Objective Four-carbon dicarboxylic acids are a class of important platform chemicals widely used in the food, pharmaceutical, and chemical industries. However, the efficiency of microbial fermentation for producing four-carbon dicarboxylic acids still faces challenges, mainly limited by insufficient central carbon metabolic flux and byproduct accumulation. Methods This study used Escherichia coli as the chassis strain and adopted a strategy combining rational metabolic engineering and non-rational modification to systematically optimize the four-carbon dicarboxylic acid synthesis capacity of E. coli. Results The non-cyclic glyoxylate shunt was reconstructed and the expression of key pathway enzymes was optimized to enhance the metabolic flux toward four-carbon dicarboxylic acids. The synthesis capacity of four-carbon dicarboxylic acids was enhanced by employing atmospheric and room-temperature plasma (ARTP) mutagenesis. The knockout of key genes in the acetate, formate, and lactate synthesis pathways effectively minimized carbon flux diversion, thereby enhancing the availability of oxaloacetate, the central precursor to four-carbon dicarboxylic acids. On this basis, through specific modification of terminal metabolic pathways, the engineering strain E. coli Fum02 for fumaric acid production were constructed. Finally, in a 5 L fermenter, the fumaric acid titer, yield, and productivity of the engineering strain E. coli Fum02 reached 45.2 g/L, 0.45 g/g, and 0.23 g/(L·h), respectively. Furthermore, by blocking the succinate dehydrogenase gene (sdhAB) and implementing fermentation optimization strategies, this platform strain could also be redirected toward efficient succinate production. Conclusion This study provides a reference for the metabolic engineering modification of bacteria to produce organic acids and also lays a foundation for the industrial biomanufacturing of four-carbon dicarboxylic acids.

Objective Fusarium wilt caused by Fusariumoxysporum f. sp. nivum is a typical soil-borne disease in watermelon production, posing significant threats. This study investigates the microbial community structures in the rhizosphere soil of healthy and Fusarium wilt-affected watermelon plants to clarify the regulatory effects of this disease on the physicochemical properties and microbial communities of rhizosphere soil. It aims to reveal the interactions between pathogen enrichment, beneficial microbial decline, and soil environmental factors, providing theoretical support for the green control of Fusarium wilt in watermelon plants by rhizosphere microbiome regulation. Methods Rhizosphere soil samples were collected from healthy plants (HT group) and Fusarium wilt-infected plants (FT group) of the watermelon variety ‘Xiaoyu No. 5’ in Shaoyang, Hunan. Physicochemical indicators including total nitrogen (TN), total phosphorus (TP), available phosphorus (AP), and available potassium (AK) were measured. Illumina high-throughput sequencing was employed to analyze the structures and diversity of microbial communities in the rhizosphere soil of healthy and disease-infected plants. Results The FT group had lower content of TP, AP, and AK in the rhizosphere soil than the HT group (P<0.05). The TN, organic matter (OM), and pH in the FT group were lower without significant differences than the HT group. The FT group had higher fungal ACE and Chao1 indices (P<0.05), higher bacterial ACE and Chao1 indices (P>0.05), and higher fungal and bacterial Simpson indices (evenness) (P<0.05) than the HT group. The abundance of Bacillota was significantly higher in the HT group than in the FT group, whereas that of Ascomycota was significantly higher in the FT group. At the genus level, the abundance of beneficial bacteria such as Neobacillus and Bacillus decreased in the FT group, while that of the pathogenic genus Fusarium increased sharply from 0.06% to 2.40%. The redundancy analysis (RDA) indicated that TN, TP, and OM were key drivers of bacterial community changes, whereas TN, OM, and AK were core regulators of fungal communities. Functional prediction suggested enhanced functions such as stress responses and energy metabolism of bacteria, alongside increased potential for functions such as plant cell wall degradation of fungi, in the diseased rhizosphere. Conclusion The occurrence of Fusarium wilt in watermelon plants leads to depletion of phosphorus and potassium in the rhizosphere soil and disrupts microbiome balance. This is manifested by the enrichment of Fusarium and the decline of beneficial bacteria (e.g., Neobacillus and Bacillus). Soil TN, OM, and AK are key environmental factors regulating this imbalance, with AK deficiency potentially serving as a pivotal link between soil environmental degradation and disease intensification. These findings provide crucial theoretical support for developing eco-friendly control strategies-potassium supplementation and stabilization alongside the targeted cultivation of beneficial microbial communities-targeting Fusarium wilt in watermelon plants.

