Abstract:Pathogenic fungi can cause not only superficial tissue infections but also invasive infections in deep tissues and organs, especially in individuals with compromised immune systems or those who are hospitalized for extended periods. Recently, the extensive use of broad-spectrum antibiotics and immunosuppressive therapies has exacerbated the severity of fungal infections and the prevalence of drug resistance, presenting a formidable challenge to global public health. Proteomics, as an extension and complement to genomics and transcriptomics, allows the systematic analysis of biological functions and regulatory networks at the protein level, serving as a powerful tool for uncovering the molecular foundations of life processes. This review comprehensively summarizes the application of proteomic technologies in studying major human pathogenic fungi, such as Candida, Aspergillus, and Cryptococcus. It highlights key discoveries in areas like drug resistance mechanisms, virulence factors, host-pathogen interactions, and clinical diagnostic biomarkers. Finally, the review discusses the potential of advanced proteomic technologies like thermal proteome profiling in research on human pathogenic fungal infection, aiming to offer new insights and intervention strategies for the precise diagnosis and treatment of fungal infections.

Abstract:Ganoderma lingzhi is a medicinal fungus of significant pharmacological value. Its bioactive components, triterpenoids, exhibit diverse therapeutic properties such as antioxidant, antitumor, and immunomodulatory activities. This review summarizes the biosynthetic pathways of G. lucidum triterpenoids and the genes encoding key enzymes involved in these pathways, with a specialized focus on the regulatory role of dissolved oxygen (DO) in mycelial growth and metabolite synthesis during liquid fermentation. Studies demonstrate that DO modulates triterpenoid synthesis efficiency through redox homeostasis regulation, enzymatic activity enhancement, and signaling pathway activation. Furthermore, this article discusses advances in enhancing the volumetric oxygen transfer coefficient (K_La) via optimized fermentation parameters to achieve high-yield triterpenoid production. Future research should integrate gene-editing technologies (CRISPR/Cas9) and omics technology to elucidate the molecular mechanisms underlying oxygen-dependent triterpenoid biosynthesis and advance industrial-scale production.

Abstract:Methicillin-resistant Staphylococcus aureus (MRSA) has become a major global public health challenge due to its complex drug resistance mechanisms and widespread transmission. In recent years, single-antibiotic therapy has been proven ineffective in controlling MRSA infections, prompting researchers to explore multidimensional combined therapeutic strategies. This review systematically summarizes novel combined therapeutic strategies against MRSA, including antibiotic combinations, phage therapy, natural medicines, phototherapy, and nanotechnology. The mechanisms of action, latest research advances, and potential clinical applications of these strategies are discussed in detail. Studies have shown that synergistic effects among different antibacterial approaches can enhance the bactericidal efficacy, reduce the emergence of resistance, and improve clinical treatment outcomes. Additionally, pharmacokinetics/pharmacodynamics (PK/PD)-based dosing optimization and targeted personalized precision medicine approaches are becoming key directions for future development. With the progress in multidisciplinary integration, the treatment for MRSA infections is shifting towards precision treatment, personalized treatment, and combined therapy. Future research should focus on elucidating resistance mechanisms, optimizing combined therapeutic strategies, and translating basic research into clinical applications to develop more effective and safer treatment solutions.

Abstract:Fructose is one of the important monosaccharides involved in metabolic processes in plants, animals, and microorganisms. Using fructose as the feedstock to synthesize value-added chemicals through microbial cell factories is the key to realize green biomanufacturing. As fructose is the second most utilized monosaccharide in carbohydrate metabolism, building microbial cell factories that can efficiently metabolize fructose is of great significance. However, the lower metabolic efficiency of fructose than that of glucose in most microorganisms limits the application of fructose. Recent advances in microbial metabolic mechanisms and modern biotechnology have driven significant improvements in fructose conversion efficiency while broadening the spectrum of value-added derivatives. This article reviews the fructose metabolic pathways in organisms and the major derivative products, summarizes the metabolic regulation strategies employed by different chassis cells for fructose utilization, and reviews the technical solutions and research advancements in synthesizing related products from fructose. Finally, this article discusses the current technical bottlenecks and outlines the future development directions in this field.

Abstract:Salt stress is one of the major constraints for plant growth and development, food security, and environmental protection. Plant growth-promoting rhizobacteria (PGPR), as beneficial microorganisms that colonize the rhizosphere soil of plant or attaches to plant roots, can improve plant salt tolerance and adaptation to saline environments. Moreover, they can remediate the salinized soil through interactions with host plants, which is an environmentally friendly and cost-effective alternative strategy to reduce the use of chemical fertilizers and pesticides in agricultural practice. The present study discusses the effects of salt stress on plants and strategies of plants to alleviate salt stress, dissects the mechanisms of plant salt tolerance enhancement and growth promotion by PGPR, and unveils the role of PGPR in improving the fertility of salinized soil. It is emphasized that new methods should be used to explore the mechanism of rhizosphere microbiome centered on excellent PGPR in the interaction between plants and saline-alkali soil in the future. Attention should be paid to the development and application of specialized microbial fertilizers in enhancing plant growth and salt tolerance in production practice. This study lays a theoretical foundation for enhancing plant growth and salt tolerance, improving the utilization of saline-alkali land, and restoring and reconstructing saline habitats.

Abstract:The carbohydrate esterase 4 (CE4) family comprises a group of enzymes with deacetylation activity, primarily catalyzing the hydrolysis of acetyl groups in carbohydrates and polysaccharides. This family includes various enzymes, such as chitin deacetylases, chitooligosaccharide deacetylases, peptidoglycan N-acetylglucosamine deacetylases, peptidoglycan N-acetylmuramic acid deacetylases, poly-β-1, 6-N-acetylglucosamine deacetylases, and acetylxylan esterases. These enzymes play crucial physiological roles in nature, particularly in plant cell wall degradation and chitin catabolism, and have potential as drug targets for bacterial and fungal pathogens. By deacetylation, CE4 family members alter the physicochemical properties and enhance the susceptibility of polysaccharides to further degradation by cellulases, hydrolases, and other enzymes. This process can thus significantly improve the efficiency of biomass conversion. However, the research on CE4 family members remains limited. The number of reported enzymes is relatively small, and those identified from different sources exhibit considerable variations in functional characteristics, spatial structures, and enzymatic properties. This review systematically summarizes recent advances in the enzymatic properties, three-dimensional structural features, catalytic mechanisms, and molecular engineering strategies of CE4 family members, aiming to provide theoretical insights and novel approaches for elucidating structure-function relationships and exploring molecular engineering strategies of these enzymes.

Abstract:Neoscytalidium dimidiatum is a fungus taxonomically belonging to Botryosphaeriaceae and closely related to many plant diseases. It is widely distributed worldwide, infecting fruit plants, shrubs, trees, and economic crops to cause diseases such as canker, dieback, wilt, leaf spot, root rot, and fruit rot. With the intensification of global warming, the prevalence and severity of diseases are increasing. This review summarizes the latest progress in the research on N. dimidiatum, including its taxonomy, host range, epidemiology, genomic characteristics, and interactions with hosts. These findings could provide a theoretical basis for the etiological research and comprehensive control of N. dimidiatum.

