Abstract Yarrowia lipolytica is a promising industrial host, yet its metabolic engineering potential remains limited by insufficient genetic tools. Here, we engineered a synthetic Transcription Activation Toolkit (TAT) based on LacI–VPRH, a chimeric protein fusing the prokaryotic DNA-binding domain LacI with the eukaryotic activation domain VPRH. Systematic optimization of LacO copy numbers and core promoter composition achieved up to 205-fold gene activation. The TAT platform was further expanded to construct bidirectional expression systems and enable multiplexed gene control. Applied to resveratrol biosynthesis via a “push-pull” strategy, CRISPR/Cas9-mediated integration of TAT-controlled synthetic promoters upregulated the shikimate pathway genes aroM10 and aroC alongside the rate-limiting enzyme ST1, achieving a shake-flask titer of 2.715 g/L─the highest reported to date. Additionally, an IPTG-inducible “turn-on” system (TAT-2.0) incorporating the antiLacI9 mutant was developed for small-molecule-responsive transcriptional control. Collectively, the modular TAT system provides a versatile strategy for precise metabolic pathway optimization in Y. lipolytica.
Abstract Ethanol-induced gastric mucosal injury (AGMI) is intrinsically linked to oxidative stress, inflammatory responses, and compromised mucosal defense. This study aimed to isolate bioactive peptides from yeast protein (YP) and elucidate these in vitro protective mechanisms against AGMI. Activity-guided multi-technique separation, nanoLC-MS/MS, and bioinformatic screening were used to identify two novel peptides, ISPALLDKL and TAADLRYF. In ethanol-injured GES-1 cells, these peptides (240 and 320 μg/mL) exhibited potent cytoprotective activity comparable to omeprazole. Peptide pre-treatment attenuated reactive oxygen species (from 31.79 to 9.52 and 10.02), restored mitochondrial membrane potential (ΔΨm increased from 0.5 to 2.78 and 2.11), and reduced apoptosis (from 28.55 to 10.52% and 12.58%). Furthermore, these peptides associated with the suppression of pro-inflammatory cytokines, decreasing TNF-α (24.9 and 21.9%), IL-1β (30.1 and 26.3%), and IL-6 (41.9 and 37.6%), while markedly enhancing the anti-inflammatory cytokine IL-10 (110.8 and 91.0%), respectively; these effects were accompanied by inhibition of NF-κB and JAK2–STAT3 phosphorylation. Collectively, ISPALLDKL and TAADLRYF protect against ethanol-induced gastric epithelial cell injury by simultaneously modulating oxidative stress, apoptosis, and inflammation, positioning them as promising natural candidates for gastric mucosal protection.
Abstract Fungal diseases remain a major constraint on global crop production, driving the search for fungicides with broader efficacy and improved selectivity. Confronting the dual challenges of structural rigidity and declining antifungal performance in conventional succinate dehydrogenase inhibitors (SDHIs), this study developed novel syringic acid derivatives. Notably, W11 exhibited remarkable broad-spectrum potency against eight phytopathogenic fungi and two oomycete species, with EC50 values of 0.679, 0.424, and 0.384 μg/mL against Gibberella zeae, Alternaria solani, and Colletotrichum fructicola, respectively. This activity significantly surpassed that of the commercial fungicide boscalid (EC50 >25.0, 0.826, and >25.0 μg/mL). Interestingly, W11 exhibited excellent succinate dehydrogenase inhibitory activity, causing a marked increase in intracellular reactive oxygen species (ROS), severe mitochondrial damage, and ultimately cell death. Overall, this work presents a structure-driven strategy that expands the antifungal spectrum of SDHIs and offers a promising scaffold for developing eco-friendly agrochemicals to address both limited antifungal activity and emerging oomycete challenges in sustainable agriculture.
