[Objective] To investigate the metabolite differences among different varieties and grades of flue-cured tobacco, as well as the impacts of nicotine-degrading bacteria on the quality of different grades of tobacco leaves. [Methods] This study employed untargeted metabolomics to identify and analyze metabolites in fermented tobacco leaves of ‘Yunyan 87’ and ‘Yunyan 97’, specifically B2F and C2F grades, while also examining the influences of nicotine-degrading bacteria on the quality of different grades of flue-cured tobacco leaves. [Results] There were significant metabolite differences between tobacco leaves of different varieties and grades. A total of 131 differential metabolites were identified between the samples of ‘Yunyan 87’ and ‘Yunyan 97’, while 138 differential metabolites were identified between B2F and C2F grades. These differential metabolites mainly included amino acids, flavonoids, alkaloids, and their derivatives. Analysis of KEGG metabolic pathways and enrichment levels for differential metabolites across different varieties and grades all indicated that flavonoid biosynthesis pathways were the most prominent. Furthermore, this study successfully isolated two bacterial strains, Pseudomonas sp. TR9 and Pseudomonas sp. TR14, from tobacco-cultivated soil, both capable of utilizing nicotine as the sole carbon and nitrogen source. Inoculation of the strain combination into different grades of tobacco leaves significantly reduced the content of nicotine, protein, and starch in lower-grade tobacco leaves. [Conclusion] This study reveals the mechanism by which varieties and grades affect the quality of tobacco leaves through the flavonoid biosynthesis pathways, and verifies the improvement effects of nicotine-degrading bacteria on low-grade tobacco leaves, providing theoretical support for the improvement of flue-cured tobacco quality and the optimization of fermentation processes.

[Objective] As soil acidification in southwestern China becomes increasingly severe, the labile phosphorus pool is transformed into a non-labile phosphorus pool, which reduces the availability of soil phosphorus, affecting crop yield and wasting phosphate fertilizer resources. In this study, we prepared a biochar-immobilized phosphorus-solubilizing bacterial agent with biochar as the carrier and a strain capable solubilizing both organic phosphorus and inorganic phosphorus as the immobilized strain and then optimized the preparation conditions. Furthermore, this bacterial agent was evaluated in terms of the stability and the solubilizing effects on insoluble phosphorus. [Methods] Selective media were used for the isolation of phosphorus-solubilizing bacteria from plant rhizosphere soil. The molybdenum-antimony colorimetric method was employed to quantify the ability of bacteria to solubilize phosphorus. The bacterial strain was identified through physiological and biochemical tests and molecular biological analysis. The immobilized bacterial agent was prepared by the adsorption method, and the preparation conditions were optimized by single factor experiments. The prepared agent was characterized by Fourier transform infrared spectrometry and scanning electron microscopy. Furthermore, the metabolic spectrum of organic acids and phosphatase activity were qualitatively and quantitatively tested by HPLC and the fluorescence method, respectively. [Results] The strain Klebsiella sp. was isolated for immobilization, and its abilities to solubilize lecithin and tricalcium phosphate were 236.5 mg/L and 200.3 mg/L, respectively. Genome analysis showed that the strain N107 carried 27 genes related to organic and inorganic phosphorus solubilization. The optimized preparation conditions were biochar addition of 30.0 mg/mL, N107 inoculation amount of 6.0%, immobilization temperature of 30.0 ℃, and immobilization time of 12.0 h. The bacterial agent prepared under the optimal conditions increased the phosphorus-solubilizing capacity for lecithin and tricalcium phosphate by 24.0% and 22.5%, respectively, compared with the free bacterial strain. The biochar-immobilized phosphorus-solubilizing bacterial agent contained more oxygen-containing functional groups, compared with the original biochar, its total specific surface area and external surface area increased by 61.9% and 165.1%, respectively. The mechanism of phosphorus solubilization by the immobilized bacterial agent was preliminarily analyzed. The results showed that the levels of tartaric acid, citric acid, and total acids changed significantly and the activities of acid and alkaline phosphatases in the culture medium were effectively improved, although the types of organic acids secreted by the agent had no obvious changes. The structural equation model showed that pH value was closely related to phosphatase activity and organic acid content, and the immobilized bacterial agent can promote the activation of insoluble phosphorus by increasing phosphatase activity and organic acid content. [Conclusion] The immobilized phosphorus-solubilizing bacterial agent prepared in this study provides a good bioremediation material for the activation of insoluble phosphorus. This study provides an innovative perspective for developing green remediation strategies based on microbiomes.

