molecular dynamics simulations revealed that these mutations widen the substrate channel and strengthen binding. To overcome UDP-glucose dependency, we designed the fusion enzyme AtSuSy-L12-UGT74AN1M2 via the iMARS platform, enabling in situ UDP-glucose generation coupled with highly efficient BIA glycosylation. Consequently, the synthesized dihydrojatrorrhizine-3-O-β-d-glucoside (1b) demonstrated superior antitumor activity compared to its aglycone through stronger proliferation inhibition and apoptosis induction. This work provides an efficient enzymatic toolkit for green BIA glycoside synthesis and identifies promising candidates for drug development.
Jun Song, Yu Qin, Lu Jin et al.· Journal of Agricultural and...· 0 citations
Engineered glycosynthases (GSs) are powerful biocatalysts for custom glycan synthesis, yet their optimization via directed evolution is severely constrained by bottlenecks in high-throughput screening for activated azido-sugar donors. Here, we demonstrate that chemical rescue (CR)—the azide-mediated restoration of hydrolytic activity in nucleophile-deficient mutants—serves as a predictive, high-throughput proxy for glycosynthase activity. Applying an azide-responsive Escherichia coli biosensor screen to a site-saturation mutagenesis library of Thermotoga maritima α-L-fucosidase (TmAfc), we established a strong rank-order correlation between CR and GS activities in both crude lysates (ρ = 0.73) and purified enzymes (ρ = 0.95). Transition path sampling and QM/MM umbrella sampling revealed that both pathways proceed through a shared oxocarbenium-ion-like transition state (ΔG‡ ≈ 8.7 kcal/mol), providing a structural and thermodynamic rationale for using CR to select for transition-state-stabilizing mutations. Biochemical characterization of top-performing variants yielded an engineered fucosynthase (TmAfc_D224G_N70D_T392S) exhibiting a nearly 100-fold enhancement in Vmax alongside altered regioselectivity. This two-tiered screening framework leverages cost-effective chemical rescue assays to streamline glycosynthase engineering for tailored glycans synthesis.
Mohit Kumar, C. Bandi, Sri Vidya Vyjayanthi Tallavajhula et al.· bioRxiv· 0 citations
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