Abstract:Fermented foods are products formed through the growth and metabolism of a diverse range of microbial communities. Lactic acid bacteria (LAB), as the core functional bacteria for regulating the quality of fermented foods, dominate the formation and deterioration of quality through their acid production, aroma generation, and metabolic imbalances. Precision control technologies based on phage-host interactions offer emerging strategies for dynamically manipulating microbial community functions and have become a research hotspot for elucidating fermentation mechanisms and developing control tools. This review outlines the impact of the triggering of LAB phages on the quality of fermented foods and elucidates the conditions under which various environmental factors (such as pH, temperature, ultraviolet light, secondary metabolites, calcium ions, and magnesium ions) in the fermentation system induce LAB phages to shift to lytic phages. Furthermore, this review summarizes the mechanisms by which LAB phages affect hosts through regulating host metabolism and lysis and the impact of LAB phages on the quality of fermented foods. These mechanisms not only affect the growth of LAB and the generation of metabolic products but also may further impact key quality factors such as flavor and texture during the fermentation process.

Abstract:[Background] Parageobacillus thermoglucosidasius is a Gram-positive facultative anaerobic bacterium. Due to the fast growth, wide spectrum of substrate utilization, and low pollution during high-temperature fermentation, P. thermoglucosidasius has become an advantageous thermophilic chassis. However, the current gene editing method of P. thermoglucosidasius requires temperature switching to facilitate the loss of temperature-sensitive editing plasmids. This precludes the completion of editing at the optimal growth temperature, rendering the existing editing methods time-consuming and laborious. [Objective] To optimize the CRISPR type Ⅰ-B system, simplify the operation steps of gene editing, and shorten the editing cycle. [Methods] We first employed the constitutive promoter P_ldh to replace the inducible promoter for the transcription of guide RNA targeting the cleavage of the target DNA, thereby simplifying the operation process. Secondly, a plasmid backbone containing a temperature-insensitive replicon was used to carry the repair template, enabling the gene editing to be rapidly completed at the optimal growth temperature of 60 ℃. Finally, a modified stringent xylose-inducible promoter P_xylAst was knocked into the genome to drive the guide RNA targeting the editing plasmid, guiding the endogenous CRISPR system to achieve plasmid curing. [Results] The optimized editing plasmid efficiently completed the knockout of the target gene without the addition of inducers, achieving the knockout efficiency up to 100%. Moreover, the time required for gene knockout was significantly reduced from 2.5 d to 0.5 d. Subsequently, a stringent inducible promoter P_xylAst was developed, which further reduced the leakage expression by 57.39% compared with that of the xylose-inducible promoter P_xylA and shortened the time required for plasmid curing from 1.5 d to 0.5 d. [Conclusion] We overcame the drawbacks, such as cumbersome operation and inability to function optimally, of the existing editing methods by synergistically optimizing the promoter, replicon, and plasmid curing system. This advancement provides an efficient editing tool for genetic manipulation of thermophilic strains.

Abstract:[Background] Soil microorganisms are important drivers of material transformation in forest ecosystems, and Huoditang in Qinling Mountains is located in the transition zone between the northern subtropical and warm temperate zones of China, with diverse forest types. [Objective] To compare the soil microbial community structures among different plantations. [Methods] We compared the physicochemical characteristics of soils among three common plantations [Pinus armandii Franch. (PA), Picea wilsonii Mast. (PW), and Betula albosinensis Burkill. (BA)] in Huoditang in Qinling Mountains and analyzed the soil microbial community structures of the three plantations by high-throughput sequencing. [Results] The soils in the three plantations were acidic, with soil pH being the lowest in the BA plantation and the highest in the PA plantation. Soil organic matter (SOM) and total nitrogen (TN) were the highest in the BA plantation, moderate in the PA plantation, and the lowest in the PW plantation. Soil microbial α diversity was higher in the PA plantation. There were significant differences in soil microbial β diversity among the three plantations. The dominant phyla in the soils of the three plantations were Proteobacteria, Acidobacteriota, Basidiomycota, and Ascomycota. Linear discriminant analysis effect size (LEfSe) showed that the soil in the PA plantation was mainly enriched with Ascomycota and Chloroflexota, and that in the BA plantation was mainly enriched with Nitrospirota, Methylomirabilota, Cortinarius, and Laccaria. Functional prediction showed that carbon cycle-related functions (chemoheterotrophy, chitinolysis, etc.) were enriched in the PA plantation, while the BA plantation was enriched with more mycorrhiza fungi (ectomycorrhizal, endomycorrhizal, and ericoid mycorrhizal) as well as soil nitrogen cycle-related functions (e.g., nitrogen fixation). [Conclusion] Our study helps to understand the relationship between soil microorganisms and ecological functions in forest ecosystems. It provides a reference for the maintenance of ecological functions and the improvement of soil microbial community structure in Huoditang in Qinling Mountains.

Abstract:[Background] Exploring functional microbial resources is a crucial approach for advancing new quality productive forces, which necessitates innovative strategies to enhance the research and development efficiency. The prevention and control of chronic non-communicable diseases are key concerns in the grand health strategy, and the identification of elite microorganisms with antioxidant functions is an essential way to achieving this strategic goal. [Objective] To investigate whether fire disturbance enhances the antioxidant functions of soil microorganisms. [Methods] We simulated different intensities of fire disturbance (high-intensity group G, medium-intensity group Z, and low-intensity group D) and designed a simulated natural fire group (R) and a control group (CK). High-throughput sequencing was employed to continuously monitor changes in antioxidant activity-related genes and bacterial community composition in the upper (0–10 cm) and lower (10–20 cm) soil layers post fire disturbance. The culture method was adopted to count the culturable bacteria and actinomycetes with antioxidant activity in natural fire-affected soil, and the soil oxidative stress levels were monitored simultaneously. [Results] The simulated fire experiments revealed that the bacterial communities in the upper soil layer in groups G and Z demonstrated decreased Shannon indexes (P < 0.05) and structural changes (P=0.001). Group G showed increased abundance of Cupriavidus and Virgisporangium, while group Z exhibited increased abundance of Sphingomonadaceae. Group R demonstrated elevated abundance of Alphaproteobacteria and Pseudomonadaceae. Post fire disturbance, 22 bacterial genes in the upper soil layer showed upregulated abundance (P < 0.05), with variations in the number and type of affected genes across different fire intensities. Groups G and Z exhibited the most upregulated antioxidant activity-related genes (15), whereas group D had the fewest (6). Field monitoring of the natural fire-affected soil indicated a substantial increase in H₂O₂ content and a rise in counts of culturable bacteria and actinomycetes with antioxidant activity (P < 0.05), which remained higher than those in non-burned soil over time. [Conclusion] Fire disturbance sustainably elevates soil oxidative stress levels, alters bacterial community structure, and enhances the abundance of antioxidant activity-related genes and culturable bacteria/actinomycetes. These findings demonstrate that fire disturbance strategies can facilitate the discovery of high-quality antioxidant microbial resources in soil, offering a novel approach for functional microbial resource exploration.