[Objective] To develop a low-cost and highly sensitive endotoxin detection reagent and detection method with recombinant horseshoe crab factor C enzymogen (rFC). [Methods] The Bac-to-Bac baculovirus expression system was used to express rFC in Sf9 cells and the activity of rFC was measured by the end-point fluorescence assay with endotoxin. The conditions of protein expression were optimized, and ion exchange was used for crude enzyme separation. An endotoxin detection method with rFC based on end-point fluorescence assay was established after the reaction conditions were optimized. Furthermore, the established method was compared with the conventional limulus amebocyte lysate (LAL). [Results] The expression level of rFC was 110.42 mg/L, increasing by 4.75 times. The linear range of endotoxin detection was 0.005-1.000 EU/mL in 1 h, with a good linearity and the limit of detection being 0.005 EU/mL. The applicability rate of this method for actual samples was 92.45%. The consistency of the detection results was 83.67%, and 89.80% of the samples had consistent detection limits with LAL. [Conclusion] This study achieves the efficient expression of rFC and establishes an endotoxin detection method with higher sensitivity than LAL, which has great potential for application.

[Objective] The soil in the vegetable plantation suffered from fertility degradation, pH decrease, and heavy metal leaching, necessitating the exploration of the mechanism by which composite bacterial agents regulate the bacterial community structure, nitrogen composition, and heavy metal availability in the vegetable plantation soil. [Methods] The heavy metal-resistant bacterial strains Ralstonia Bcul-1 (R-B) and Bacillus cellulasensis Zn-B (BC-Z) were prepared with biochar as an immobilized bacterial agent and then applied to the acidic soil (pH 5.6) of a vegetable plantation under long-term tomato rotation. High-throughput sequencing of soil bacteria and the determination of soil composition were conducted to analyze the bacterial diversity, soil pH, nitrogen-carbon content, and heavy metal chemical speciation, on the basis of which the effects of the biochar composite bacterial agent on the bacterial community structure, nitrogen-carbon supply, and heavy metal activity in the soil were analyzed. [Results] Biochar immobilization facilitated the growth of exogenous bacteria R-B and BC-Z in the vegetable plantation soil contaminated with heavy metals and maintained long-term coexistence of R-B and BC-Z with the original highly resistant Bacillus (10.18%-11.88%) in the soil. Accordingly, it effectively improved the bacterial community structure, adjusted the distribution of differential bacteria (biomarkers), and restoratively increased the relative abundance of abundant bacteria (such as Streptomyces, Geopathophilus, and Nocardioids) in the soil. In addition, soil bacterial genera, partial abundant bacteria, and the exogenous bacterial strain R-B were closely related to heavy metal chemical speciation and nitrogen-carbon components. The application of biochar bacterial agents (BI+R-B, BI+BC-Z, and BI+R-B+BC-Z) increased the pH, EC, total nitrogen, nitrate nitrogen, organic matter, and total organic carbon of the soil by up to 0.41, 20.74%, 18.96%, 24.77%, 10.26%, and 21.56%, respectively, while decreasing the ammonium nitrogen residue by 13.91%, maintaining the nitrogen-carbon supply capacity of the soil. BI+R-B and BI+R-B+BC-Z reduced the content of exchangeable, reducible, and oxidizable heavy metals (Cd, Cr, Pb, Cu, and Zn) by 0.18%-12.33%, but increased the residual content of these heavy metals by 0.16%-14.59%, effectively passivating heavy metals in the soil. [Conclusion] The biochar composite bacterial agent (BI+R-B+BC-Z) improved the bacterial community structure, promoted R-B growth, increased the abundance of abundant bacteria, and maintained the long-term coexistence of exogenous bacteria R-B and BC-Z with the original highly resistant Bacillus in the vegetable plantation soil with heavy metal compound pollution. Moreover, it increased soil pH, EC, total nitrogen, nitrate nitrogen, total organic carbon, and organic matter, while reducing ammonium nitrogen residue and passivating soil heavy metals (Cd, Pb, and Cu). Therefore, it effectively regulated the bacterial community activity, exogenous bifunctional bacterial growth, nitrogen-carbon supply, pH, and heavy metal chemical speciation, with the potential to maintain the fertilizer supply capacity and control heavy metal compound pollution of vegetable plantation soil.

