Dynamically Coupled Network-Guided Engineering of Amine Dehydrogenase With Cofactor Recycling for Efficient Biosynthesis of (R)-3-Aminobutanol.

Structure-guided multi-domain engineering of glucose dehydrogenase for enhanced catalytic efficiency and cofactor regeneration.

Glucose dehydrogenase (GDH) is a key enzyme for NAD(P)H cofactor regeneration in industrial biocatalysis. However, conventional engineering approaches are frequently constrained by limited catalytic efficiency and excessive enzyme loading, which collectively compromise process economics and hinder large-scale deployment. In this study, we performed structure-guided rational design by targeting three functionally discrete domains of GDH, including the substrate-binding region, the cofactor-binding pocket, and the interdomain communication interface. An engineered variant GDH-M6 was constructed, which manifests a 35-fold enhancement in catalytic efficiency relative to the wild-type enzyme. In the biocatalytic synthesis of the pivotal chiral intermediate for R-lipoic acid, GDH-M6 reduced enzyme loading by more than 90% and allowed a doubling of the substrate concentration. As a result, overall reaction productivity was substantially increased and the GDH-M6 outperformed wild-type GDH as well as all previously reported mutants under comparable conditions. Notably, the domain-engineering paradigm established herein provides a broadly applicable toolkit for augmenting the catalytic performance of dehydrogenases, and offers a structural blueprint for resolving analogous kinetic bottlenecks that commonly arise in NAD(P)⁺-dependent enzymes utilized for industrial cofactor regeneration.

Structure-Guided Engineering of Glycosyltransferase UGT73-327-2 Coupled with UDP-Glucose Regeneration Enables Highly Efficient Biosynthesis of Mogroside VI.

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.

Construction of a Self-Assembled Multi-Enzyme Cascade for Efficient D-Allulose Biosynthesis.

d-Allulose is a valuable low-calorie rare sugar with diverse physiological benefits. Although phosphorylation-dephosphorylation-based multi-enzyme cascades enable efficient d-allulose biosynthesis, the free-enzyme format limits substrate channeling and promotes intermediate diffusion, resulting in byproduct accumulation and reduced cascade efficiency. In this study, peptide-mediated assembly strategies were employed to construct a dual-enzyme complex using d-allulose 6-phosphate epimerase (A6PE) and d-allulose 6-phosphate phosphatase (A6PP) as model enzymes, to mitigate reversible epimerization. Among them, the ReverseTag/ReverseCatcher system was selected due to its positive impact on enzyme activity, as evidenced by the 2.1‑fold and 27.5% increases in activity observed for RCA6PE and RTA6PP, respectively. Successful complex assembly was confirmed by dynamic light scattering and transmission electron microscopy. A five-enzyme complex (RFE) was further constructed by integrating α-glucan phosphorylase, phosphoglucomutase, phosphoglucose isomerase, A6PE, and A6PP to spatially organize an artificial in vitro d-allulose biosynthetic pathway. With 10 g/L maltodextrin as the substrate, the RFE system achieved a d-allulose yield of 63.1%, representing a 37.2% increase over the free-enzyme system. These findings demonstrate that ReverseTag/ReverseCatcher-mediated covalent assembly improves multi-enzyme cascade efficiency and provides a modular platform for engineering artificial in vitro biosynthetic systems.

Engineering O-methyltransferase and SAM-ATP cofactor cycle for high-efficiency melatonin biosynthesis in Escherichia coli.

Multilevel metabolic and enzyme engineering enables efficient biosynthesis of cyanidin-3-O-glucoside from dihydroquercetin in Escherichia coli.

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.

Engineering the Stability of Sucrose Synthase to Enable Efficient Coupled Production of UDP-Glucuronic Acid.

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.