Food-derived bioactive peptides have emerged as promising functional ingredients for hyperuricemia management. However, multienzyme hydrolysis strategies remain underexplored because of inefficient screening methods. Herein, a large language model (LLM)-guided strategy integrating deep learning-assisted enzyme selection with experimental validation was developed to generate antihyperuricemic peptides from chickpea proteins. The optimal enzyme combination (Flavourzyme-Pepsin-Pancreatin) produced a hydrolysate (MGI) with strong xanthine oxidase (XO) inhibitory activity (94.1% at 10 mg/mL), outperforming single-enzyme treatments. MGI retained 91.5% activity after simulated digestion and significantly reduced intracellular uric acid, oxidative stress, and inflammation in HK-2 cells. Molecular docking identified four tripeptides (LLF, GFM, FSF, and SWL) with favorable binding to XO through hydrogen bonding and hydrophobic interactions. This study provides a practical strategy for enhancing peptide bioactivity and supports the development of chickpea-derived peptides for hyperuricemia management.

Abstract CRISPR‐Cas systems have far‐reaching implications for genome editing and molecular diagnostics, and are emerging as important tools in biomedical research and clinical practice. These systems enable precise genetic manipulation and highly sensitive nucleic acid detection, capabilities that are invaluable for therapeutic development and pathogen surveillance. Nevertheless, native CRISPR‐Cas systems possess inherent limitations that constrain their broader utility. These limitations in genome editing include insufficient editing efficiency, a restricted range of targets due to protospacer adjacent motif constraints and non‐negligible off‐target effects. In molecular diagnostics, the remaining problems include detecting low‐abundance targets, achieving high specificity, and ensuring effective compatibility with isothermal amplification. To address these limitations, researchers have proposed many enhancement strategies that mainly center around four aspects: engineering of Cas effector protein, optimizing guide RNA designs, developing advanced reporter probes, and utilizing chemical additives. This review summarizes the current developments in these areas and highlights their significant contribution to increasing the fidelity of editing and diagnostic sensitivity. In addition, we discuss future prospects for enhanced CRISPR‐Cas systems, focusing on their accelerating clinical translation and expanding biomedical applications.

This study investigates the development of a novel starch-based functional ingredient derived from small-granule Agriophyllum squarrosum starch (ASS), whose molecular chains can form inclusion complexes with butyric acid (BA) via non-covalent interactions. The effects of ionic liquids and ultrasonication pretreatments on the structure and physicochemical properties of regenerated ASS were systematically examined, along with their combined impact on the functionality of ASS-BA complexes. The results indicated that appropriate pretreatment an ionic liquids-water system (8:2, w/w) combined with ultrasonication promoted the formation of hydrophobic cavities within ASS chains, facilitating subsequent complexation with BA. The resulting ASS-BA complexes exhibited a significantly increased resistant starch content, enhanced thermal stability, and improved BA retention during in vitro digestion. Molecular docking and quantum chemical analyses revealed the non-covalent interactions responsible for stabilizing the complexes, while Pearson correlation analysis further linked the structural properties of regenerated ASS to the functional performance of ASS-BA complexes. In a murine colitis model, the ASS-BA complex exhibited superior protective effects than ASS alone, underscoring its potential for mitigating intestinal inflammation. Overall, these findings provide new insights into the formation and functionality of small-granule starch-BA inclusion complexes and highlight their potential as a novel starch-based health food ingredient.