Abstract:[Background] Anthracnose, a major disease attacking Camellia oleifera, is mainly caused by Colletotrichum fructicola. [Objective] To study the biological functions of the histone methyltransferase CfSet2, and provide a theoretical basis for deciphering the molecular mechanism underlying the pathogenicity of C. fructicola. [Methods] Overlap PCR was utilized to construct the CfSet2-deleted fragment, and the deletion mutant ΔCfset2 and complemented strain ΔCfset2-C were constructed by homologous recombination and PEG-mediated protoplast transformation. [Results] CfSet2 was located in the nucleus. Compared with the wild type and ΔCfset2-C, ΔCfset2 showcased significant decreases in the colony size, conidial production, and appressorium formation. The differences were extremely significant regarding turgor pressure, expression of melanin-related genes, H3K36me2 level, and endoplasmic reticulum sensitivity. Moreover, ΔCfset2 demonstrated diminished pathogenicity to C. oleifera leaves. [Conclusion] CfSet2 is involved in regulating the growth, development, expression of melanin-related genes, stress responses, and pathogenicity of C. fructicola.

Abstract:[Background] Plant endophytes and their hosts engage in diverse mutualistic interactions. [Objective] To investigate the composition and functional characteristics of endophytes in typical recalcitrant seeds of Pentadesma butyracea Sabine (Pb). [Methods] We employed the culture method to study the culturable endophytes in pre- and post-germination Pb seeds and examined the plant growth-promoting effects of the isolated endophytes. [Results] Compared with pre-germination seeds, the post-germination seeds exhibited significantly increased diversity (Shannon index) of endophytic bacteria and decreased diversity of endophytic fungi. The endophytic bacterial genera such as Bacillus, Brucella, and Chimaeribacte were isolated only from pre-germination seeds, whereas genera such as Pseudomonas, Acinetobacter, and Alcaligenes were isolated only from post-germination seeds. Endophytic bacteria of Stenotrophomonas demonstrated high abundance in both pre- and post-germination seeds. The endophytic fungi in pre-germination seeds were dominated by Neopestalotiopsis, Fusarium, and Pestalotiopsis, while those in post-germination seeds were dominated by Xenoacremonium, Pseudocosmospora, and Aureobasidium. Among the endophytic bacteria isolated from Pb seeds, we identified five strains with phosphorus- solubilizing ability, five strains with nitrogen-fixing ability, and seven strains with siderophore- producing function, of which five bacterial strains have both phosphorus-solubilizing and nitrogen-fixing abilities. Strain PBS3-48 (Stenotrophomonas), with both phosphorus- solubilizing and nitrogen-fixing functions, promoted the germination of Pb seed tissue blocks. [Conclusion] The results reveal significant disparities in the culturable endophytic microbiota between pre- and post-germination seeds, and multiple endophytic bacterial strains exhibiting plant growth-promoting effects are successfully isolated. This study provides insights into the characterization of endophytic microbiota in recalcitrant seeds and lays the groundwork for targeted exploration of functional endophyte resources.

Abstract:[Background] The application of fertilizers affects soil quality and sustainable agricultural development. [Objective] To screen and identify efficient potassium-solubilizing bacteria from the soil to provide elite strain resources for green and efficient agricultural production. [Methods] Selective media were used to screen potassium-solubilizing and cigar tobacco growth-promoting bacteria from the rhizosphere soil of cigar tobacco. The isolates were identified through 16S rRNA gene sequence analysis. Their physiological and biochemical characteristics were determined by indole test, methyl red test, Voges-Proskauer test, citrate test, hydrogen sulfide test, and Gram staining. Subsequently, the bacteria were characterized regarding their capabilities to solubilize potassium and phosphorus, fix nitrogen, produce indole-3-acetic acid, inhibit fungi, and tolerate salt, acids, alkali, and drought. The bacteria were then formulated into a microbial inoculum for field trials, and their effect on cigar tobacco growth was evaluated. [Results] Strain SCUEC18 was screened out and identified as Bacillus zanthoxyli. This strain exhibited strong potassium-solubilizing ability and had the capabilities of solubilizing phosphorus, fixing nitrogen, producing indole-3-acetic acid (IAA), and inhibiting Alternaria solani and Fusarium oxysporum. Moreover, it can tolerate alkali, salt, and drought to some extent. The results of field trials showed that compared with the treatment without the inoculum of B. zanthoxyli SCUEC18, the root irrigation with this strain increased the growth indices such as plant height, stem girth, number of leaves, leaf length, and leaf width of cigar tobacco. The results suggested that SCUEC18 had a growth-promoting effect on cigar tobacco. [Conclusion] The potassium-solubilizing and growth-promoting bacterium, Bacillus zanthoxyli SCUEC18, was screened out, serving as elite germplasm for solving the insufficient potassium and phosphorus in the soil for plant utilization.

Abstract:[Background] Astragalus membranaceus var. mongholicus is a genuine medicinal herb in Longxi County, mostly grown in arid and infertile soil. The growth environment determines that the endogenous nitrogen-fixing bacteria of A. membranaceus var. mongholicus have strong stress resistance. [Objective] To explore the endophytic nitrogen-fixing bacterial resources of the new lines of A. membranaceus var. mongholicus, screen strains for efficient joint nitrogen fixation with wheat, mitigate the effects of continuous cropping obstacles on wheat, and reduce the application of chemical fertilizers. [Methods] The seedlings of two A. membranaceus var. mongholicus lines ("Mengqi 07-22-1" and "Mengqi 07-22-2") bred in Longxi, Gansu were used for the isolation of endophytic bacteria by tissue milling and plate streaking. The Ashby solid medium was used for screening of nitrogen-fixing bacteria with full colonies. Strain identification was performed by colony morphology observation combined with 16S rRNA gene sequence analysis. Multifunctional identification media were used to determine the nitrogen-fixing, phosphorus-solubilizing, potassium-solubilizing, indole-3-acetic acid (IAA)- producing, and siderophore-synthesizing abilities of nitrogen-fixing bacteria. Finally, the ability of nitrogen-fixing bacteria to promote the growth of wheat in continuous cropping was tested by pot experiments with and without the application of organic fertilizers. [Results] Two and three excellent strains of nitrogen-fixing bacteria were obtained from "Mengqi 07-22-1" and "Mengqi 07-22-2", respectively. Strains G2B58, G2B59, G2B65, G1B66, and G1B89 were identified as Shinella kummerowiae, Agrobacterium radiobacter, Rhizobium sphaerophysae, Bosea vestrisii, and Mycobacterium frederiksbergens, respectively. Five strains of endophytic nitrogen-fixing bacteria were screened out for their functions of nitrogen fixation, phosphorus solubilization, potassium solubilization, IAA production, and siderophore production. Pot experiments with wheat seedlings in continuous cropping showed that the inoculation with G2B58, G2B59, and mixed strains promoted wheat growth. The plant height, stem diameter, fresh weight per plant, and dry weight per plant were significantly increased by 11.65%, 9.61%, 13.24%, and 18.92%, respectively, in the G2B58 treatment under fertilization. [Conclusion] All the five strains of endophytic nitrogen-fixing bacteria demonstrate good plant growth-promoting properties, providing high-quality strain resources for the development of microbial agents. Inoculation with G2B58 has the best plant growth-promoting effect and significantly improves the growth indexes of wheat seedlings in continuous cropping. In the case of applying microbial agents in the soil with continuous cropping, moderate application of organic fertilizer can enhance the plant growth-promoting effects of the endophytes of A. membranaceus var. mongholicus.