Abstract The gem-difluoroalkylthio (−SCF2−) moiety has achieved great success in medicinal chemistry, but its application in agrochemicals remains underexplored because of synthetic challenges. Herein, we report a series of 1,2,4-triazole derivatives bearing the gem-difluoromethylthio group and evaluate their antifungal activities against ten phytopathogenic strains. Compound 7n exhibits potent broad-spectrum activity, with EC50 values of 0.007, 0.121, 0.122, and 0.068 μg/mL against Botrytis cinerea, Phytophthora capsici, Curvularia lunata, and Alternaria solani, respectively, outperforming tebuconazole. In vivo, 7n shows protective and curative efficacy comparable to tebuconazole on apple fruits, tomato fruits, and Suzhou Qing leaves. Mechanistic studies, including scanning electron microscopy (SEM), transcriptomics, qRT-PCR, and molecular docking, indicate that 7n disrupts mycelial morphology, interferes with membrane-associated processes, and inhibits sterol biosynthesis by targeting CYP51. These findings highlight the −SCF2– group as a promising scaffold for novel agricultural fungicides.
Shell ginger (Alpinia zerumbet) is a perennial species widely utilized as both an edible spice and a medicinal plant. Phytochemical investigation of its rhizomes yielded 33 terpenoids, of which 12 are previously undescribed compounds, comprising both diterpenes and sesquiterpenes. Notably, compound 1 represents a rare, highly oxygenated isospongiane-type diterpenoid with a novel skeleton. The anti-inflammatory potential of all isolates was assessed using lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages and BV2 microglial cells. Subsequent mechanism-oriented assays demonstrated that compounds 2 and 4 attenuated neuroinflammation in BV2 cells through an effect attributable to inhibition of nuclear factor kappa-B (NF-κB) nuclear translocation, reduction of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), and signal transducer and activator of transcription 3 (STAT3) protein expression, and downregulation of the mRNA levels of iNOS, interleukin-1β (IL-1β), COX-2, and tumor necrosis factor-α (TNF-α).
Bemisia tabaci is a destructive agricultural pest with a remarkable capacity to exploit diverse host plants. Horizontally transferred genes (HTGs) have recently been implicated in this adaptive success, yet the functions of most HTGs in the whitefly remain unclear. Feeding behavior is crucial for nutrient acquisition and reproduction in piercing-sucking insects, but whether HTGs contribute to host adaptation by regulating feeding remains largely unknown. Here, we identified BtHCYP, a plant-derived HGT gene in B. tabaci encoding a cysteine protease. Biochemical and in vivo assays confirmed that BtHCYP retained cysteine protease activity. RNA interference-mediated silencing of BtHCYP significantly reduced whitefly fecundity. Electrical penetration graph on cotton plants analyses further revealed that BtHCYP knockdown impaired phloem feeding. Collectively, these findings demonstrate that a plant-derived HTG can enhance whitefly fecundity and efficient phloem feeding, as BtHCYP silencing reduced oviposition by 27.6%, highlighting a potential molecular target for whitefly management.
Abstract A broad-spectrum fluorescent LFIA was developed for the rapid group-selective screening of walnut and/or hazelnut protein residues in foods, with positive results indicating the presence of walnut and/or hazelnut protein residues but not distinguishing between the two sources. Under optimized conditions, the assay was completed within 10 min. The visual limits of detection for walnut and hazelnut proteins were 25 and 250 ng/mL, respectively, while the instrumental limits of detection were 12 and 156 ng/mL. The assay showed no obvious cross-reactivity with 20 nontarget food ingredients. In biscuit, sausage, and soymilk, the minimum detectable levels were 0.5 mg/kg for walnut protein and 5 mg/kg for hazelnut protein. These results indicate that the proposed assay is a rapid, sensitive, and practical tool for group-selective screening of walnut and/or hazelnut protein residues in complex food matrices.
Abstract Agricultural applications of nanotechnologies inevitably introduce nanoparticles and pesticides into agroecosystems as complex mixtures, with unknown implications along terrestrial food chains. We investigated the joint effects of ZnO-NPs and imidacloprid (IMI) along a lettuce–snail food chain over 21days of uptake and 10 days of depuration, focusing on IMI biotransformation and the associated changes of gut microbiota. Coexposure to ZnO-NPs increased the uptake rate constants and the kinetic trophic transfer factor of IMI from lettuce to snail soft tissues by 1.98–2.14-fold and 1.78–1.92-fold, respectively. Higher biotransformation rate constants and 5-hydroxy-imidacloprid (5-OH-IMI) concentrations in mixture treatments suggested enhanced IMI metabolism under coexposure. IMI predominantly accumulated in soft tissues, whereas 5-OH-IMI was primarily excreted via feces, and Zn was retained in viscera. Coexposure was accompanied by larger shifts in gut bacteria and predicted xenobiotic-metabolism-related functional changes, which correlated with IMI and 5-OH-IMI toxicokinetic patterns. These findings underscore the relevance of nanoparticle–pesticide–microbiota–host interactions to agroecosystem risk assessment.
