Yeast β-glucan is a well-established immunomodulatory agent. Studies have demonstrated that compared with vegetative yeast cells, β-glucan exposed on the surface of mutant yeast spores exhibits enhanced immunostimulatory effects. [Objective] To screen and identify yeast mutants with enhanced ability to activate the mammalian immune system. [Methods] Two yeast mutants (osw1Δ and mum3Δ) exposing β-glucan on the spore wall were constructed, and their immunostimulatory activity was evaluated via the macrophage stimulation assay and a mouse model. The role of the Dectin-1 signaling pathway was verified through small interfering RNA-mediated gene knockdown experiments. [Results] The spore lysates of osw1Δ and mum3Δ induced the production of inflammatory cytokines including interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) in macrophages. Their immune activation was mainly dependent on the Dectin-1-mediated signaling pathway. In vivo studies showed that after oral administration of the osw1Δ spore lysate, the levels of IL-6 and TNF-α in the mouse serum increased by 1.8-fold and 1.7-fold, respectively, compared with those after oral administration of the wild-type spore lysate. Moreover, the osw1Δ spore lysate exerted a significant tumor-suppressive effect. [Conclusion] The spores of osw1Δ exhibit strong immune-activating effects. This mutant could be a candidate for the development of novel immunomodulators.

[Objective] The heat-labile enterotoxin (LT) is widely recognized as a potent mucosal immunoadjuvant. However, the mechanisms underlying its interaction with host epithelial cells remain incompletely understood, which makes it difficult to separate its toxicity from its adjuvant activity. This limitation has severely hampered its clinical application. Therefore, this study aims to elucidate the differential regulatory effects of LT and its A (LTA) and B (LTB) subunits on epithelial cells, as well as the mechanism underlying the initiation of their initial adjuvant activity. [Methods] The biologically active LTB, LTA, and its mutant LTA(R192G) were prepared via prokaryotic expression. Using the human small intestinal epithelial cell line FHs 74 Int and a mouse jejunal ex vivo intestinal segment model, we examined cell viability and apoptosis, analyzed inflammatory cytokine expression, and investigated changes in the NF-κB and NLRP3 inflammasome pathways. [Results] The three prepared proteins all possessed biological activity and could be effectively internalized by cells. LTB primarily induced early apoptosis, significantly up-regulated the expression of IL-6, IL-8, IL-1β, and TNF-α, and activated the NF-κB pathway. LTA triggered significant pyroptosis, specifically up-regulated the expression of IL-18 and IL-1β, and activated the NLRP3 inflammasome pathway. The mutant LTA(R192G) exerted weakened effects but could still activate the NLRP3 pathway. [Conclusion] The adjuvant activity of LT stems from its subunits activating immune responses in epithelial cells through different mechanisms: LTB primarily promotes pro-inflammatory cytokine production via the NF-κB pathway, while LTA mainly relies on the NLRP3 pathway to induce IL-1β/IL-18 secretion and pyroptosis. This discovery not only reveals the initial molecular events of LT-initiated mucosal immunity, but more importantly, lays a solid theoretical foundation for developing novel, safe, and efficient mucosal vaccine adjuvants through targeted modification of LTA and LTB subunits.

[Objective] Ergothioneine (EGT), a sulfur-rich derivative of histidine, is utilized in the food, pharmaceutical, and cosmetic industries. However, large-scale production of EGT faces challenges due to the high costs and inefficiency of conventional chemical synthesis and extraction techniques. This study aims to engineer Saccharomyces cerevisiae to provide a microbial platform for EGT biosynthesis. [Methods] The biosynthetic pathway for EGT was reconstructed in S. cerevisiae by heterologously expressing Egt1 from Neurospora crassa and Egt2 from Claviceps purpurea. To overcome the metabolic bottlenecks related to precursor supply, we optimized the upstream pathways for histidine, cysteine, methionine, and S-adenosylmethionine to enhance the flux toward EGT synthesis. Fermentation performance of the engineered strain was assessed in both shake flasks and a 5-L bioreactor. [Results] The engineered S. cerevisiae strain produced 312.8 mg/L of EGT in shake flask fermentation. In a 5-L bioreactor, the strain achieved the EGT titer of 1 312.2 mg/L after 168 h, with the productivity of 7.8 mg/(L·h). [Conclusion] This study presents a metabolic engineering strategy for producing EGT in S. cerevisiae. The approach not only significantly improves EGT biosynthesis but also serves as a reference for microbial production of other compounds.