Ectoine, a cyclic amino acid derivative synthesized via the aspartate pathway, has emerged as a promising bioactive compound in the functional food industry owing to its exceptional protective effects on cellular structures and macromolecules. Despite its natural synthesis by halophilic microorganisms such as Halomonas, large-scale industrialization remains constrained by suboptimal yields and the technical complexities of hypersaline fermentation. To address these challenges, this structured narrative review outlines recent breakthroughs in the efficient biomanufacturing of ectoine. This review first outlines its biochemical properties and native biosynthetic mechanisms, followed by a comprehensive analysis of metabolic engineering strategies employed in both wild-type halophiles and engineered industrial chassis. At the molecular level, emphasis is placed on the precise regulation of the ectABC gene cluster, precursor pool expansion, and dynamic flux regulation to bypass feedback inhibition. At the process level, this article evaluates the impact of diverse fermentation optimization approaches—such as osmotic titration and sustainable feedstock utilization—and reviews advanced downstream separation and purification techniques. Finally, to overcome existing barriers to large-scale bio-manufacturing, this review prospects several critical future directions: the AI-driven intelligent design of microbial strains, dynamic metabolic regulation via biosensors, the development of novel microbial chassis and unconventional carbon sources, the digital and intelligent control of fermentation processes, and the advancement of continuous integrated downstream processing. Addressing these technological frontiers, alongside navigating food safety regulations, will facilitate the transition of ectoine toward efficient, green, and sustainable industrial production.

Lipase is a valuable biocatalyst in food processing due to its ability to catalyze reactions like transesterification. However, its industrial application is hindered by its instability in non-natural environments such as organic solvents, extreme pH, and its frequent inefficiency with non-natural substrates. To overcome these limitations, lipase modification is employed to enhance its properties for practical use. Current modification strategies are primarily divided into two categories: protein engineering (rational design, directed evolution, semi-rational design) and in vitro methods (chemical modification, enzyme immobilization). This paper reviews recent advances in these areas, discussing the advantages and disadvantages of each approach. As a single method often fails to improve all desired properties, the future trend points toward the strategic combination of protein engineering and in vitro modification. This integrated approach allows for targeted structural changes to meet specific industrial demands, facilitating the development of advanced, versatile lipase food biocatalysts.

(2 R ,3 R )-butanediol dehydrogenases (BDHs) are promising catalysts for the production of α -hydroxy ketones, which are highly valuable compounds in the synthesis of fine chemicals and pharmaceuticals. However, (2 R ,3 R )-BDHs display limited stereoselectivity, thus restricting wider applications. In this study, we engineered a (2 R ,3 R )-BDH from Bacillus subtilis ( Bs BDH) to enhance and invert its stereoselectivity toward 1,2-cyclohexanediol (1,2-CHD) for the production of chiral 2-hydroxycyclohexanone. The hot spots 115, 118, 293 of Bs BDH were initially identified using the protein language model ESM-1v. Subsequently, to obtain a stable scaffold to engineer stereoselectivity, we devised a strategy of position analysis and source search, achieving a true-positive rate of 88.2% in designing thermostable single variants. Furthermore, iterative saturation mutagenesis was applied to the hot spots of the thermostable variant 6M2, and obtained a trans -CHD preference variant LTF ( ee > 99%) and a cis -CHD preference variant 10M ( ee > 99%). Several high-activity variants were also obtained, including 6M2/F115C/L118F and 6M2/F115L/L118M, which demonstrated the activity improvements toward 25 substrates, with the highest enhancement reaching 5183.1-fold. Additionally, molecular dynamics (MD) simulations and the incorporation of non-canonical amino acids (ncAAs) were utilized to elucidate the mechanisms underlying the variants. The engineered Bs BDH variants exhibit promising potential for the biocatalytic production of α -hydroxyketones.

ABSTRACT Butyrate, a valuable short‐chain fatty acid, serves as a natural preservative, flavor enhancer, and gut health modulator in the food and feed industries. However, its sustainable microbial production is constrained by the low yield and selectivity inherent to conventional fermentation processes, primarily due to intracellular redox and energy limitations. To address this, we developed a light‐driven probiotic system by integrating in situ biomineralized cadmium sulfide (CdS) nanoparticles with metabolically engineered Clostridium tyrobutyricum . This hybrid interface enables direct injection of photogenerated electrons into cellular metabolism, boosting the intracellular NADH/NAD + ratio by 51% and effectively reprogramming the redox state. Consequently, the system achieved a 33.2% increase in butyrate yield (0.437 vs. 0.328 g/g) and elevated product selectivity from 89.5% to 95.7%. This work demonstrates a green and energy‐efficient strategy for enhancing the bioproduction of food‐grade butyrate, offering a novel platform for advancing light‐powered fermentation in food biotechnology.

