Jul 2026· Journal of Agricultural and Food Chemistry· Vol 74 30, pp.
23649-23661
· 0 citations· 38 references
Medicine
Abstract
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.
Trehalose is a functional disaccharide widely used in the food, pharmaceutical, and cosmetic industries. It is industrially produced via a dual-enzyme process involving maltoligosaccharide trehalose synthase (MTSase) and maltoligosaccharide trehalose hydrolase (MTHase), with Escherichia coli (E. coli) serving as the expression host. Bacillus subtilis (B. subtilis) is an ideal host for industrial trehalose production due to its generally recognized as safe (GRAS) status and low phage susceptibility. However, engineered B. subtilis strains often exhibit slow growth, low heterologous protein expression, and high fermentation costs, thereby limiting their industrial application. To address these challenges, this study employed a synergistic strategy that combined chassis modification, expression element optimization, and knockout of substrate-competition pathways. First, a tryptophan-independent strain was constructed by reverting the trpC2 mutation to shorten the growth cycle. Next, knockout of flgD, yueB, and integration of E. coli-derived glutamate dehydrogenase (gdhA) significantly enhanced biomass accumulation. Expression of MTSase and MTHase was markedly improved through tandem strong promoters (PHpaII-P36) and ribosome-binding site (RBS) optimization (RBS1), achieving a 10.87-fold and 4.22-fold increase in enzyme activity, respectively. Finally, disruption of the amyE gene reduced non-specific substrate degradation. Using maltodextrin as substrate, the final trehalose conversion rate reached 76%. This study constructed B. subtilis chassis cells that highly express MTHase and MTSase respectively, laying a foundation for subsequent industrial trehalose production.
Jianghua Chen, Yu-Jue Wang, Qiang Wang et al.· Fermentation· 0 citations
Deoxynivalenol (DON), a trichothecene mycotoxin commonly found in cereal grains and their derived products, poses significant risks to human and animal health. In previous work, a fusion enzyme composed of the dehydrogenase DADH and the aldo-keto reductase AKR13B3 was engineered to convert DON into the non-toxic 3-epi-DON in a single step. However, the poor thermal stability of this fusion enzyme limited its industrial application. In this study, EVcouplings and the GRAPE-WEB platform were utilized to identify key amino acid residues governing the thermal stability of the fusion enzyme AKR13B3–DADH. Through single-point mutation screening and the combination of beneficial mutation sites, a triple mutant M361L/T508Y/Y603F (M1) was obtained. The half-life of M1 at 50 °C reached about 500 min, representing a 16.4-fold increase compared with the wild type, while its catalytic activity increased by 2.7-fold. The apparent melting temperature increased by approximately 4 °C. Molecular dynamics simulations verified that the improved thermostability results from reduced conformational flexibility in key regions, enhanced structural packing, and a strengthened hydrogen bond network. These results demonstrate the successful development of a thermostable DON-detoxifying fusion enzyme and provide a practical basis for its industrial application.
Yi-Ting Pan, Hao Zhu, Qing-Wei Jiang et al.· International Journal of Mol...· 0 citations
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.
Zheming Wu, Yu Lu, Jin-Chao Zhang et al.· Biotechnology and Bioenginee...· 0 citations
Epilactose is a promising functional disaccharide, but its biomanufacturing is limited by insufficient enzyme activity, poor thermostability, and costly catalyst preparation. We first used the REME platform to computationally evaluate candidate enzymes. Among them, cellobiose 2-epimerase from Caldicellulosiruptor saccharolyticus (CsCE) showed the highest epilactose synthesis activity. We therefore developed an integrated strategy combining computational design, SpyTag/SpyCatcher-mediated cyclization, and ethanol-permeabilized whole-cell catalysis. By combining enzyme ligand binding energy analysis, protein stability prediction, and catalytic constant prediction, the V52N variant was obtained. Its epilactose synthesis activity was 3.45 times that of the wild type, while lactulose formation was reduced to 14.8% of the wild-type level. Cyclized CCT increased the optimum temperature to 80 °C and extended the half-life at 85 °C by 5.52-fold. The optimized whole-cell process produced 65.81 g/L epilactose from 200 g/L lactose within 20 min, corresponding to 32.90% conversion. This strategy provides a practical route for efficient epilactose biomanufacturing.
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
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.