Abstract:[Background] Endophytic bacteria hold potential application value in the biocontrol of plant diseases. [Objective] To isolate and screen endophytic bacteria inhibiting Botrytis cinerea (the pathogen of ginseng gray mold) and Alternaria alternata (the pathogen of ginseng black spot) and optimize the culture conditions of the endophytic bacteria by response surface methodology. [Methods] Endophytic bacteria were isolated from adventitious roots of ginseng by the tissue culture method. The isolated bacteria were identified by morphological observation, physiological and biochemical assays, and molecular biological techniques. The culture conditions of the endophytic bacteria were optimized by single factor experiments, Plackett-Burman design, and Box-Behnken design. [Results] The identified strain G was Bacillus altitudinis, with GenBank accession number PQ498692. The optimized culture conditions for strain G were as follows: beef extract 0.70%, yeast extract 0.10%, MgSO₄ 0.07%, pH 7.5, temperature 36 ℃, rotation speed 180 r/min, and inoculum volume 5.0%. The OD₆₀₀ value of strain G cultured under the optimal conditions was 2.59±0.01, and the effective colony count was (1.96±0.14)×10⁹ CFU/mL. Strain G cultured under the optimal conditions exhibited the inhibition rates of (80.12±1.69)% against mycelia of B. cinerea and (67.57±1.27)% against mycelia of A. alternata. In in vitro experiments, strain G showed inhibition rates of (73.35±1.99)% and (66.93±11.52)% (P < 0.05) against ginseng gray mold and ginseng black spot, respectively. In pot experiments, strain G demonstrated the inhibition rates of (59.86±1.91)% and (74.45±8.60)% (P < 0.05) against ginseng gray mold and ginseng black spot, respectively. [Conclusion] B. altitudinis strain G, an endophyte strain of ginseng, has inhibitory effects on the pathogens causing ginseng gray mold and ginseng black spot, showcasing development and application value in the prevention and treatment of the two diseases.

Abstract:[Background] Morchella is a rare fungus that is both edible and medicinal. These mushrooms can only grow in soil, which may contain bacteria that influence their development. [Objective] To isolate bacteria that enhance the growth of Morchella and to optimize the culture conditions for creating microbial agents that can help overcome the continuous cropping obstacle of Morchella. [Methods] The bacteria promoting the mycelial growth and sclerotium formation of Morchella were isolated from the soil and fruiting bodies of Morchella. The isolates were identified based on morphological traits, physiological and biochemical properties, and 16S rRNA gene sequences. To identify the components of the culture medium and optimize the culture conditions, we conducted both single-factor experiments and orthogonal experiments. The cultivation experiments were conducted to clarify the effects of the strain on the mycelial growth and sclerotium formation of Morchella eximia KS5 and determine its appropriate concentration. [Results] The isolated strain T3 was identified as Peribacillus simplex. The optimal concentration of T3 for enhancing the mycelial growth and sclerotium formation of KS5 was 4×10³ CFU/mL. The ideal culture medium for T3 consisted of 12 g/L tryptone, 6.25 g/L yeast extract powder, and 5 mg/L ferrous sulfate. The optimal culture conditions were initial pH 7.5, 25 ℃, a liquid loading volume of 30 mL in a 250-mL flask, and a rotation speed of 200 r/min. The co-culture of T3 added at an amount of 6.67×10⁴ CFU/g with KS5 significantly increased the mycelial growth rate by 10.53% compared with the control. When the addition amount of T3 was 6.67×10⁵ CFU/g, the number of sclerotia formed by KS5 was significantly higher than that of the control. [Conclusion] Strain T3 at an appropriate concentration demonstrates a clear promoting effect on the mycelial growth and sclerotium formation of Morchella, which provides technical support for the development of microbial agents promoting Morchella growth.

Abstract:[Background] Ethyl acetate is a crucial aroma component in Maotai-flavor Baijiu, and its content directly impacts the flavor characteristics. [Objective] To overcome the functional degradation of single strains for enhancing ethyl acetate synthesis in the practical application, we employed directional domestication to enhance the ethyl acetate synthesis of microorganisms at the community level. [Methods] We thoroughly analyzed the gene expression abundance to identify the differential nutrients in substrates for microbial metabolism. According to the pathway of microbial synthesis of ethyl acetate, we determine three key carbon sources: glucose, fructose, and arabinose, which were then used for the directional domestication of the microbial community. [Results] The microbial biomass in groups S1 (1.37 mg/mL), S2 (1.40 mg/mL), and S3 (1.57 mg/mL) was lower than that in the CN control group (1.87 mg/mL). After 14 rounds of directional domestication, the microbial diversity showed a downward trend and the structure of the formed fungal community was similar. The relative abundance of Wickerhamomyces anomalus increased remarkably, surging from 0.04% to 65% and even higher (P < 0.05). The ability of fermentation microorganisms to synthesize ethyl acetate decreased by about 44% after continuous dilution and subculture. The microbial community after directional domestication can avoid the reduction of ethyl acetate synthesis and increase the ethyl acetate accumulation by 20%-70% without affecting ethanol production. The domesticated microbial community was then blended with the original Daqu for simulated fermentation. The results showed that the microbial fermentation agent prepared by the semi-synthetic method without microbial extraction (EG1) or the total synthesis method after extraction and mixing (EG2) increased the yield of ethyl acetate by more than 210% during the fermentation process. [Conclusion] The microbial community capable of efficiently synthesizing ethyl acetate was successfully constructed by the directional domestication strategy.