Abstract The efficacy of synbiotic (“probiotic + prebiotic”) strategies against soil-borne diseases is often limited by the low specificity of conventional prebiotics, which may also stimulate pathogens. Here, we developed a precision synbiotic system using a sugar alcohol mixture (SAs) as a selective substrate for the probiotic yeast Saitozyma podzolica (SP), while being poorly utilized by the root rot pathogen Ilyonectria vredehoekensis (I1). In Panax notoginseng, the synbiotic system simultaneously enhanced root biomass and reduced root rot incidence. Multiomics analyses revealed dual mechanisms underlying these effects. The synbiotics reshaped the fungal community by increasing network stability and suppressing pathogen abundance while also activating host growth- and defense-related pathways, including the tryptophan-dependent auxin pathway (TAA1, ALDH) and the phenylpropanoid pathway (CYP73A, CAD). These findings provide a mechanistic framework for precision synbiotics that integrate microbiome modulation with host metabolic regulation for sustainable disease management and crop growth promotion.
Abstract Residual hypochlorite/hypochlorous acid (ClO–/HClO) is widely used in disinfection, yet simple assays integrating visual readout with quantitative fluorescence analysis remain limited. Herein, we report a single-atom-switching strategy for constructing a six-member, full-spectrum library of ClO–-responsive probes. Aqueous screening identified Scou, Scou-Ph, and Scou-CN as selected candidates based on pronounced spectral responses and visual contrast. HRMS supports the ClO–-triggered oxidative desulfurization of the thiocarbonyl moiety to the corresponding carbonyl fluorophore, while DFT calculations rationalize the associated spectral changes. Detailed evaluation of the three probes revealed distinguishable fluorescence responses over pH 2–10 and enabled determination of ClO– in tap water and lake water, affording recoveries of 98.3–111% with RSD values of 0.22–6.1%. This work expands the structural diversity of ClO– probes and offers a practical workflow for molecular-library screening and selected-probe evaluation.
Abstract The carboxyl/choline esterase (CCE) superfamily plays critical roles in insect xenobiotic detoxification, yet its involvement in Aphis glycines tolerance to abamectin remains poorly understood. In this study, 26 AgCCE genes were identified genome-wide and classified into six phylogenetic clades. Transcriptomic and RT-qPCR profiling showed that AgBe10 was most strongly induced under sublethal abamectin exposure. Functional validation using RNA interference revealed that silencing AgBe10, but not AgAce1, AgBe8, AgNeu3, or AgUn1, significantly increased aphid mortality under sublethal abamectin treatments. Molecular docking simulations based on the predicted amino acid sequence of AgBe10 indicated stable binding to abamectin, while high-performance liquid chromatography assays confirmed that recombinant AgBe10 mediates the in vitro metabolic depletion of abamectin. These findings elucidate the molecular and biochemical roles of AgBe10 in abamectin detoxification, providing insights into the molecular and biochemical mechanisms of early metabolic adaptation, thereby supporting sustainable pest management strategies.
Abstract Endophyte-based coatings enhance plant resistance and offer a sustainable alternative to chemical insecticides; however, their efficacy under nonsterilized soil conditions remains unclear. Here, we examined how nonsterilized soil conditions influence aphid resistance in wheat conferred by the endophytic fungus Beauveria bassiana strain YC. YC inoculation reduced Rhopalosiphum padi populations in both sterilized and nonsterilized soils, with stronger resistance observed under nonsterilized conditions, as evidenced by reduced aphid performance and altered feeding behavior. These effects were accompanied by shifts in the rhizosphere bacterial and fungal communities. Integrated transcriptomic and metabolomic analyses revealed coordinated activation of plant defense responses, particularly phenylpropanoid metabolism, including enhanced flavonoid accumulation and the upregulation of lignin and cell wall biosynthesis-related genes (e.g., PAL, 4CL, CCR). Overall, nonsterilized soil conditions modulated the magnitude of endophyte-mediated resistance, suggesting that soil environments should be considered when evaluating and applying endophyte-based strategies for enhancing crop resistance against insect herbivores.
