Objective The mesophilic salt-tolerant xylanase XynRBM26, a member of the GH10 family, holds significant application value in industrial fields such as animal feed. This study improved the thermostability of this enzyme by protein modification via rational design, aiming to lay a foundation for the industrial application of XynRBM26 preparations. Methods The bioinformatics software FoldX was used to conduct positionscan of the 3D structure predicted by AlphaFold 2.0 for XynRBM26. The mutants with free energy changes less than -0.5 kcal/mol were selected to construct an initial electronic library. According to the Pro effect and screening principles for potential mutants, an electronic library composed of Pro mutations was subsequently established. Finally, site-directed mutagenesis was employed to construct mutant genes, and positive mutants were screened after heterologous expression, purification, and experimental verification. Results After screening of the initial potential mutants, a small and precise mutant library consisting of 21 Pro effect mutants was constructed. All the mutants were experimentally validated, and positive single-point mutants D222P, V182P, D344P, and A352P with significantly increased T_m values were screened out. Through subsequent stacking screening, a three-point stacked Pro effect mutant with superior properties was obtained. The combination of this mutant with the experimentally screened positive mutant G115D produced the most stable mutant M4 (G115D-D222P-D344P-A352P). Compared with wild-type XynRBM26, M4 showed increases of 6.5 °C and 5 °C in T_m and optimal temperature, respectively. Moreover, M4 presented the half-life (t_1/2) at 55 °C 7.5-fold longer than the wild type, and its relative activity at the optimal temperature was 3.44 folds that of the wild type. Conclusion Screening thermostable mutants of the salt-tolerant xylanase XynRBM26 of the GH10 family based on the Pro effect and two different effect superimposing strategies is an effective approach.

Pseudomonas aeruginosa (PA) is a major cause of hospital-acquired infections. It can survive in a variety of extreme environments, and is highly resistant to antibiotics. PA can attack immunocompromised populations to cause severe infections, being a major cause of mortality in clinical practice. Therefore, rapid diagnosis of PA is critical for infection control. Conventional detection techniques such as plate assays, immunoassays, and nucleic acid assays have excellent accuracy and sensitivity, while they are costly and time-consuming. In recent years, novel biosensing technologies have achieved ultrasensitive and precise detection of PA by integrating various biorecognition elements and signal enhancement strategies, providing an efficient, convenient, and cost-effective solution for the rapid identification, treatment, and control of PA. In this paper, we systematically review the principles, application progress, and challenges of magnetic separation biosensing technologies based on electrochemical, optical, CRISPR/Cas12a system, and magnetic nanoparticles, and provide future research directions, aiming to promote the innovation and clinical application of the technologies for rapid detection of PA.

SnoaL family proteins belong to a class of the nuclear transport factor 2 (NTF2)-like superfamily, which are characterized by a distinctive α+β barrel fold structure. To date, proteins of this family have been demonstrated to catalyze a variety of reactions, such as aldol reaction, hydroxylation, decarboxylation, epoxide hydrolysis, and cycloaddition reaction, playing crucial roles in the biosynthesis of complex bioactive natural products. These proteins are widely distributed in various organisms including bacteria and fungi. However, research on proteins of this family is relatively limited at present. We reviewed the research progress on proteins of SnoaL family. The review not only provides beneficial guidance for the subsequent functional identification and mechanism research of related proteins but also helps to discover novel biocatalytic tools, laying a foundation for the research of bioactive molecule biosynthesis.

[Objective] To investigate the pathogenicity of Lichtheimiaramosa LYSF001 and ITS resistance to common fungicides, providing experimental evidence for the diagnosis and treatment of fungal infections in animals. [Methods] The strain was identified by morphological characterization and molecular biology techniques. PCR was employed to detect virulence genes in strain LYSF001, and an animal infection model was established to evaluate the pathogenicity of the strain. Histopathological examination, Grocott’s methenamine silver (GMS) staining, and ITS sequences amplification were employed to analyze pathological changes in the livers and kidneys of infected animals. Additionally, antimicrobial susceptibility tests were conducted to assess the sensitivity of strain LYSF001 to caspofungin, amphotericin B, and itraconazole. [Results] Strain LYSF001 was identified as L. ramosa. PCR analysis revealed that strain LYSF001 carried five virulence genes (CalA, PKP2, LaeA, Alp2, and AspF1). Animal experiments demonstrated that the strain led to the mortality of 40% and caused visceral hyperemia in mice, with the most significant pathological changes (inflammatory cell infiltration and tissue necrosis) observed in the livers and kidneys. Severe infections led to animal mortality. GMS staining revealed the presence of dark-colored hyphae in the heart and liver, and ITS sequences amplification further confirmed fungal infection. The antimicrobial susceptibility test results indicated that strain LYSF001 was resistant to caspofungin but sensitive to amphotericin B and itraconazole. [Conclusion] L. ramosa LYSF001 exhibits strong pathogenicity, capable of causing severe infections and even death in animals. Additionally, the strain showed resistance to caspofungin but sensitivity to amphotericin B and itraconazole. The findings provide important experimental evidence and technical support for the diagnosis and treatment of fungal infections in animals.