Abstract:[Background] Amylase-producing microorganisms may cause oyster sauce thinning, while their species and characteristics remain unclear. [Objective] To isolate and identify amylase-producing microorganisms associated with oyster sauce thinning, investigate their growth characteristics, and provide a theoretical basis for controlling these microorganisms during oyster sauce production. [Methods] Target strains were isolated from thinned oyster sauce by enrichment culture, transparent circle method, and re-inoculation experiments. Subsequently, the strains were identified based on molecular biological evidence and studied regarding their growth characteristics and heat tolerance. [Results] Two bacterial strains, named OYS1 and OYS3, were obtained. Through physiological and biochemical tests, 16S rRNA gene sequencing, and phylogenetic analysis, they were identified as Paenibacillus cookii and Bacillus velezensis, respectively. Both strains exhibited optimal growth at 37 ℃ and could grow at 50 ℃. The optimal pH values for the growth of strains OYS1 and OYS3 were 6.5 and 5.5, respectively, and both strains grew well at pH 5.0–7.0. Complete inhibition of strains OYS1 and OYS3 required 8% and 17% NaCl, respectively. Potassium sorbate at the concentration above 0.7 g/L effectively inhibited both strains. Both strains showed strong heat tolerance, surviving temperatures below 100 ℃ but being sterilized by the treatment at 110 ℃ for 5 min. [Conclusion] P. cookii and B. velezensis can produce amylase and cause oyster sauce thinning. Their growth can be inhibited by low temperatures, high salt, and preservatives, while the treatment at the temperatures above 110 ℃ achieves a good sterilization effect. This study provides guidance for optimizing oyster sauce production and sterilization process, ensuring product stability during the shelf life.

Abstract:[Background] The abuse of antibiotics has exacerbated the drug resistance of pathogens, and the research on the use of phages in the treatment of multidrug resistant pathogens has attracted extensive attention. [Objective] This study aims to isolate new Escherichia coli phages and give insights into the application of phage therapy in the treatment of foodborne E. coli infections. [Methods] With multidrug resistant E. coli 6-3 as the host bacterium, the double-layer agar plate method was employed to isolate phages from the sewage of the farm. Plaque counting was conducted to determine the optimal multiplicity of infection (MOI), one-step growth curve, host range, thermal stability and other biological characteristics of phages. Next-generation sequencing was employed to obtain the whole genome sequences of phages, and the genomic and comparative genomics analyses were performed. [Results] The E. coli phage EcoM_GaoY6-3C had an optimal MOI of 1, an incubation period of 5 min, and stable potency at -70 ℃ to 50 ℃ and pH 3.0–11.0. The results of host profiling showed that this phage could infect pathogenic E. coli O157:H7. The genome assay results revealed that its genome was double-stranded circular DNA, with a length of 171 703 bp and repeat sequences of 111 bp at both ends. The genome had the G+C content of 40.28% and included 91 protein-coding genes and 2 tRNA-coding genes. The comparative genomics analysis revealed that EcoM_GaoY6-3C was a novel E. coli phage. Preliminary application experiments indicated that EcoM_GaoY6-3C effectively killed host bacteria in milk at 4 ℃, demonstrating promising practical applications. [Conclusion] EcoM_GaoY6-3C is a new E. coli phage and can be used for food safety control.

Abstract:[Background] Japanese encephalitis virus (JEV) is a mosquito-borne zoonotic pathogen that induces severe neurological disorders, posing a substantial threat to human and animal health. In recent years, CRISPR/Cas9 gene editing technology has been extensively utilized in antiviral research, offering a novel approach for investigating host disease resistance mechanisms. DNAJB6, a prominent member of the heat shock protein 40 family, is integral to protein folding and cellular stress responses, exerting a significantly regulatory influence on viral replication through its molecular chaperone function. Studies have demonstrated an interaction between DNAJB6 and the NS3 protein of JEV via yeast two-hybrid screening. However, the impact of the host factor DNAJB6 on JEV replication in the PK15 cell line remains unclear. [Objective] To investigate the influence of DNAJB6 on JEV replication, we developed a pig kidney PK15 cell line (PK15-DNAJB6-KO) with knockout of DNAJB6 by CRISPR/Cas9 gene editing technology, thereby offering a novel research avenue for elucidating the molecular mechanisms underlying viral infection. [Methods] Initially, CRISPR/Cas9 technology was employed to engineer a lentiviral vector containing porcine DNAJB6 (LentiCRISPR-V2-DNAJB6-sgRNA). This vector was subsequently packaged into a lentivirus, which was then used to transfect PK15 cells. A monoclonal cell line deficient in DNAJB6 (PK15-DNAJB6-KO) was successfully established via puromycin selection and the limiting dilution assay. The knockout efficacy was assessed via Western blotting and sequencing. The replication of JEV in PK15-DNAJB6-KO and PK15 cells were compared by Western blotting, RT-qPCR, plaque assay, and indirect immunofluorescence assay. [Results] Sequencing data revealed an insertion of a single base in the exon of DNAJB6 in PK15-DNAJB6-KO monoclonal cells compared with PK15 cells. Western blotting confirmed the complete absence of DNAJB6 protein in PK15-DNAJB6-KO cells. Furthermore, the results from Western blotting, RT-qPCR, plaque assay, and indirect immunofluorescence assay indicated that JEV load was markedly elevated in PK15-DNAJB6-KO cells relative to PK15 cells. [Conclusion] We successfully construct a PK15 cell line with knockout of DNAJB6 and demonstrate that DNAJB6 plays an inhibitory role in JEV replication in PK15 cells.

Abstract:[Background] The porcine myxovirus resistance Ⅱ (Mx2) protein, a member of the Mx protein family, is an interferon-induced GTPase that plays a pivotal role in mediating the antiviral immune responses of the host. [Objective] To investigate the functions of porcine Mx2 protein, we aim to establish a PK-15 cell line stably expressing the porcine Mx2 protein by using a lentiviral vector system. [Methods] We designed specific primers based on the nucleotide sequence of the porcine Mx2 gene in GenBank. The target gene was amplified, and the recombinant lentivirus plasmid was constructed. Then, HEK293T cells were transfected with a three-plasmid system for lentivirus packaging and the obtained recombinant lentivirus was used to infect PK-15 cells. After puromycin screening, the PK-15 cell line stably expressing porcine Mx2 protein was obtained, and its viability was detected by the CCK-8 method. The expression level of porcine Mx2 protein in the constructed cell line was analyzed by Western blotting, and the effect of the cell line on the replication of pseudorabies virus (PRV) was evaluated by real-time fluorescent quantitative PCR, Western blotting, and tissue culture infectious dose 50% (TCID₅₀). [Results] The PK-15 cell line expressing porcine Mx2 protein was successfully constructed. Compared with the wild-type PK-15 cells, the constructed cell line showed significantly up-regulated expression of porcine Mx2 protein and inhibited the replication of PRV. [Conclusion] This cell line serves as a valuable tool for delving into the functions and antiviral mechanism of porcine Mx2 protein.