Objective To investigate the effects of combined application of organic and inorganic fertilizers on soil nutrient content and microbial community structures and functions in soybean fields, thus providing a scientific basis for rational fertilization and high-quality, high-yield soybean production. Methods Four fertilization treatments—control (CK: no fertilization), inorganic fertilizer (CF: compound fertilizer), organic fertilizer (OF: dry chicken manure), and combined organic-inorganic fertilizers (OCF: dry chicken manure+compound fertilizer)—were established. During the experiment, soil organic matter (SOM), alkali-hydrolyzed nitrogen (AN), available phosphorus (AP), available potassium (AK), and microbial community structures and functions were measured to investigate the relationships between microbial communities and soil nutrients. Results Different fertilization treatments influenced soil nutrients in soybean fields. Compared with CK, the OCF treatment increased the soil SOM, AN, AP, and AK by 60.67% (P<0.05), 68.09% (P<0.001), 15.18 folds (P<0.001), and 59.54% (P<0.01), respectively, and it also increased soil pH. Amplicon sequencing indicated that different fertilization measures did not alter the community composition of soil microorganisms but changed the relative abundance of different phyla and genera. Compared with CK, the OCF treatment increased the relative abundance of Basidiomycota and Mortierellomycota by 5.62 folds and 4.51%, respectively, while decreasing that of Ascomycota by 38.35%. The OCF treatment reduced fungal community richness and diversity (P<0.05). The alpha diversity analysis revealed that both bacterial and fungal diversity decreased after organic fertilizer application, with fungal alpha diversity showing the most significant reduction (P<0.05). Functional prediction indicated that amino acid metabolism exhibited the highest relative abundance among metabolic pathways in bacterial communities, suggesting that the OCF treatment promoted metabolic processes centered on nitrogen assimilation and protein synthesis, facilitating bacterial participation in soil nutrient transformation. Under the OF treatment, symbiotropic fungi exhibited the highest relative abundance, which suggested that organic fertilizer promoted the ecological functions of fungi in soil nutrient cycling. Conclusion Combined application of organic and inorganic fertilizers modulates soil pH, mitigates soil acidification, and enhances soil nutrient content. Fungal communities exhibit greater sensitivity to organic fertilizer application, which significantly reduces their diversity and stimulates the proliferation of certain pathogenic fungi. Conversely, inorganic fertilizer suppresses the relative abundance of pathogenic fungi. Thus, the combined application of organic and inorganic fertilizers demonstrates distinct advantages in balancing soil nutrient supply with microbial community structure and optimizing soil microbial functional composition. This approach provides theoretical foundations and practical guidance for achieving efficient, green, and sustainable fertilization management in soybean fields.

Listeria monocytogenes, as a major causative agent of foodborne illness outbreaks, poses a serious threat to food safety and public health. In complex foodborne pathogen environments, the specific and effective detection methods for L. monocytogenes are crucial. Objective To develop a novel recombinase-aided amplification-exonuclease (RAA-exo) assay for detecting L. monocytogenes. Methods Multiple RAA primer and probe sets targeting hly were designed, and the optimal primer set was selected based on nucleic acid amplification efficiency. The reaction system was rigorously optimized, focusing on the concentrations of A buffer, B buffer, and RAA primers and probe. Results The optimized RAA-exo assay showed a limit of detection (LOD) of 0.5 copies/μL for recombinant plasmids and 10 CFU/mL for L. monocytogenes suspensions. The assay demonstrated high specificity, selectively detecting L. monocytogenes without cross-reactivity to other common foodborne pathogens, including Salmonella, Escherichia coli, Staphylococcus aureus, Bacillus cereus, or other Listeria species (L. ovinae, L. seeligeri, and L. innocua). In a validation study using 44 pork samples, the RAA-exo assay results were in complete agreement with those of the real-time fluorescence PCR internal standard method outlined in the industry standard SN/T 5224—2019. Conclusion The developed RAA-exo assay exhibits high sensitivity and specificity, requires minimal hands-on time, and achieves detection within 20 min at 37 °C. Therefore, the assay is suitable for rapid, on-site testing, serving as an efficient and convenient tool for L. monocytogenes detection, with promising applications in food safety monitoring.