Copper-based electrocatalysts are regarded as highly efficient electrocatalysts due to their low cost and environmental friendliness. However, their practical application in water splitting remains severely limited by intrinsic drawbacks such as inferior electron transfer kinetics and moderate catalytic activity. Herein, we synthesized copper oxysulfide (CuS x O1-x ) flake-like nanoflowers through a redox reaction and explored their application as efficient electrocatalysts for the oxygen evolution reaction. As expected, compared to the original CuS, the copper oxysulfides exhibited superior OER activity and stability, showing a remarkably low overpotential of 206 mV at a benchmark current density of 10 mA cm-2 for the OER. This study elucidates the reconstruction of copper oxysulfide electrocatalysts during the oxygen evolution reaction through in situ ATR-IR measurements. The facilitated formation of reaction intermediates and accelerated reconstruction kinetics during the structural evolution of copper oxysulfide synergistically contribute to its enhanced oxygen evolution reaction performance.

Radiation resistance in bacteria is a critical trait with implications for biotechnology, medicine and environmental science. Deinococcus species possess unique genomic features that enable their extraordinary survival in extreme environments. However, the diversity of radiation resistance mechanisms across bacteria remains poorly explored. Here, we analysed the genomic diversity and functional gene repertoire of Deinococcus and extended the analysis to a broader set of radiation-resistant bacteria. We found that highly resistant strains tend to have smaller genomes and harbour more genes positively associated with radiation resistance, highlighting their potential roles in sustaining cellular homeostasis under radiation-induced stress. By integrating machine learning with curated genomic datasets, we identified key resistance-associated gene signatures and developed a predictive tool to quantify bacterial radiation resistance. This study offers new insights into the genomic basis of radiation resistance and provides a practical framework for its assessment in environmental and microbial engineering applications.

Diabetic chronic wounds represent a significant clinical challenge, characterized by bacterial biofilms, impaired vascularization, and persistent oxidative stress. While photothermal therapy (PTT) has emerged as a potential antibacterial approach, its efficacy is often constrained by limited photothermal conversion efficiency and unintended aggravation of oxidative stress. Drawing inspiration from the coordinated energy conversion and photoprotective mechanisms of natural photosynthesis, we developed a biomimetic two-dimensional H₂/O₂-producing photothermal (2D-HPT) nanosheets. This nanosheets integrate liquid-phase exfoliated CaSi₂ nanosheets functionalized with palladium nanoparticles and polyvinylpyrrolidone to execute an artificial photosynthetic cycle. Under near-infrared (NIR) light irradiation, the nanosheets exhibit an enhanced photothermal effect and hydrolytic H2 generation, thereby achieving efficient biofilm disruption. Crucially, the continuous release of H₂ and in situ catalytic production of O₂ not only mimic natural photosynthetic outputs but also programmatically activate a cell-protective mechanism. This process, analogous to non-photochemical quenching in plants, results in scavenging of reactive oxygen species (ROS), stabilization of mitochondrial membrane potential, and suppression of apoptosis. These coordinated actions significantly downregulate pro-inflammatory cytokines (IL-6/TNF-α) and promote VEGF/CD31-mediated angiogenesis, collectively accelerating the healing of infected diabetic wounds. Our work thus provides an effective strategy for chronic wound repair. STATEMENT OF SIGNIFICANCE: This study presents a biomimetic strategy for treating diabetic chronic wounds, which are clinically challenging due to bacterial biofilms and persistent oxidative stress. Inspired by natural photosynthesis, the designed two-dimensional H₂/O₂-producing photothermal nanosystem not only disrupts biofilms via enhanced photothermal effects and hydrolytic H₂ generation under NIR irradiation, but also continuously releases H₂ and catalytically produces O₂ to activate a cell-protective mechanism. This coordinated action alleviates oxidative stress, reduces inflammation, and promotes VEGF/CD31-mediated angiogenesis, collectively accelerating wound healing. The work offers an effective innovative therapeutic approach for chronic wound management.