Abstract Prunella vulgaris polysaccharide (PVP) possesses multiple bioactivities, yet its role in ulcerative colitis (UC) remains undefined. In this study, the effects of PVP were examined in mice with a DSS-induced colitis. In this colitis model, PVP treatment markedly reduced colonic inflammation and maintained the tight junction integrity. Meanwhile, PVP-enriched beneficial gut microbiota. Further research has revealed that PVP-modulated microbiota alone are sufficient to relieve UC symptoms. Mechanistically, PVP-enriched Ligilactobacillus agilis (Lig. agilis) subsequently enhanced tight junction protein expression and attenuated inflammation. In summary, these data suggest that PVP mitigates DSS-driven colitis, an effect mediated by enriching Lig. Agilis to suppress inflammation and maintain intestinal barrier integrity, and such effects point to the therapeutic value of PVP for treating UC.
Abstract Tropomyosin is a pan-allergen in crustaceans known to cross-react with homologous proteins from edible insects. However, epitope-level cross-reactivity in edible insects remains poorly defined. This study aimed to identify IgE-binding epitopes of tropomyosin and to evaluate their cross-reactivity between shrimp and the edible two-spotted cricket. Here, sera from shrimp-allergic patients were screened for IgE-binding to proteins from Pacific white shrimp (Penaeus vannamei) and two-spotted cricket (Gryllus bimaculatus). Candidate tropomyosin epitopes from the two-spotted cricket were predicted. The cross-reactivity of tropomyosin was assessed using an inhibition dot blot assay, inhibition ELISA, and LAD2 cell degranulation assay. Approximately 40% of shrimp-allergic patients showed IgE reactivity to crude proteins from shrimp and the two-spotted cricket. Four identified peptide epitopes induced β-hexosaminidase release from LAD2 cells. These findings provide insights into the molecular basis of tropomyosin cross-reactivity essential for the development of biomarker-based tools for allergen risk assessment in the food industry.
Abstract Tannases can reduce haze and astringency in tea beverages, but their application is often limited by poor thermal stability and catalytic efficiency. A tannase from Paraburkholderia pallida (Tanpp) was engineered using active-site mutagenesis and computational hotspot prediction. The double mutant F384T/N307A showed the best performance, with a 2.7-fold increase in catalytic efficiency and an 11-fold longer half-life at 50 °C than wild-type Tanpp. Structural modeling and molecular dynamics simulations suggested that N307A widened the substrate-entry region, whereas F384T compacted the catalytic pocket. The double mutant remained more stable at 323.15 K. In green tea clarification assays, F384T/N307A produced the lowest turbidity and promoted conversion of galloylated catechins into non-galloylated products and gallic acid. It retained superior performance across the tested enzyme dosages and treatment times. These findings show that coordinated active-site remodeling can improve both catalytic performance and thermal durability, supporting F384T/N307A as a promising tannase for tea clarification.
Abstract Cytochrome P450 enzymes are versatile monooxygenase biocatalysts. Members of the bacterial CYP226 family are hypothesized to catalyze the oxidation of diterpenoid resin acids as the first step in their catabolism. Two CYP226 enzymes (CYP226A3 and CYP226A31) from the thermophilic bacterium Zestomonas thermotolerans were characterized. Both could bind the diterpenoids in gum rosin. Oxidation metabolites were formed using peroxide-driven reactions with the wild-type enzymes and variants with modifications at the dioxygen-binding groove. The CYP226A31 enzyme was used to enantioselectively hydroxylate dehydroabietic acid to the 7β-hydroxy metabolite, enabling its isolation and characterization. The crystal structure of the CYP226A31 enzyme was determined with dehydroabietic acid bound, demonstrating a binding mode consistent with the stereoselective product formation and the unusual UV–visible substrate-bound spectrum. This work establishes the role of these enzymes in the oxidation of diterpenoids and provides a foundation for the valorization of complex mixtures of resin acids from renewable bark biomass.