Trees can form mutualistic symbionts with mycorrhizal fungi. Different mycorrhizal types affect the community structure of endophytic fungi by regulating tree physiology and root microenvironment, thus becoming a key link driving the interaction network between soil and microorganisms in tropical forests. However, the mechanisms by which different mycorrhizal types regulate the diversity and community composition of endophytic fungi in tropical tree roots are still not fully understood. Objective To explore the effects of different mycorrhizal types on the diversity and community structure of root endophytic fungi in tropical trees, as well as their key driving factors, systematically clarifying how mycorrhizal types affect the composition and diversity of endophytic fungal communities by regulating root traits and rhizosphere environment, and identifying the key driving factors. Methods On the basis of 3 773 sets of soil and root data collected from three research sites of Chinese Ecosystem Research Network (CERN) in Xishuangbanna tropical forest, China, we integrated and constructed a dataset at the tree species level. This dataset encompassed data of the root traits, soil physical and chemical properties, and the operational taxonomic unit (OTU) abundance of endophytic fungi in the roots of 119 trees (54 species) with arbuscular mycorrhizas (AM) and 31 trees (12 species) with ectomycorrhizas (ECM), and it was then used for the research. Results The alpha diversity of endophytic fungi in the roots of AM trees was higher than that of ECM trees (P<0.05). Mycorrhizal types affected the dominant groups of root endophytic fungi. Ascomycota had the highest relative abundance (43.17%) in the roots of AM trees, and Basidiomycota had the highest relative abundance (65.17%) in the roots of ECM trees. The co-occurrence network analysis showed that the endophytic fungal network was denser in the roots of AM trees and more modular in roots of ECM trees. Soil properties were the dominant driving factors for the endophytic fungal communities in the roots of AM trees, while the endophytic fungal communities in the roots of ECM trees were regulated jointly by root traits and soil properties. Soil phosphorus was a key factor affecting the endophytic fungal communities in the roots of AM and ECM trees. Conclusion In tropical forest ecosystems, AM drives trees to form species-rich and closely interacting endophytic fungal communities in the roots, and the assembly process is mainly regulated by soil factors. ECM trees form a specialized symbiotic fungal system, whose construction is regulated by both root traits and soil factors. In addition, soil phosphorus is the core factor driving the formation of endophytic fungal communities in the roots of the two types of trees.

Objective Transient receptor potential vanilloid 4 (TRPV4), a non-selective cation channel, is deeply involved in the physiological and pathological regulation of multiple organ systems, while the comprehensive influencing mechanism of its mutation on animal intestines and intestinal flora is not clear. This study explored the regulatory effects of Trpv4 exon 8 c.1491+1G>A mutation on intestinal barrier integrity and flora-metabolic microenvironment in mice, aiming to provide an experimental basis for analyzing the interaction mechanisms between host genes and intestinal flora. Methods Trpv4 exon 8 c.1491+1G>A gene-edited mice previously constructed in our laboratory were taken as the research objects, and the expression levels of Trpv4 and TRPV4 in the intestinal tissue were determined by qPCR and Western blotting, respectively. Pathological sections were prepared for observation of the structural changes of the intestinal tissue. The 16S rRNA gene high-throughput sequencing was conducted to reveal the structural differences of intestinal flora. Non-targeted metabolomics based on LC-MS was employed to examine the changes of fecal metabolites, and the correlations between flora and metabolites were analyzed. Results Trpv4 editing led to the abnormal expression of Trpv4 and TRPV4 in the intestinal tissue of mice, which resulted in the structural abnormality of the intestinal tissue and the impairment of intestinal barrier function. In addition, the gene-edited mice exhibited an imbalance in intestinal flora, with significantly increased relative abundance of Bacteroidota, a significantly decreased Bacillota/Bacteroidota (F/B) ratio, and reduced abundance of common commensal bacteria such as Staphylococcus. Metabolomic analysis indicated that the gene-edited mice presented disordered lipid metabolism and abnormalities in immune-related metabolites. The abundance of Bacteroidota was positively correlated with lipid metabolites, while that of Desulfovibrio and Enterobacter was negatively correlated with lipid and immune metabolites. Conclusion Trpv4 exon 8 c.1491+1G>A gene-edited mice exhibited impaired intestinal barrier function, along with alterations in intestinal flora structure and the metabolic microenvironment. This study provides basic data for elucidating the interactions between specific gene mutations and the gut microbiota and offers theoretical support for the development of diagnostic and therapeutic strategies for Trpv4-related diseases.