Raspberry ketone (RK) is a high-value natural aromatic compound widely used in food, flavors and fragrances, and daily chemical products. Traditional plant extraction methods are constrained by raw material availability and high separation costs, while chemical synthesis suffers from high environmental burden and difficulties in meeting market demand for natural-source products. Biocatalytic synthesis represents the core direction for its green manufacturing. However, existing systems remain limited by bottlenecks such as insufficient enzyme activity and substrate tolerance, poor stability of cofactor regeneration systems, and product titers insufficient for industrial needs. [Objective] To construct an efficient, stable, and scalable biocatalytic synthetic system for RK, overcoming existing technical bottlenecks. [Methods] A flavin-independent ene-reductase from Arabidopsis thaliana, AtQOR, was screened and coupled with formate dehydrogenase from Lactobacillus buchneri, LbFDH, to construct an NADPH self-recycling dual-enzyme cascade system. By optimizing vector copy number and gene expression order, an engineered strain co-expressing both enzymes was constructed, and the optimal strain, Escherichia coli 02, was selected. Key reaction conditions for whole-cell biocatalysis were systematically optimized, and a scale-up experiment was performed in a 1 L fermenter using a fed-batch strategy. The product was quantitatively analyzed and structurally verified using gas chromatography (GC) and nuclear magnetic resonance (NMR) spectroscopy. [Results] The selected AtQOR exhibited a specific activity of 10.35 U/mg in catalyzing the conversion of p-hydroxybenzylidene acetone to RK. Under optimal reaction conditions, E. coli 02 produced 37.88 g/L of raspberry ketone from 40 g/L p‑hydroxybenzylidene acetone in 12 h, with a conversion rate of 93.54%. Using a fed‑batch strategy in a 1 L fermenter, the RK titer reached 54.32 g/L in only 10 h, with a conversion rate of 89.43%, and the product was confirmed to be a high-purity target compound. [Conclusion] The dual-enzyme cascade catalytic system constructed in this study substantially overcomes the existing titer bottleneck in RK biosynthesis, providing an efficient, stable, and scalable technological solution for its industrial green manufacturing.

Aeromonas veronii is a Gram-negative pathogenic bacterium that causes various diseases in aquatic animals and humans, posing a serious threat to aquaculture and public health. The type Ⅵ secretion system (T6SS) is a key virulence factor determining the pathogenicity of A. veronii. It is known that the bacterial enhancer-binding protein (bEBP) VasH is responsible for regulating the T6SS function, while whether this regulatory relationship exists in A. veronii remains unknown. [Objective] To elucidate the impacts of VasH on T6SS expression and function in A. veronii, thereby providing a theoretical basis for deciphering the mechanism of T6SS-mediated pathogenicity of A. veronii and for the subsequent prevention and control of A. veronii infections. [Methods] With A. veronii C4 as the wild-type strain, the VasH-deficient mutant strain ΔvasH and the complemented strain ΔvasH/p-vasH were constructed via a homologous recombination strategy. RT-qPCR was employed to measure the relative expression levels of T6SS genes in each strain to clarify the effect of VasH on T6SS gene expression. Growth curve establishment, transmission electron microscopy (TEM) for observing bacterial morphology, and the crystal violet assay for biofilm quantification were performed to determine the influences of VasH on pathogenic characteristics. An in vitro bacterial competition assay and a zebrafish model for determining the median lethal dose (LD₅₀) were employed to assess the comprehensive effects of VasH on bacterial competitiveness and pathogenicity. [Results] The VasH-deficient mutant strain ΔvasH and the complemented strain ΔvasH/p-vasH were successfully constructed. The deletion of VasH resulted in a decrease (P<0.05) in the expression of genes encoding both T6SS structural and effector proteins, indicating that VasH was responsible for regulating T6SS gene expression. The deletion of vasH did not significantly affect the growth or surface morphology/structure of A. veronii. However, it led to a significant increase in the biofilm formation and a significant decrease in in vitro bacterial competitiveness. The zebrafish infection assay showed that the LD₅₀ of ΔvasH was 1.58×10¹¹, which was 2.15 times that (7.34×10¹⁰) of the wild type. Moreover, at an infection concentration of 5×10⁸ CFU/mL, the death rate of zebrafish decreased from 33.3% in the wild-type infection group to 0 following infection with ΔvasH, indicating that deletion of vasH attenuated the virulence of A. veronii. [Conclusion] In A. veronii, VasH positively regulates T6SS gene expression. It may not be involved in regulating the growth state or external morphology of the pathogen but significantly influences the biofilm formation and competitiveness of A. veronii, thereby modulating the overall virulence during infection of the zebrafish host. This study provides essential research tools and lays a preliminary foundation for further exploration of the molecular mechanisms by which VasH mediates pathogenicity through regulating T6SS activity and function in A. veronii.