[Objective] To construct an Escherichia coli mutant strain that accumulates pyruvate by genetic modification guided by the genome-scale metabolic network model. [Methods] Using a genome-scale metabolic network model as a guide, we simulated pyruvate production of E. coli, screened key genes in metabolic pathways, and developed gene editing procedures accordingly. We knocked out the acetate kinase gene ackA, phosphate acetyltransferase gene pta, alcohol dehydrogenase adhE, glycogen synthase gene glgA, glycogen phosphorylase gene glgP, phosphoribosyl pyrophosphate (PRPP) synthase gene prs, ribose 1,5-bisphosphate phosphokinase gene phnN, and transporter encoding gene proP. Furthermore, we knocked in the transporter encoding gene ompC, flavonoid toxin gene fldA, and d-serine ammonia lyase gene dsdA. [Results] A shake flask process with the genetically edited mutant strain MG1655-6-2 under anaerobic conditions produced pyruvate at a titer of 10.46 g/L and a yield of 0.69 g/g. Metabolomic analysis revealed a significant increase in the pyruvate level in the fermentation broth, accompanied by notable decreases in the levels of certain related metabolic byproducts. Through 5 L fed-batch fermentation and an adaptive laboratory evolution, the strain finally achieved a pyruvate titer of 45.86 g/L. [Conclusion] This study illustrated the efficacy of a gene editing strategy predicted by a genome-scale metabolic network model in enhancing pyruvate accumulation in E. coli under anaerobic conditions and provided novel insights for microbial metabolic engineering.

[Objective] To identify and develop a phage-derived lyase that can be heterologously expressed with high activity and stability and determine its optimal working conditions. [Methods] We employed the turbidity reduction assay to evaluate the bacteriolytic activity and identify the optimal parameters. [Results] Genome annotation and protein prediction of the Vibrio alginolyticus phage phiV208 showed that ORF30 encoded a lyase, named lysV208. This enzyme demonstrated soluble expression in Escherichia coli BL21(DE3), reaching a purified concentration of 204 μg/mL after 16 h induction with 0.25 mmol/L IPTG. Its bacteriolytic activity (turbidity reduction rate) increased from 24.2% to 68.0% in the presence of 0.5 mmol/L EDTA. Enzymatic characterization revealed that lysV208 exhibited the maximum bacteriolytic activity (75.6%) at 45 ℃ while maintaining high activity (52.8%-71.9%) within the temperature range of 25-37 ℃, which is typical for bacterial disease outbreaks in aquatic and terrestrial animals. The enzyme showed the maximum activity at pH 7.0 and retained substantial bacteriolytic activity (44.0%-63.2%) under alkalescence conditions (pH 7.0-9.0), demonstrating adaptability to marine and freshwater aquaculture environments. Divalent metal ions including Zn²⁺, Mg²⁺, Mn²⁺, and Fe²⁺ at 0.1-1.0 mmol/L moderately enhanced the bacteriolytic activity of lysV208, whereas those at 10.0 mmol/L reduced the activity (P<0.01). In addition, lysV208 displayed broad-spectrum lytic effects, showing the bacteriolytic activity of 59.7% against V. alginolyticus V039, 68.9% against Vibrio vulnificus H1, 65.8% against Vibrio parahaemolyticus GH32, and 38.0% and 65.6% against Vibrio harveyi TY13 and G1, respectively. [Conclusion] The recombinant lyase lysV208 demonstrates robust and stable in vitro bacteriolytic activity and a broader spectrum than its source phage. These findings highlight its potential for the control of bacterial infections and the development of phage-lyase synergistic agents.

[Objective] Porcine circovirus type 3 (PCV3) is associated with the development of reproductive disorders in sows, piglet dermatitis, and myocarditis. However, due to the difficulty in stably passaging PCV3 in vitro, the animal infection model construction and specific pathogenicity of PCV3 remain to be investigated. This study aims to solve the above problems. [Methods] A PCV3 strain (PCV3-HK) was successfully isolated by inoculating PK-15 cells with lymph node samples from PCV3-positive weaned piglets. The strain was identified using PCR, indirect immunofluorescence assay, and transmission electron microscopy, and its complete genome was sequenced. The whole genome of the strain was sequenced. The pathogenicity of the strain was evaluated based on the clinical symptoms, body weight changes, virus excretion, and pathological changes of 21-day-old non-weaned piglets after inoculation with the 15th-passage viral cell culture at a dose of 10^6.4 TCID₅₀/mL. [Results] PCV3-HK had a genome length of 2 000 bp and belonged to PCV3c subtype. The viral particles were spherical, non-enveloped, about 15-20 nm in diameter, and capable of reacting specifically with the monoclonal antibody against PCV3 Cap protein. The results of infection in piglets showed that the PCV3-HK infected pigs developed obvious symptoms such as dermatitis, slow weight gain and even wasting, as well as anatomical changes such as lymph node edema and interstitial pneumonia. The viral nucleic acid was detected in oral swabs and anal swabs on day 7-21 post infection, and viremia was observed. The virus infected multiple organs, including the heart, lungs, kidneys, and lymph nodes. Notably, inflammatory cell infiltration and aggregation of necrotic cells were observed in the kidneys and lymph nodes. [Conclusion] We isolate a PCV3 strain with classical circovirus morphology and establish a piglet infection model, which provides key materials for pathogenic mechanism study and vaccine development regarding PCV3.