Objective The rapid increase in wastewater discharge from animal husbandry has caused severe environmental pollution. Identifying efficient heterotrophic nitrifying-aerobic denitrifying bacteria and investigating their denitrification mechanisms are of great theoretical and practical importance for mitigating nitrogen pollution in the wastewater. Methods A strain exceling in heterotrophic nitrification-aerobic denitrification (HN-AD) was isolated and from activated sludge in pig farms. Culture conditions were optimized by response surface methodology. We evaluated the inorganic nitrogen-transforming capacity of the strain by assessing its utilization efficiency of single and mixed nitrogen sources and through nitrogen balance analysis. The completeness of the denitrification process was confirmed via gas chromatographic measurements of N₂ and N₂O. Finally, the nitrogen removal pathways and underlying mechanisms were elucidated through whole-genome analysis. Results The successfully isolated strain Klebsiella sp. WH-E exhibited excellent HN-AD capabilities. The growth conditions of the strain were optimized as follows: sodium citrate as the carbon source, 34.18 °C, initial pH 7.1, a C/N ratio of 14.53, and a shaking speed of 159.59 r/min. When the strain was cultured with ammonium, nitrate, or nitrite as the sole nitrogen source, the nitrogen removal rates were 99.80%, 81.54%, and 80.00%, respectively. Furthermore, when ammonium was the sole nitrogen source, 35.84% and 35.91% of nitrogen were converted into cellular nitrogen and gaseous nitrogen, respectively. When ammonia nitrogen was combined with nitrate nitrogen as mixed nitrogen sources, the nitrogen removal rate was 100.00%; When ammonia nitrogen was combined with nitrite nitrogen as mixed nitrogen sources, the ammonia nitrogen removal rate was 100.00%, and the nitrite nitrogen removal rate was 91.97%, respectively. Whole-genome sequencing identified several nitrogen metabolism-related functional genes, including glnB, norVWR, narGHI, nasBC, and nirBD. Conclusion Klebsiella sp. WH-E possesses three nitrogen metabolism pathways: ammonium assimilation, nitrification-denitrification, and nitrate assimilation and dissimilation. This study confirms the applicability of Klebsiella sp. WH-E for nitrogen removal from full-scale piggery wastewater and establishes a solid theoretical foundation for its engineering applications.