[Objective] To investigate the effects of luxS deletion on the biofilm formation and stress tolerance of Listeria monocytogenes and to elucidate the role of luxS in regulating environmental adaptability of this pathogen. [Methods] With Listeria monocytogenes ATCC 19112 as the parental strain, a luxS-deleted mutant, a complemented strain, and an empty-vector control strain were constructed. Growth curve analysis, environmental stress tolerance assays and biofilm formation assays were performed to comparatively analyze phenotypic differences among the wild-type, mutant, and complemented strains. [Results] Compared with the wild-type strain, the luxS-deleted mutant exhibited reduced adaptability under adverse conditions, including low temperature, high osmotic pressure, oxidative stress, and acid stress. Its biofilm-forming ability decreased after 24 h and 48 h of incubation (P<0.01), accompanied by a reduction in the production of extracellular polymeric substances in the biofilm matrix. Further transcriptional analysis revealed that the expression levels of agrA, agrB, lmo2504, and sigB were downregulated (P<0.05), whereas that of ladR was upregulated (P<0.05) following luxS deletion. Under disinfectant stress, MTT assay showed that the metabolic activity of the luxS-deleted strain was lower than that of the wild-type and complemented strains after 48 h of biofilm cultivation (P<0.05). After 72 h, no significant differences were observed among the strains. [Conclusion] These findings demonstrate that luxS plays a critical role in regulating the environmental adaptability of L. monocytogenes and provide a novel theoretical basis for the development of future control and prevention strategies targeting this pathogen.

[Objective] To characterize the successional dynamics of bacterial and fungal communities during leaf litter decomposition and elucidate the associations between keystone taxa and substrate component losses, thereby advancing our understanding of microbial regulation during litter decomposition in forest ecosystems. [Methods] We established an indoor microcosm with the leaf litter from Alnus cremastogyne to track microbial community succession during the litter decomposition. High-throughput sequencing, chemical composition analysis, and bacterial-fungal cross-domain co-occurrence network analysis were integrated to characterize microbial community dynamics, identify keystone taxa, and test their associations with loss rates of major chemical components across four decomposition stages (initial, 45%, 75%, and 90% mass loss). [Results] At the phylum level, bacterial and fungal community composition showed only minor changes in relative abundance across stages, with Pseudomonadota (relative abundance of 50%- 80%) dominating bacterial communities and Ascomycota (>95%) dominating fungal communities. In contrast, pronounced stage-dependent succession was observed at the genus level. Decomposition stage accounted for substantial variations in bacterial (R²=0.573, P<0.001) and fungal (R²=0.377, P<0.001) community structures. With the progression of decomposition, cross-domain networks exhibited increased nodes and connectivity and shifted from loose to modular structures, with positive correlations consistently exceeding negative correlations. The number of keystone taxa increased over time and became progressively dominated by fungi. During the first three decomposition stages, the number of bacterial nodes accounted for approximately 75% of total nodes, whereas at the 90% decomposition stage, the number of fungal nodes increased markedly (from 103 to 320), resulting in comparable proportions of bacterial and fungal nodes. At the 45% decomposition stage, keystone bacterial taxa were primarily associated with losses of non-structural components and hemicellulose, whereas at the 75% and 90% decomposition stages, keystone fungal taxa were more closely associated with cellulose and lignin losses. Functional predictions further supported this stage-specific division of labor, indicating stronger potential of structural carbon degradation at the late stages of decomposition. [Conclusion] Bacteria and fungi jointly participate in A. cremastogyne leaf litter decomposition with temporally differentiated contributions. Bacteria play a more prominent role during early and middle stages, whereas fungi become increasingly important at late stages. These findings indicate coordinated shifts in microbial community structure and resource utilization rather than simple taxonomic replacement, contributing new insights into microbial regulation during litter decomposition.

