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

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.

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

(R)-3-Aminobutanol is a valuable chiral amino alcohol widely used in the pharmaceutical industry. The amine dehydrogenase (AmDH)-catalyzed synthesis with inorganic ammonia as the amine donor represents one of the most promising routes for its production. However, this approach is limited by the low catalytic efficiency of AmDHs toward the unnatural substrate 4-hydroxy-2-butanone, as well as the high cost associated with coenzyme requirements. To address these issues, a structure-guided engineering strategy integrating loop remodeling with dynamically coupled network analysis was employed to modify an AmDH derived from leucine dehydrogenase (GKGB-AmDH). A pentamutant was obtained with a 7.8-fold increase in catalytic efficiency (kcat/Km) and a 6.8-fold increase in specific activity. Molecular dynamics simulations were performed to elucidate the molecular mechanism underlying the improved catalytic performance. A dual-enzyme co-expression system for GKGB-AmDH-M5 and formate dehydrogenase (FDH) was established for in situ NADH regeneration. As a result, the catalytic efficiency toward 4-hydroxy-2-butanone was enhanced, and the conversion reached 83.8% at a substrate concentration of 300 mM, with an enantiomeric excess (e.e.) of > 99.99%. These results demonstrate the feasibility of this engineering strategy and provide a theoretical basis for the efficient and green biomanufacturing of chiral amino alcohols.

Computationally Guided Engineering of Multi-Enzyme Cascades Enables Efficient Trehalose Biosynthesis.

Trehalose is a nonreducing disaccharide widely used for its biomolecule-protective properties. However, multienzyme cascade production remains limited by low enzyme expression and suboptimal catalytic performance. To address this, thermostable maltooligosyltrehalose synthase (TreY) and trehalohydrolase (TreZ) from Arthrobacter ramosus were individually expressed intracellularly in Bacillus subtilis, and the crude lysates were combined for trehalose biosynthesis, achieving 281.4 g/L trehalose and a yield of 0.7 g trehalose/g maltodextrin. Integrated computational screening identified MalQ-3 from Cyanobacterium stanieri as a suitable 4-α-glucanotransferase for soluble expression. Subsequent semirational engineering generated MalQ-3-M2 (S54P/V472F), with enhanced activity and stability associated with improved substrate-pocket dynamics, thereby facilitating glucan-chain rearrangement and short-chain reutilization. MalQ-3-M2 was separately expressed in B. subtilis and incorporated into the crude-lysate cascade, increasing the trehalose titer to 338 g/L and the yield to 0.85 g trehalose/g maltodextrin. Overall, this work establishes a scalable B. subtilis platform for efficient trehalose production.

De novo L-(+)-tartaric acid biosynthesis in multi-modular engineered yeasts

L-(+)-tartaric acid (L-TA) is a high-value chiral organic acid essential for food and pharmaceuticals. Despite its industrial importance, sustainable green production is constrained by the lack of a fully defined biosynthetic pathway. Here, we report the de novo biosynthesis of L-TA in Saccharomyces cerevisiae through reaction-guided enzyme mining, experimental validation, and Enzyme Commission-specific Catalytic Hybrid Optimizer (ECHO)-assisted enzyme prioritization. We first elucidate the elusive two-step conversion from precursor 5-keto-D-gluconic acid (5-KGA) to L-TA, catalyzed by transketolase (TK) and succinate semialdehyde dehydrogenase (SSDH). To optimize this critical step, we develop the ECHO. This multimodal framework integrates sequence, substrate, and pocket-aware structural information to identify high-performance TK-SSDH pairs. By integrating this pathway with de novo precursor synthesis, cofactor engineering, and semi-rational protein engineering, a final L-TA titer of 6.59 mg L−1 was achieved in a 5-L bioreactor. By connecting computational mining and metabolic assembly through a multi-module engineering strategy, our study establishes a green platform for L-TA production and demonstrates an effective workflow for synthetic pathway design. L-(+)-tartaric acid (L-TA) is a high-value chiral organic acid for food and pharmaceuticals. Here the authors produce L-TA in S. cerevisiae through reaction-guided enzyme mining and Enzyme Commission-specific Catalytic Hybrid Optimizer (ECHO)-assisted enzyme prioritization.

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