The enzymatic degradation of poly(ethylene terephthalate) (PET) offers a sustainable route for plastic recycling but is often hindered by limited enzyme adsorption on hydrophobic surfaces. Inspired by carbohydrate-binding modules (CBMs), which enhance enzyme performance on insoluble substrates, we developed a machine-learning-assisted pipeline to discover PET-binding modules from natural protein architectures. Integration of profile hidden Markov model-based homology searching with a supervised PET hydrolase machine-learning model (PETML) revealed that CBMs belonging to family 13, typically known for glycan recognition, were the most abundant CBMs associated with putative PET hydrolase homologs in the screened dataset. From 197 non-redundant candidates, high-throughput docking and molecular dynamics simulations prioritized tCBM13-1 (WP_357125140.1), which exhibited stable interfacial binding via cooperative aromatic and polar interactions. When fused to sfGFP, tCBM13-1 demonstrated superior adsorption (∼70%) and surface retention (∼90%) on PET powder at 37 °C and 45 °C, outperforming a benchmark CBM2. Co-displayed with FAST-PETase on the Escherichia coli (E. coli) surface using a dual-anchor system (OmpA and EhaA), tCBM13-1 enhanced PET film depolymerization by ∼ 43%, achieving a rate of 2793  μg/(d·cm2). The whole-cell catalyst retained > 64% of its initial activity after 10 cycles, indicating robust recyclability. This work integrates machine-learning-guided module mining with synthetic biology to engineer efficient, reusable biocatalysts for PET degradation, offering a scalable strategy for polymer biorecycling.

Polyurethanes (PU) are difficult to recycle because of their thermoset-like cross-linked structure and robust urethane linkages, yet they contain both polyols and aromatic diamine precursors that are attractive targets for circular valorization. Glycerol-based glycolysis of PU has been reported previously, mainly with an emphasis on polyol recovery, whereas the composition and potential of the diamine-rich lower phase have remained insufficiently characterized. Here, we combine product speciation, phase-behavior analysis, and density functional theory (DFT) calculations to elucidate why glycerol is particularly effective at driving TDI-based PU foams toward high-yield aromatic diamine recovery. Under tin catalysis at 200 degrees C and 1 atm N2, glycerol mediates efficient depolymerization of model and commercial TDI-based foams, affording near-quantitative toluenediamine (TDA) yields in the lower phase together with a polyether-polyol-rich upper phase. Comparative experiments with diethylene glycol and a series of C5 alcohols show that glycerol uniquely combines high overall PU conversion with markedly enhanced TDA selectivity. DFT calculations indicate that secondary-hydroxyl participation lowers the rate-determining barrier relative to typical diols. Using crude glycerol as both reagent and reaction medium, kilogram-scale glycolysis of waste car seat cushions affords a diamine-rich lower phase and a polyether-polyol-rich upper phase that closely match the speciation trends observed at bench scale, demonstrating a diamine-targeted, mechanistically guided alternative to existing polyol-centric glycolysis processes.

Covalent bond-forming peptide tagging systems have emerged as powerful and versatile tools across a broad spectrum of biological and biotechnological applications. This review systematically summarizes the origins, molecular mechanisms of intramolecular covalent bond formation, major classes, and design strategies of peptide tagging systems. Based on their underlying chemistry, current systems are primarily categorized into isopeptide-bond-based and ester-bond-based platforms, both of which have demonstrated prominent utility in protein cyclization as well as in vivo and in vitro multi-enzyme assembly. Beyond these applications, isopeptide-bond-forming systems have been widely adopted as robust purification tags, whereas ester-bond-based systems offer unique opportunities for pH-responsive modulation of enzyme activity. Collectively, peptide tagging systems based on either isopeptide or ester bond formation represent an expanding and highly efficient toolkit for biotechnology. Continued advances in their design and application are expected to further broaden their functional scope and provide innovative solutions for future developments in protein engineering and related fields.