[Objective] The gut microbiota plays crucial roles in host energy metabolism, immune regulation, and environmental adaptation. However, seasonal variations in the structure, function, and assembly mechanisms of the gut microbiota in the plateau zokor (Eospalax baileyi) remain poorly understood. [Methods] This study employed Illumina high-throughput sequencing technology to perform 16S rRNA (V3-V4 region) sequencing on 17 gastrointestinal content samples of plateau zokors, thus investigating the effects of season (spring vs. winter) on the diversity, function, and assembly processes of the gut microbiota. [Results] The alpha diversity and richness of the gut microbiota in plateau zokors were higher in spring than in winter (P<0.05), and the gut microbiota structure in winter exhibited extremely significant differences (P<0.001). At the phylum and genus levels, the relative abundance of certain dominant microbial taxa differed between seasons (P<0.05). PICRUSt functional prediction revealed that at Level 1, metabolism was the dominant pathway, with the genetic information processing, environmental information processing, human diseases, and organismal systems pathways being enriched in the spring group compared with the winter group (P<0.05). Further analysis of metabolic pathways at Level 2 indicated that pathways related to carbohydrate metabolism, amino acid metabolism, nucleotide metabolism, lipid metabolism, metabolism of other amino acids, and metabolism of terpenoids and polyketides were higher in spring than in winter (P<0.05). The neutral community model (NCM) showed more extensive dispersal of the gut microbiota among individuals in winter (Nm=228.75) than in spring (Nm=216.68). The normalized stochasticity ratio (NST) was less than 0.5 in the spring group, indicating dominance by deterministic processes, while it was greater than 0.5 in the winter group, suggesting dominance by stochastic processes, with significant differences between groups (P<0.001). Further iCAMP analysis revealed that drift and dispersal limitation were the primary ecological processes governing microbial community assembly in both seasons, and the relative contributions of these ecological processes differed between spring and winter (P<0.001). [Conclusion] Season significantly affects the structure, composition, function, and assembly mechanisms of the gut microbiota in plateau zokors. During winter, plateau zokors may save energy by reducing the metabolic activity and information-processing functions of the gut microbiota. In addition, plateau zokors may adjust taxa abundance of the gut microbiota to cope with environmental changes and maintain intestinal homeostasis. These findings provide important insights into the mechanisms by which wild plateau rodents adapt to high-altitude and cold environments from the gut microbiota.

Microbial antioxidant functions hold significant application potential in the development of holistic health resources, yet their spatial distribution patterns across continuous ecological gradients remain poorly understood. [Objective] To analyze the diversity and spatial distribution characteristics of antioxidant function-related genes in prokaryotes within the Lasha Mountain watershed. [Methods] A spatially exhaustive nine-grid sampling strategy was employed, with 117 sampling points established across the watershed and its 12 sub-basins. PICRUSt2 was used for functional prediction based on high-throughput sequencing data of the 16S rRNA gene. [Results] A total of 38 genes associated with antioxidant compound synthesis were identified and categorized into 12 functional groups. The diversity and total abundance of antioxidant function-related genes were unevenly distributed within the Lasha Mountain watershed, with the downstream region exhibiting higher gene diversity and abundance. The genes associated with non-enzymatic antioxidant compounds dominated both in functional category richness (7 categories) and abundance (0.005 270-0.007 657), with glutathione and thioredoxin genes exhibiting the highest abundance. The overall abundance variation of individual genes (CV=125.85) primarily originated within sub-basins (CV=77.23). After the antioxidant function-related genes were classified by synthesized compounds, the pattern of variation reversed. [Conclusion] The antioxidant function-related genes of prokaryotes exhibit spatial distribution differences in the watershed, and the downstream area—characterized by multiple coupled stress conditions—is a hotspot for the diversity and total abundance of such genes. This study provides an ecological basis for the targeted screening of high-quality antioxidant strains.

[Objective] To explore genes related to the regulation of yeast spore germination, we previously screened for mutants defective in maintaining the quiescent state of spores. [Methods] Using the temperature-sensitive yeast mutant J33 with defective spore quiescence maintenance as the research material, heterozygous diploid strains were constructed through tetrad dissection and haploid fusion, and genetic segregation analysis was performed to clarify the genetic regulatory pattern of mutant phenotypes. The yeast genomic library complementation screening was applied to identify candidate mutant genes, and gene cloning and sequence alignment were conducted to confirm the mutation site. Furthermore, recombinant plasmids carrying wild-type and mutant TIP20 were constructed for functional complementation assays, and site-directed mutagenesis was used to verify the correlation between TIP20 mutation and strain phenotypes. [Results] Functional complementation experiments demonstrated that introduction of the wild-type TIP20 restored the growth at 37 ℃ and the normal spore germination phenotype of J33, while the mutant TIP20 and empty vector had no such effect. Site-directed mutagenesis analysis further confirmed that this single-base mutation was T1219G, a direct cause of the TS phenotype and defective spore germination of J33. [Conclusion] TIP20 is involved in the regulation of yeast spore germination. TIP20 encodes a subunit of the tethering complex involved in endoplasmic reticulum-Golgi retrograde transport. However, TIP20 is likely a multifunctional protein, and its mammalian homologue RINT1 has been reported to regulate the cell cycle checkpoint. Thus, TIP20 may also be involved in regulating the quiescent state of yeast cells.