Abstract As a thermotolerant bacterium, Bacillus licheniformis is an attractive chassis for high-temperature biomanufacturing. Here, we identified a novel temperature-responsive promoter, PycgM, which maintained strong transcriptional activity at 37–52 °C. In a promoter–mCherry reporter system, PycgM exhibited 2287.3-fold higher activity than P2 at 52 °C, demonstrating excellent compatibility with a thermotolerant host. Truncation analysis identified a 150-bp core functional region responsible for optimal activity under induction and heat stress. When applied to drive glutamate decarboxylase expression at 50 °C, PycgM enabled γ-aminobutyric acid production of 391.67 g/L with a 98.69% conversion rate, representing a 275% increase over 37 °C fermentation. The whole-cell biocatalyst retained 86% activity after five reuse cycles, and SEM analysis indicated acceptable structural stability despite moderate morphological changes. These results demonstrate that PycgM is a robust, high-temperature genetic element for efficient enzyme and metabolite production in thermotolerant hosts.
Abstract Glycosylation of natural compounds modulates their bioactivity as well as their physicochemical properties, making glycodiversification a valuable strategy for identifying glycosidic derivatives with improved functional profiles. However, the use of glycosyltransferases (GTs) for their synthesis is constrained by stringent sugar nucleotide donor selectivity, which limits the structural diversity of accessible glycosides. The chimeric NB1//CC1, constituted of 2 self-assembled domains originating from plant glycosyltransferases, was previously demonstrated to transfer glucose onto a wider range of natural compounds than its parental enzymes. This study demonstrates that this chimeric enzyme also exhibits a relaxed selectivity toward sugar nucleotide donors. This donor promiscuity can further be enhanced by the Mg2+ cation, which increases the sugar transfer rate for the chimeric enzyme. NB1//CC1 can therefore be used as a single biocatalyst to generate a library of glycosidic variants of a given natural glycosylated compound.
Abstract Sclerotinia stem rot caused by Sclerotinia sclerotiorum severely constrains global crop production. This study explored the regulatory role of checkpoint kinase SsChk2 in tebuconazole resistance. Bioassays indicated that ΔSsChk2 mutants showed reduced sensitivity to three demethylation inhibitors (DMIs), compared with wild-type strain UF-1, while maintaining normal biological phenotypes. Transcription of SsChk2 was significantly suppressed in five UV-induced tebuconazole-resistant mutants and inhibitor-treated UF-1. Independent of SsCYP51 amino acid substitutions, the ΔSsChk2 strain exhibited elevated SsCYP51 expression, ergosterol accumulation, and cellular structural stability under tebuconazole stress. RNA-seq analyses revealed that SsChk2 deletion upregulated key ergosterol biosynthesis genes and ABC1/MFS2 transporters and activated coordinated defensive pathways. These findings reveal that loss or downregulation of SsChk2 stimulates ergosterol biosynthesis to confer DMI resistance and uncover a promising target for sustainable control of Sclerotinia stem rot.
Abstract Cacao fruit maturation directly influences the chemical composition of tissues that are central to chocolate production and byproduct valorization, yet its lipid remodeling remains unexplored. Here, an untargeted UHPLC–Orbitrap–MS lipidomics workflow was used to map lipid profile changes in cacao seeds and cacao pod husk (CPH) across three maturation stages (green, intermediate, and ripe). In seeds, 290 lipid species were annotated, with most lipid classes showing lower relative abundance at the mature stage. Major cocoa butter triacylglycerols exhibited selective accumulation, revealing that maturation involves targeted lipid reorganization rather than a generalized increase in storage lipids. In CPH, 224 lipid species were annotated, with limited variation at the class level but pronounced species-specific changes; steryl esters, diacylglycerols, hydroxy fatty acids, and acylated sterol glycosides increased during ripening. These trends indicate distinct metabolic roles for storage and protective tissues during cacao development.