Abstract:[Background] Autophagy is essential in the interaction between intracellular bacteria and macrophages, and its role in pathogen infections have garnered great attention. Edwardsiella tarda is a major facultative intracellular pathogen with a broad host range and it can replicate and survive in host macrophages. However, the precise role of autophagy in the E. tarda-macrophage interaction remains unclear. [Objective] To determine whether E. tarda infection can induce the autophagy of RAW264.7 macrophages and establish a cell model of autophagy for exploring the effect of autophagy on the inflammatory response of macrophages infected by E. tarda, thus providing new ideas for the prevention and treatment of Edwardsiellasis. [Methods] LC3, the marker of autophagy, was detected by immunofluorescence and immunoblotting to evaluate the autophagy activity. Autophagosome formation in the cells transfected with the GFP-LC3 plasmid was observed. Cells were pretreated with different concentrations of the autophagy inhibitor 3-methyladenine (3-MA) and activator rapamycin (Rapa), and the optimal working concentrations and time were determined with the cell viability and LC3-Ⅱ/Ⅰ ratio as indicators. The labeling with the GFP-RFP-LC3 fusion protein was employed to monitor the autophagy flow. The cell model of autophagy was treated with 3-MA and Rapa, and the dynamics of inflammatory cytokines secreted by infected cells were measured. The levels of tumor necrosis factor-α (TNF-α), nitric oxide (NO), and reactive oxygen species (ROS) were determined by ELISA, the nitrate reductase method, and the DCFH-DA fluorescent probe, respectively. RT-qPCR was conducted to determine the mRNA levels of other inflammatory factors. [Results] The expression level of LC3 was upregulated in the cells infected with E. tarda. LC3 aggregated in dots, with significantly increased fluorescent spots. E. tarda recruited a large number of LC3 molecules to localize and form autophagosomes. The constructed autophagy model showed that pretreatment of cells with multiplicity of infection=100, 3-MA (5 mmol/L) and Rapa (200 nmol/L) for 2 h and 6 h, respectively, could achieve good autophagy intervention effects. E. tarda induced the autophagy of RAW264.7 macrophages in dose-dependent and time-dependent manners. As the time after infection extended, the relative expression level of LC3-Ⅱ gradually rose and reached the peak at 9 h. Autolysosomes began to be formed at the time point of 12 h after infection and the autophagy flow began to enter the late stage at 24 h. The secretion of inflammatory factors by E. tarda-infected macrophages were dynamically measured after autophagy intervention. The results showed that autophagy exerted varied effects on various inflammatory factors of macrophages at the early stage after infection. However, the effects tended to be consistent at 24 h after infection. That is, 3-MA treatment significantly downregulated the secretion of inflammatory factors, while Rapa had the opposite effect. [Conclusion] E. tarda infection could trigger autophagy of RAW264.7 cells and induced complete autophagy flow. Our findings provide preliminary evidence that autophagy is involved in the regulation of macrophage inflammatory response activated by E. tarda, promoting the expression of various inflammatory factors at the late stage after infection.

Abstract:[Background] Since 2016, gout caused by goose astrovirus (GAstV) infection in goslings has spread extensively among goose farms throughout China. [Objective] To investigate the pathogenicity of two endemic GAstV strains isolated and identified, thereby providing foundational data for further research on GAstV. [Methods] Two tissue samples, tested positive for goose astrovirus type 1 (GAstV-1) and goose astrovirus type 2 (GAstV-2) via PCR, were selected for further analysis. Goose embryos were used for virus isolation. Following sequencing verification, the whole genome sequences of the two GAstV strains were amplified. The pathogenicity of the two isolated GAstV strains was evaluated by an animal regression test. The clinical symptoms, pathological manifestations during necropsy, and histopathological changes of goslings were observed after intramuscular injection of GAstV suspensions. [Results] In this study, one isolate each of GAstV-1 and GAstV-2 were successfully obtained and named GDYJ-21-01 (GenBank: OP776630) and GDZJ-21-01 (GenBank: OP776629), respectively. Whole genome sequence alignment results indicated that GDYJ-21-01 clustered within the GAstV-1 branch and shared 89.70% to 98.60% similarity with GAstV-1. GDZJ-21-01, on the other hand, was situated within the GAstV-2 branch and shared 97.10% to 99.30% similarity with GAstV-2. After GAstV infection, goslings showed signs of emaciation. Necropsy of deceased goslings revealed urate deposits in their organs. Histopathological examinations demonstrated small focal necrosis of myocardial fibers, congestion and dilation of hepatic sinusoids in the liver, splenic congestion, as well as disruption of the original structure of some renal tubules, renal tubule dilation, and mild hyperplasia of the interstitial connective tissue in the kidney tissue. [Conclusion] We successfully isolated the GAstV-1 strain GDYJ-21-01 and the GAstV-2 strain GDZJ-21-01. Both strains were proven capable of infecting goslings and inducing gout.

Abstract:[Background] Riemerella anatipestifer (RA) causes infectious serositis in poultry, posing a serious threat to the global poultry industry. The continuous evolution of its serotypes has attracted widespread attention. [Objective] To investigate the serotype, antibiotic resistance, virulence, and whole genome sequence information of strain RAf950. [Methods] We employed agar gel diffusion, antimicrobial susceptibility tests, and animal experiments to examine the serotype, antibiotic resistance, and virulence, respectively, of strain RAf950. Illumina sequencing and assembly were carried out to obtain the genome of the strain. GeneMarkS and other software were used to analyze the components of the genome. Finally, the genome sequence was compared with the known sequences in NR, COG and other databases to predict gene functions. [Results] RAf950 was classified as a serotype 5 strain, which was sensitive to 9 antibiotics (including azithromycin, florfenicol, and cephalexin) and resistant to 14 antibiotics (including polymyxin B, compound sulfamethoxazole, amikacin, tigecycline, and enrofloxacin). The median lethal dose (LD₅₀) of RAf950 to 14-day-old Muscovy ducks was 6.6×10⁵ CFU. The genome of the strain was 2 115 628 bp in length, containing 1 977 coding genes, of which 50 antibiotic resistance genes, 2 virulence genes, and 3 mutated genes leading to enhanced pathogenicity were annotated. [Conclusion] We analyze the antibiotic resistance, virulence, and whole genome sequence of a serotype 5 strain of RA, providing a reference for further research on the antibiotic resistance and pathogenic mechanisms of RA.

Abstract:[Background] Citrobacter freundii is a zoonotic opportunistic pathogen, and the emergence of multidrug-resistant strains poses a major threat to global public health. In September 2024, the Affiliated Hospital of Yangzhou University isolated a bacterial strain from pus of human palm soft tissue infection. [Objective] To identify the isolate, explore its biological characteristics, and conduct whole genome sequencing. [Methods] Gram staining, 16S rRNA gene sequencing, and whole-genome sequencing were performed for the identification. The biological characteristics were explored through growth curve analysis, antimicrobial susceptibility testing, and pathogenicity assessment. Illumina PE150 and PacBio were used for whole genome sequencing, and sequence analysis was carried out with the aid of various bioinformatics tools. The expression of iron uptake-related virulence factors under iron restriction conditions was determined by RT-qPCR. [Results] The isolated strain was identified as C. freundii. Growth curve analysis revealed that the strain entered the exponential phase at the time point of 1 h, reaching peak OD₅₉₅ at the time point of 7 h. Antimicrobial susceptibility testing showed that the strain was resistant to eight antimicrobials (including cefradine, amikacin, and gentamicin) and sensitive to eleven antimicrobials (including carbenicillin, cefazolin, and ceftriaxone). Pathogenicity assessment demonstrated that the strain was highly virulent in the zebrafish infection model. The genome of the strain was 4 938 581 bp in length, with the G+C content of 51.77%, encoding 4 576 predicted genes, which included 24 antimicrobial resistance genes and 44 virulence factors. The genome of this bacterium was predicted to contain 27 mobile genetic elements, 13 regulatory RNA elements, and 27 strain-specific genes. RT-qPCR revealed that the iron uptake-related virulence factors of this strain were significantly upregulated under iron restriction conditions. [Conclusion] The strain isolated from pus of human palm soft tissue infection was C. freundii. This bacterium exhibits strong antimicrobial resistance and high pathogenicity, and its genome harbors multiple antimicrobial resistance genes and virulence factors, which underscores the need for attention in clinical practice.