Microbial contamination is a major challenge in industrial fermentation, while conventional sterilization is energy-intensive and costly. Here, an anti-contamination system was developed in Clostridium tyrobutyricum for non-sterile production of butyl butyrate (BB). C. tyrobutyricum L319 grow on unconventional nitrogen sources, namely formamide (50 mM) and urea (20 mM), as well as phosphite (50 mM) as the sole phosphorus source. Heterologous expression of phosphite dehydrogenase (ptxD) further enhanced phosphite utilization and raised the intracellular level of reducing equivalents. The engineered strain maintained up to 88% population dominance under non-sterile conditions. In 3 L bioreactors, the engineered strain produced 0.5 g/L BB under non-sterile batch fermentation. This proof-of-concept strategy has the potential to reduce energy input and production costs by eliminating the need for sterilization and antibiotics. These findings represent an initial step toward low-cost, antibiotic-free biomanufacturing using C. tyrobutyricum as cell factory.

The CRISPR/Cas system is a powerful tool for molecular diagnostics, but its reliance on linear amplification constrains sensitivity, particularly for in situ imaging. Here, we discovered that phosphorothioate (PS)-modified activators can modulate Cas enzyme conformation via hydrophobic anchoring. By adjusting the PS modification sites, we achieved precise control over Cas activation and trans-cleavage resistance. Guided by this mechanism, we proposed a tailored design strategy featuring a "scattered" PS modification to engineer a linear "Coordinator" probe. This design effectively decouples Cas enzyme activation from substrate trans-cleavage resistance, enabling the construction of a Scattered PS Nucleic Acid-driven Cas Autocatalytic system (SACA). SACA achieves exponential amplification without external enzymes, enhancing Cas12a and Cas13a sensitivity by 50 000-fold and 10 000-fold, respectively. Furthermore, the superior biostability and structural simplicity of these linear probes endow SACA with excellent compatibility, facilitating precise in situ imaging of HPV16 and HPV18 mRNA in cervical cancer cells. This study not only advances the understanding of Cas enzyme regulation by chemically modified nucleic acids but also establishes a new paradigm for precise and efficient molecular diagnostics.

Microbial conversion of renewable feedstocks into biofuels is gaining interest due to its sustainability. In this research article, we develop a carbon- and cost-efficient fermentation route in Clostridium tyrobutyricum to produce the biofuel butyl butyrate (BB) from abundant, low-cost agrifood waste. Using iterative, multimodule strain engineering-promoter engineering, multienzyme colocalization, nonoxidative glycolysis-driven carbon conservation, and cofactor engineering-we achieved high-selectivity BB production from rice straw hydrolysate and shrimp shell waste. The best strain delivered a 49.5-fold improvement, reaching 31.16 g/l BB with 98.4% selectivity and a productivity of 0.325 g/l/h in 5-l batch fermentations. Techno-economic and carbon-footprint analyses indicate that, compared with conventional sugar biorefineries, agrifood-waste biorefineries cut feedstock costs by 53.6% and life-cycle carbon emissions by 63.4% per ton of BB produced. These results show that engineered C. tyrobutyricum enables carbon-efficient upcycling of agrifood waste into high-yield BB and provides a blueprint for the biosynthesis of other biofuels.