Monascus, as a genus of edible fungi used in fermentation, are widely used in various industries such as wine making, food colorants, and pharmaceuticals due to their abundant secondary metabolites. McrA, a global regulator discovered in Aspergillus nidulans, has the function of regulating the growth and secondary metabolism of filamentous fungi. We had identified and cloned mcrA in Monascus purpureus in the previous study. [Objective] On the basis of transcriptome analysis, we mined the differentially expressed genes (DEGs) of ΔmcrA and trpC:mcrA strains to explore the function of mcrA. [Methods] The knockout strain ΔmcrA and overexpression strain trpC:mcrA of M. purpureus were constructed by homologous recombination. The colonies and microscopic morphology on different media were observed. The yields of Monascus pigments and citrinin were determined. The metabolic pathways involving DEGs were analyzed by transcriptome sequencing. [Results] The yields of Monascus pigments and citrinin of ΔmcrA decreased. Transcriptome sequencing results showed that the ΔmcrA strain up-regulated 111 genes and down-regulated 47 genes. The metabolic pathways involving the DEGs of ΔmcrA were mainly glycolysis, pyruvate metabolism, fatty acid synthesis, tyrosine metabolism and so on. The trpC:mcrA strain up-regulated 1 199 genes and down-regulated 867 genes. The main metabolic pathways involving the DEGs of trpC:mcrA were tryptophan metabolism, sucrose and starch metabolism, arginine and proline metabolism, fatty acid degradation, etc. [Conclusion] McrA is a global transcriptional regulator, and the knockout and overexpression of its gene will affect carbohydrate, lipid, and amino acid-related metabolic pathways, thus affecting the production of secondary metabolites.

[Objective] To investigate the interactions between coral-associated Symbiodiniaceae and bacteria in mediating heat stress adaptation of corals. [Methods] Using Pocillopora damicornis harbouring distinct Symbiodiniaceae clades, we performed a laboratory-controlled heat stress simulation experiment to examine the dynamics of symbiotic bacterial community shifts via 16S rRNA gene amplicon sequencing. [Results] Bacterial alpha diversity exhibited a transient increase during the initial stress, followed by a significant decrease under prolonged stress, in P. damicornis harbouring clade C (Cladocopium spp.) or clade D (Durusdinium spp.) algal symbionts (i.e., PdC versus PdD holobionts). Compared with PdD, PdC demonstrated enhanced bacterial community shifts, alongside progressively diminished network stability and complexity with prolonged heat stress. Analysis of bacterial abundance at the class level revealed divergent trajectories of the two holobionts, with the abundance of Alphaproteobacteria increasing in both PdC and PdD, whereas that of Cyanobacteriota increasing in PdC but decreasing in PdD over the course of the experiment. During the later stage of heat stress, Cladocopium spp. in PdC showed increased sensitivity, coinciding with the enrichment of potentially opportunistic pathogens, whereas Durusdinium spp. in PdD were thermotolerant, coinciding with elevated abundance of bacteria possibly involved in photosynthesis, quorum sensing, calcification, and ABC transport. [Conclusion] These findings suggest that different clades of Symbiodiniaceae might interact with bacteria to differentially regulate the P. damicornis response to heat stress. Thermal sensitive Cladocopium spp., combined with the proliferation of potential opportunistic pathogens, may exacerbate the risk of thermal bleaching in PdC, whereas resilience could be strengthened in PdD via thermotolerant Durusdinium spp. coordinating with beneficial bacteria with supportive metabolic potential (e.g., photosynthesis, calcification, and quorum sensing). This algal-bacterial interaction mode provides critical insights into the microbially-mediated thermal bleaching mechanisms and an important reference for the practice of reef restoration in the context of global climate change.

Numb-associated kinases (NAKs) are a family of evolutionarily conserved serine/threonine kinases, encompassing adaptor-associated kinase 1 (AAK1), cyclin G-associated kinase (GAK), bone morphogenetic protein 2-inducible kinase (BMP2K), and serine/threonine kinase 16 (STK16). NAKs are widely involved in various physiological processes, such as endocytosis, intracellular transport, cell differentiation, autophagy, and signal transduction. In recent years, studies have shown that NAKs play a key role in different life cycle stages including virus entry, assembly, release, and immune escape of various viruses. Furthermore, small-molecule inhibitors targeting NAKs have been applied in clinical research and treatment of related physiological or viral infectious diseases. This article systematically reviews the primary physiological functions of NAKs and their roles in viral infection, aiming to provide a theoretical foundation for elucidating the pathogenic mechanisms of viruses and developing novel therapeutic drugs targeting NAKs.