Abstract:[Background] Actinomycins are a class of chromopeptide lactones with significant anticancer and antibacterial activities. Among them, actinomycin X2 stands out due to its excellent biological activity, showing great potential for applications in medicine, dyes, and agriculture. However, among the reported actinomycin-producing strains, those primarily producing actinomycin X2 are rare, and there are few reports on using genetic engineering methods to increase the yield of actinomycin X2. [Objective] To discover new strains primarily producing actinomycin X2 and to employ genetic engineering strategies to enhance the yield of actinomycin X2. [Methods] AntiSMASH predicted that Streptomyces xanthochromogenes JCM 4594 had the potential to produce actinomycin X2, which was further confirmed by high-resolution electrospray ionization mass spectrometry (HRESIMS) and nuclear magnetic resonance (NMR) techniques. We enhanced the yield of actinomycin X2 by optimizing the culture medium, overexpressing acxD and acxO, and inserting a strong promoter kasOp*-SF14. [Results] Culture medium screening, fermentation, and metabolite analysis confirmed that the primary component of the actinomycin-like compounds produced by S. xanthochromogenes JCM 4594 was actinomycin X2. After insertion of a bidirectional strong promoter into the actinomycin X2 biosynthesis gene cluster, the yield of actinomycin X2 increased by seven folds, reaching 12.6 mg/L. After overexpression of acxO and acxD, the yields increased by 21.5 and 23.8 folds, reaching 38.7 mg/L and 43.8 mg/L, respectively. Additionally, for the first time, the inhibitory effect of actinomycin X2 on the human liver cancer cell line HuH-7 was reported, with an IC₅₀ value of 6.8 nmol/L. [Conclusion] This study identified and characterized a new strain primarily producing actinomycin X2 and elucidated its biosynthetic gene cluster. Through genetic engineering, the yield of actinomycin X2 was significantly increased, demonstrating that high expression of core biosynthetic enzymes, positive regulatory factors, and important auxiliary proteins are effective strategies to enhance the yield of this compound. This study provides new elements, a new strain, and a theoretical foundation for constructing strains for actinomycin X2 production, which may facilitate the further application and development of actinomycin X2.

Abstract:[Background] The ocean, as a vast reservoir of biological resources, contains rich and diverse rare actinomycetes. Mining structurally novel natural products from the secondary metabolites of marine rare actinomycetes plays a crucial role in drug development and disease treatment. [Objective] To identify new species of marine rare actinomycetes and evaluate their potential for producing secondary metabolites. [Methods] Rare actinomycetes were identified based on 16S rRNA gene sequence similarity, average nucleotide identity (ANI), and digital DNA-DNA hybridization (dDDH). Their main metabolites were determined through fermentation medium optimization. [Results] A marine rare actinomycete strain, Actinoplanes sp. G11-F43, was identified. It exhibited the highest 16S rRNA gene sequence similarity (98.68%) with Actinoplanes xinjiangensis DSM 45184. The maximum ANI of this strain with the most similar reference strain, Actinoplanes couchii DSM 45050, was 86.87%, which is below the 95% threshold for new species identification, confirming this strain as a novel species. Similarly, the maximum dDDH value with the most similar reference strain, Actinoplanes derwentensis DSM 43941, was 31%, below the 70% threshold for new species identification. Through fermentation medium optimization, the strain was found to produce several polycyclic tetramate macrolactams with 5/5 bicyclic and 5/5/6 tricyclic structures. [Conclusion] This study successfully identified a novel marine rare actinomycete strain, Actinoplanes sp. G11-F43, enriching the marine microbial resources. Additionally, fermentation medium optimization enabled the production of multiple polycyclic tetramate macrolactams. This strain serves as a new producer for further studies on such bioactive natural products.

Abstract:[Background] Euchresta tubulosa Dunn, an ethnic medicinal herb with high reputation, being rich in alkaloids, flavonoids and other effective components. However, due to the limited wild resources and difficult artificial cultivation, this plant is facing resource depletion. Endophytic fungi in plants can produce the same or similar secondary metabolites as their hosts. The endophytic fungi in E. tubulosa are expected to be potential resources for the screening of similar natural products as their host and new lead compounds. [Objective] To study the activities and components of fermentation products of endophytic fungi in E. tubulosa and screen the high-activity fermentation products. [Methods] We employed tissue culture and rDNA ITS sequence analysis to isolate and identify the endophytic fungi in E. tubulosa. The inhibitory activities of the liquid fermentation broth of the endophytic fungi against Staphylococcus aureus, Escherichia coli, and Candida albicans were determined by the filter paper method, and the minimum inhibitory concentration (MIC) was determined. The endophytic fungal strains with strong activities were screened based on the scavenging abilities against (2,2-diphenyl-1-picrylhydrazyl, DPPH), hydroxyl, and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) free radicals and the inhibitory activities against α-glucosidase and α-amylase. The chemical components in the fermentation products of high-activity strains were analyzed by LC-MS. [Results] The antimicrobial, antioxidant, and glucose-lowering activities of secondary metabolites produced by seven fungal strains isolated from E. tubulosa were studied. The fermentation products of strains No.1 and No.4 had high activities, and they contained organic acids, flavonoids, terpenoids, phenylpropanoids, and alkaloids. [Conclusion] The endophytic fungi from E. tubulosa have good biological activities, which provides a basis for further separation of secondary metabolites and study of pharmacological effects.

Abstract:[Background] The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system is widely employed for genome editing in Escherichia coli. However, the editing speed of existing editing tools based on CRISPR-Cas9 remains to be accelerated. [Objective] To enable rapid iterative gene editing, it is necessary to optimize the operational steps of CRISPR-Cas9 editing tools and reduce processing time, thereby improving editing efficiency. [Methods] Firstly, the tool plasmids in the accelerated iterative gene editing (AIGE) system developed in this study used non-thermosensitive replication origins, which enabled Escherichia coli to be edited at optimal growth temperatures. Secondly, the CRISPR-Cas9 system was employed to target and cleave antibiotic resistance genes in sgRNA expression plasmids, permitting rapid iterative editing through sequential plasmid transformations. Finally, a homing endonuclease gene and its cognate recognition sequence were introduced, enabling single-step induction for simultaneous curing of both tool plasmids. [Results] Without any pre-modification of host genomes, the system achieved efficient iterative editing in E. coli K-12 strains BW25113, DB3.1, and Crooks strain ATCC 8739. The total editing time was shortened after optimization of the editing temperature to match the optimal temperature of E. coli. Meanwhile, post-editing plasmid-curing duration decreased from approximately 72 h to 30 h, with both tool plasmids efficiently eliminated via a single induction step. [Conclusion] AIGE accelerates the gene editing of E. coli by not limiting the culture temperature, iterative curing of sgRNA plasmids, and simplifying the previously reported two-step method of curing tool plasmid to one-step method, providing a rapid iterative gene editing platform for the genetic engineering of E. coli.