Anaerobic biotechnology represents a promising pretreatment strategy for N, N-dimethylformamide (DMF)-containing wastewater. However, the low tolerance of microorganisms to high DMF toxicity is a bottleneck in its application. Therefore, we initiated anaerobic DMF degradation system seeded with conventional digested sludge via a gradient stepwise increase in influent DMF concentration, thereby investigating microbial community succession and ecological functional responses under sustained DMF loading conditions. The system maintained steady performance for 126 days, achieving a 87.3 % DMF removal efficiency at an organic loading rate (OLR) of 0.33 kg/(m3·d) with a 1 g/L influent DMF concentration. Initial DMF exposure reduced microbial richness and evenness, indicating resistance-driven community streamlining. Under prolonged exposure, taxonomic diversity recovered, whereas co-occurrence network complexity decreased and positive interactions intensified, suggesting stabilization through structural simplification and strengthened mutualism. Rhodobacter, Methyloversatilis, and Methanomethylovorans were identified as keystone taxon underpinning DMF degradation. Analysis of intermediate metabolites and methanogenic succession confirmed that DMF was mineralized predominantly via the hydrolytic-methanogenic pathway. Overall, this study reveals the resistance-resilience succession strategy of microorganisms under DMF stress, and provides a mechanistic basis for optimizing the start-up and operation of full-scale anaerobic DMF treatment systems.

Chitosan oligosaccharides (COS) are bioactive compounds with promising applications in functional foods, but their enzymatic production is hindered by high costs and operational instability. To address this, we developed a food-compliant magnetic bimodal mesoporous silica (Fe3O4@BMS) biocatalyst for sustainable COS synthesis via cellulase immobilization. The hierarchically structured carrier combined small (2-5 nm) and large (20-40 nm) mesopores, offering a large surface area of 851 m2/g. The immobilized cellulase demonstrated superior stability, maintaining 83.60 ± 3.32% residual activity at 80 °C, alongside exceptional pH adaptability (3-9). Magnetic Fe3O4 integration facilitated rapid biocatalyst recovery, retaining about 70% of catalytic efficiency after 10 repeated cycles. In a scaled-up stirred-tank reactor, the system achieved a high yield of 316.33 ± 9.39 mg/g reducing sugar from chitosan after 5 h under optimized conditions. This work offers an industrially scalable route for COS production, aligning with green chemistry and food safety standards.

Deoxynivalenol (DON) is one of the most prevalent mycotoxins in cereals, posing significant risks to humans and animals worldwide. While conventional strategies, including physical and chemical approaches, can mitigate DON contamination, microbial degradation through enzymatic reactions has become one of the most promising approaches due to its efficiency, specificity, and environmental friendliness. Currently, advancements in microbial and enzymatic degradation have been made in eliminating DON in the food and feed industries. Therefore, this review comprehensively summarizes the status of DON contamination and recent trends in detoxification methods, with particular focus on degradation pathways, microorganisms, including bacteria, fungi, and microbial consortia, as well as enzymes. Additionally, the challenges and future perspectives in microbial and enzymatic degradation of DON are also discussed. These insights suggest the potential of applying biocontrol agents to mitigate DON contamination in the food and feed industries.

Short-chain fatty acids (SCFAs), as key metabolites produced by the intestinal microbiota from the fermentation of dietary fiber and other substrates, play a crucial role in maintaining intestinal barrier function, regulating immune balance, and promoting systemic health. This review explores strategies for utilizing engineered food microorganisms (particularly probiotics) as novel “bio-factories” to achieve efficient and precise synthesis of SCFAs. This review outlines the core selection criteria for ideal engineered chassis strains, including their safety for application, robustness in the intestinal environment, and genetic tractability. It then details strategies for significantly enhancing the efficiency of SCFA synthesis in food microorganisms such as discovery and engineering of rate-limiting enzymes, intelligent enzyme engineering modifications, and artificial intelligence-driven metabolic network optimization. Furthermore, this review discusses how to integrate biosensors and clustered regularly interspaced short palindromic repeats-based dynamic regulation systems to achieve on-demand and precise modulation of SCFAs within the intestinal microecology. The application prospects of engineered food microorganisms are discussed in two major directions: First, in vitro food industrial biomanufacturing, i.e., utilizing engineered strains to efficiently produce SCFAs in fermentation systems as food ingredients or additives; this pathway exhibits relatively high technical maturity and a relatively clear regulatory framework. Second, in vivo live biotherapeutics, i.e., direct ingestion of engineered probiotics to in situ synthesize SCFAs in the gut. This pathway holds significant potential for personalized nutritional intervention and gut health management, but faces greater challenges in safety assessment and regulation.