Objective In view of the production and environmental issues caused by excessively high nicotine content in upper tobacco leaves, this study aims to decipher the molecular mechanism of nicotine degradation by an efficient nicotine-degrading strain Pu17 screened out in the previous study via genomic approaches. Methods The taxonomic status of the strain was determined by average nucleotide identity (ANI) analysis. Whole genome sequencing and annotation were employed to clarify the nicotine metabolic pathway. Key intermediates during degradation were detected by MS/MS. Live plant trials were conducted to explore the optimal application method for nicotine reduction. Results Phylogenetic analysis revealed an ANI value of 96.51% between Pu17 and Peanarthrobacter ureafaciens, identifying Pu17 as a strain of P. ureafaciens. The genome of Pu17 was 4.47 Mb in length, with the G+C content of 63.34%, encoding 4 155 proteins. Functional annotation and comparative genomics identified unique gene clusters related to heavy metal resistance, cell surface synthesis, and metabolic potential in Pu17, which constituted its environmental adaptation strategy. Metabolite analysis detected key intermediates such as 6-hydroxypseudooxynicotine. This result, combined with that of genomic analysis, confirmed that Pu17 degraded nicotine via the pyridine pathway, with key genes (e.g., nboR, mao, and 6-hlno) primarily located on plasmids. Efficacy evaluation demonstrated that the Pu17 fermentation broth effectively reduced nicotine content in tobacco plants through both foliar spraying and root irrigation, achieving a maximum degradation rate of 14.00% in live leaves. Conclusion This study systematically elucidates the molecular mechanism and application potential of P. ureafaciens Pu17 for nicotine degradation from genomic, metabolomic, and application perspectives. It provides a theoretical basis and microbial resources for the development of bioremediation technologies for tobacco waste and harm reduction.

γ-Aminobutyric acid (GABA), a key inhibitory neurotransmitter in the central nervous system, plays a vital role in physiological functions such as promoting sleep, relieving tremors, and regulating blood pressure. Currently, a variety of microorganisms capable of synthesizing GABA have been identified, offering diverse strategic options for the biosynthesis of GABA through different metabolic pathways. This review provides a detailed summary of the major pathways—the GABA shunt pathway and the putrescine pathway—for GABA synthesis in various microorganisms. It systematically outlines the key synthases and metabolites involved in the two pathways, while comparing their synthesis efficiency and respective advantages. Furthermore, this study delves into the regulatory mechanisms underlying GABA biosynthesis in different microorganisms, along with key regulatory targets for enhancing synthesis efficiency. The work aims to establish a theoretical framework for the regulatory mechanisms of microbial-derived GABA synthesis and to provide a scientific basis for improving the efficiency of GABA biosynthesis.

Objective To explore plant growth promoting rhizobacteria (PGPR) resources from the rhizosphere soil of maize in a maize-soybean rotation system and elucidate their roles in promoting the growth of maize and soybean, thus providing a theoretical basis and practical support at the microbial level for the sustainable development of agriculture. Methods Actinomycetes strains were isolated from the rhizosphere soil of maize via the dilution plating method with Gauze’s Synthetic Medium No. 1. The strains capable of secreting protease, producing siderophores, and fixing nitrogen were selected out. The isolated strains were identified by means of morphological observation and 16S rRNA gene sequence analysis. After optimization of the fermentation conditions and tests of stress tolerance, a synthetic microbial consortium (SMC) was prepared. Its growth-promoting effects on maize and soybean were evaluated through seed germination tests and pot experiments. Results A total of 105 Actinomycetes strains were isolated, five of which simultaneously exhibited the abilities of siderophore production, protease secretion, and nitrogen fixation. These strains were identified as Arthrobacter pokkalii (JM-18, JM-21), A. oryzae (JM-24), A. ginsengisoli (JM-47), and A. pascens (JM-48). They were mixed in equal proportions to form a SMC. Growth promotion assays showed that the SMC significantly improved maize seed germination and maize plant growth in pots. Specifically, the SMC increased the root length and shoot length in the seed germination assay by 120.22% and 20.94%, respectively, and it also significantly increased the plant height, root length, fresh weight, and dry weight of maize plants in pots. Moreover, the SMC markedly promoted soybean development, increasing soybean shoot length by 42.08% during seed germination. For potted soybean plants, the SMC increased the plant height, root length, fresh weight, and dry weight by 39.40%, 93.31%, 161.14%, and 163.57%, respectively. Conclusion We successfully identified five Actinomycetes strains capable of secreting protease, producing siderophores, and fixing nitrogen. The SMC constructed from these strains significantly enhances the growth of both maize and soybean. This study offers promising microbial resources for the development of efficient and environmentally friendly biofertilizers.