Abstract:[Background] Feline infectious peritonitis (FIP) caused by feline infectious peritonitis virus (FIPV) is a deadly disease with high mortality. Antibody-dependent enhancement (ADE) of infection is a prominent feature of this disease. [Objective] To establish ADE infection models in vivo and in vitro for the clinical isolate FIPV SD01. [Methods] We employed indirect immunofluorescence and RT-qPCR to compare the enhancement factor (EF) and expression levels of TNF-α and IL-6 in macrophages between different infection groups. The FIPV SD01-inactivated whole virus vaccine was used for vaccination of coronavirus-negative cats, which were then orally infected with 1×10⁶ tissue culture infectious dose 50% (TCID₅₀) FIPV SD01 after 42 days. The clinical symptoms, body temperature, pathological changes, peripheral blood lymphocyte count, and TNF-α and IL-6 expression levels were compared between different infection groups. [Results] The macrophages infected with the antibody-virus complex showed the EF greater than 10 and higher expression levels of TNF-α and IL-6 than the viral infection group (P < 0.01). The animal experiment showed that compared with the control infection group, the vaccination-challenge group exhibited typical infection enhancement, which was characterized by fever and lymphopenia within 2–4 days post challenge, severer systemic granulomatous lesions upon necropsy, and pronounced immunohistochemical changes. In addition, the expression levels of TNF-α and IL-6 were up-regulated (P < 0.01). [Conclusion] This study established ADE models in vivo and in vitro for the clinical isolate FIPV SD01, providing a model reference for research on the pathogenic mechanism and vaccine evaluation of FIPV.

Abstract:[Background] Lactic acid bacteria (LAB) are widely used in industrial production and medical fields due to their safety and probiotic properties. However, conventional LAB surface display systems still face challenges in terms of display efficiency, safety, and scope of application. [Objective] To establish a novel Lactococcus lactis surface display platform based on SpyTag/SpyCatcher (ST/SC). [Methods] Fistly, we employed homologous recombination to engineer the chassis strain NZ9000-SC. The binding ability of superfolder green fluorescent protein (sfGFP) and monomeric Cherry red fluorescent protein (mCherry) modified with ST at the C-terminus to NZ9000-SC was verified in vitro. Taking ST-sfGFP as a representative model, we systematically investigated critical parameters influencing display efficiency, including induction initiation phase, temporal induction window, inducer concentration gradient, and cross-linking conditions. Finally, we selected laccase (LA) from Bacillus vallismortis for surface display to evaluate the application potential of this novel display platform. [Results] The chassis strain NZ9000-SC for surface display was successfully established. The confocal laser scanning microscopy detected signals from both fluorescent proteins after coupling. By regulation of the inducible plasmid within NZ9000-SC, the expression of ST-sfGFP was controlled within the range of 0.4 μg/mL to 17.0 μg/mL. Moreover, the system could stably display ST-sfGFP under physiological conditions and different incubation conditions. Additionally, the growth of L. lactis ligated with ST-sfGFP on the surface was not affected. NZ9000-Spy-LA not only exhibited superior resistance to protease hydrolysis but also retained more than 65.0% of its initial activity in the fifth use. In a mouse model of intraperitoneal tetracycline treatment, gavage of the live LA-displayed L. lactis specifically reduced the tetracycline level in the intestinal tract without affecting the blood tetracycline level, outperforming gavage of the enzyme-cell mixture. [Conclusion] The L. lactis surface display platform based on Spy chemistry demonstrates efficient, safe, and universal enzyme immobilization, enabling probiotic functionalization with enhanced enzymatic stability. This strategy pioneers novel approaches for probiotic engineering.

Abstract:Microbiology is an irreplaceable professional core course for the cultivation of innovative talents majoring in life sciences in agricultural universities. To cultivate top-notch innovative talents who are knowledgeable about agriculture, have enthusiasm for farming and agriculture, the teaching team of Microbiology at Huazhong Agricultural University has actively explored the teaching reform of this course with a five-in-one approach (encompassing textbook compilation, curriculum design, teaching methods, assessment and evaluation, and teachers' ethics improvement), based on the cultivation needs regarding the talents' knowledge, ability, and value and the university characteristics. The specific reform measures included text compilation based on the educational goals, curriculum design integrating moral, intellectual, physical, aesthetic, and labor education, teaching method innovation in the context of all-round education, improvement of the evaluation system in multiple dimensions, and enhancement of the professional ethics and conduct of teachers to build a new type of teacher–student relationship. These measures give full play to the educational role of the Microbiology course and improve the quality of talent cultivation. This teaching reform can provide reference and implications for the course development at other universities.

Abstract:In the past two years, the rapid advancement of intelligent technology has significantly influenced various aspects of the society. The utilization of artificial intelligence (AI) in undergraduate education has become a widely discussed topic. Our team introduced the AI teaching assistant system into the Microbiology course to help students develop a self-assessment and learning system. By collaboratively utilizing knowledge graphs and goal graphs, students can progress through their learning in a goal-oriented manner and then perform self-assessment through questions linked to the relevant knowledge points. Furthermore, students engage in targeted learning with the AI teaching assistant and establishing connections among knowledge points on their own. In our practice, students exhibit an open and positive attitude toward the application of the intelligent system. With the support of the AI teaching assistant, the frequency of questions asked has increased, which promotes students' initiative and learning efficiency. Our work employed the AI teaching assistant in the teaching of Microbiology, enabling students to develop self-regulated learning skills within a digital environment. This paper aims to provide new ideas and methods for the application of intelligent technology in teaching.

Abstract:Comprehensive design-oriented experiments are a top priority in the experiment teaching for undergraduates and are one of the main measures to cultivate undergraduates' comprehensive practical and innovative abilities. However, currently, most comprehensive design-oriented experiments are designed by teachers, and students complete them according to predetermined steps, which is not conducive to the cultivation of students' ability of independent innovation. Taking the microbial detection project in the Environmental Microbiology Experiment course as an example, we guide students to independently select topics, design experimental schemes, conduct experiments following standard procedures, and evaluate experimental results according to relevant national and industry standards, based on artificial intelligence (AI) and oriented towards solving actual scientific problems. Moreover, the scientific report paradigm is introduced in the experiment report to help students establish scientific thinking and independent inquiry abilities. Teachers employ AI to correct students' experimental schemes, experimental reports, etc. and establish a whole-process, multi-dimensional evaluation system for experimental projects, guiding students to cultivate their comprehensive innovation ability and teamwork spirit during the practical process. This case provides experience and reference for AI-enabled teaching of experiments.