Citrus reticulata cv. Gonggan in Deqing is a national geographical indication product of China, and its quality formation is intricately linked to microbial ecology. In-depth exploration of the microbial resources associated with Gonggan is of great significance for enhancing the quality and increasing the yield of this fruit. [Objective] To isolate and culture bacteria from the rhizosphere soil and phyllosphere surface of Gonggan in Deqing and systematically evaluate their plant growth-promoting functions, thus providing applicable bacterial resources for the eco-friendly cultivation of Gonggan in Deqing. [Methods] Samples of rhizosphere soil and leaves of Gonggan in Deqing were collected. Bacteria within the samples were isolated via the dilution plating method. Bacterial strains were identified based on the homology of their 16S rRNA gene sequences. The functional media for phosphate solubilization, potassium release, nitrogen fixation, and siderophore production were used to evaluate the plant growth-promoting ability of bacterial strains, and the strains with strong plant growth-promoting functions were screened out. Ultimately, pot experiments were conducted to validate the plant growth-promoting functions of the selected strains. [Results] A total of 240 bacterial isolates were obtained, including 96 strains from the rhizosphere soil and 144 strains from the phyllosphere. These strains belonged to 51 genera, 29 families of 4 phyla, where Pseudomonadota was the dominant phylum and Bacillaceae was the dominant family. Functional characterization revealed that 230 (95.83%) strains exhibited at least one plant growth-promoting function, while 123 strains (51.25%) possessed three or more such functions. Given the extended growth cycle of Gonggan in Deqing, we utilized tomato as a model plant to evaluate the plant growth-promoting functions of eight bacterial strains possessing at least two functions through pot experiments. The results demonstrated that the tested strains significantly increased both plant height and fresh weight of tomato plants. [Conclusion] The rhizosphere soil and phyllosphere of Gonggan in Deqing harbor abundant functional microbial resources. This study successfully screened multiple strains of plant growth-promoting bacteria, and pot experiments demonstrated their significant ability to promote tomato growth. These findings provide a solid theoretical foundation for exploiting microbial resources of Gonggan in Deqing and advancing eco-friendly cultivation practices.

α-ketoglutarate is an important short-chain organic acid that is widely used in various fields such as food, medicine, cosmetics, and animal feed. However, the efficiency of producing α-ketoglutarate through biological fermentation remains to be improved, primarily due to the limitations in the synthetic capacity of microbial metabolic pathways. [Objective] To address the above issues, we developed an engineered Escherichia coli that can efficiently produce α-ketoglutarate, thereby providing theoretical support for the large-scale production of α-ketoglutarate in the future. [Methods] We employed an efficient approach combining rational and irrational modifications to overcome the constraints of endogenous metabolic pathways and enhance the biosynthesis efficiency of α-ketoglutarate. [Results] The oxidative TCA pathway was reconstructed to improve α-ketoglutarate production through expressing pyruvate carboxylase, citrate synthase, aconitase, and isocitrate dehydrogenase. The metabolic network for α-ketoglutarate biosynthesis was irrationally optimized and strengthened to enhance its biosynthesis capability by atmospheric pressure room temperature plasma mutagenesis. To improve the supply efficiency of the precursor for α-ketoglutarate biosynthesis, we reduced the dissipation of carbon flux in the pyruvate node by knocking out genes related to the accumulation of lactate, acetate, and formate. Furthermore, we knocked out the genes related to the degradation pathway of α-ketoglutarate to achieve the retention of carbon flux at α-ketoglutarate node and improve its production. Through the optimization of fermentation conditions, the fermentation in a 5 L fermenter with the engineered strain E. coli KA29 achieved the α-ketoglutarate titer, yield, and productivity of 28.7 g/L, 0.29 g/g, and 0.48 g/(L·h), respectively. [Conclusion] The research strategies mentioned above lay a foundation for the development and application of strains with high production of α-ketoglutarate and provide a reference for metabolic engineering to produce other organic acids.

[Objective] Birds, with unique life history characteristics, are ideal models for studying gut microorganisms. The niche overlap between wild birds and poultry increased the risk of interactive transmission of pathogens. This study focused on the community characteristics of gut fungi and pathogens in wild birds (crested myna, tundra swan, and common coot) and sympatric poultry (domestic duck and domestic chicken) in Chaohu Lake. [Methods] High-throughput sequencing (Illumina MiSeq) was employed to analyze the fungal communities in guts of wild birds and sympatric poultry in Chaohu Lake of China, and the characteristics of gut pathogens of each species were particularly studied. [Results] The gut fungal diversity of domestic duck and common coot was significantly higher than that of domestic chicken, crested myna, and tundra swan. There were significant differences in gut fungal community composition among different species. Due to grain-based diets, the guts of poultry were significantly enriched with the fungal taxa related to grain degradation, such as Ascomycota, Mortierellomycota, and Kazachstania. Tundra swan is herbivorous waterfowl. The genus Cladosporium, efficient plant-degrading fungi, dominated in the gut of tundra swan. The gut of tundra swan maintained higher relative abundance of plant saprotroph. The fungal community assembly in guts of wild birds was dominated by deterministic processes, which indicated that wild birds had a stronger gut filtering capacity. In addition, wild birds had lower diversity and relative abundance of pathogens. [Conclusion] The characteristics of gut fungal communities in wild birds and domestic poultry showed significant host specificity. Due to grain-based diets, the guts of poultry were significantly enriched with fungal groups related to grain degradation. The guts of wild birds had a stronger filtering capacity, which reduced the diversity and relative abundance of pathogens.