[Objective] To investigate the effects of fertilization regime alterations on microbial communities in paddy soils. [Methods] We conducted a long-term field experiment with three treatments: chemical fertilizer (H) and H converted to a conventional amount of organic fertilizer (HC), a conventional amount of organic fertilizer (C) and C converted to chemical fertilizer (CH), and a high amount of organic fertilizer (G) and G converted to chemical fertilizer (GH). Metagenomic sequencing was combined with bioinformatics analysis to assess the structures, diversity, and co-occurrence networks of microbial communities in paddy soils. [Results] Compared with the H treatment, the HC treatment increased the soil organic carbon, dissolved organic carbon, total nitrogen, and alkali-hydrolyzable nitrogen (P<0.05). The CH and GH treatments exhibited lower soil carbon and nitrogen levels than C and G treatments, respectively. The HC treatment markedly altered the relative abundance of Acidobacteriota, Nitrospirota, Candidatus Rokubacteria, Mucoromycota, and Thaumarchaeota. The CH treatment showed no significant changes in microbial composition at the phylum level, whereas the GH treatment significantly modified the relative abundance of Nitrospirota and the archaeal community structure. Although fertilization regime alterations showed no significant effect on microbial alpha diversity (P>0.05), the beta diversity differed across treatments (P<0.05). Co-occurrence network analysis demonstrated that the HC treatment enhanced the network complexity relative to the H treatment, with increased nodes (181), edges (2 935), average degree (16.215), modularity (0.757), and clustering coefficient (0.495). The CH treatment showed more edges (3 894) and higher average degree (21.514) but lower modularity (0.599) than the C treatment (0.751). Conversely, the GH treatment diminished all network topology parameters relative to G. Redundancy analysis identified dissolved organic carbon (22.1%), soil pH (16.8%), total nitrogen (15.6%), ammonium nitrogen (14.6%), available potassium (11.8%), and available phosphorus (10.6%) as the primary drivers of microbial community variations. [Conclusion] The findings indicate that fertilization regime alterations influence the microbial community structure and network characteristics in paddy soils by modifying soil physicochemical properties. Transitioning from chemical to organic fertilization enhances microbial community stability and soil ecological functions, while replacing organic with chemical fertilization reduces soil carbon and nitrogen availability, potentially compromising microbial network complexity and resilience.

[Objective] Microorganisms play a critical role in the treatment of food waste. Elucidating their community structure and functional characteristics is essential for optimizing treatment processes and improving operational efficiency. [Methods] We employed high-throughput 16S rRNA gene amplicon sequencing to investigate the microbial community structures and potential functions in three systems: the anaerobic digestion system, the wastewater treatment system, and the air purification system (including biofilters and deodorization towers), in a food waste treatment facility. [Results] The microbial communities differed significantly in diversity and composition across systems. The anaerobic digestion system exhibited lower microbial diversity and richness, primarily influenced by environmental factors such as temperature, pH, and biochemical oxygen demand (BOD). Functional analysis indicated that organic matter degradation was the dominant microbial function across the facility. Specifically, the anaerobic system was enriched with genera such as Defluviitoga, Methanothermobacter, and Lentimicrobium, which were involved in hydrolysis, fermentation, and methanogenesis. The wastewater treatment system was dominated by Candidatus Anammoximicrobium, Nitrolancea, and Truepera, which drove organic matter degradation and nitrogen transformation processes, including anaerobic ammonium oxidation (anammox), nitrification, and denitrification. The air purification system was enriched with Chryseobacterium and Paracoccus, which were associated with biofilm formation and the degradation of volatile organic compounds and sulfur-containing compounds. [Conclusion] The microbial community characteristics in each treatment system was closely associated with system-specific operational conditions. The enrichment of functionally distinct microbial taxa reflected their ecological roles. In the anaerobic digestion system, methanogens and syntrophic bacteria cooperatively facilitated organic matter degradation and methane production. In the wastewater treatment system, nitrogen-cycling bacteria ensured efficient nitrogen removal. In the air purification system, the enrichment of heterotrophic degraders and sulfur compound-metabolizing genera (e.g., Sphingobium and Flavobacterium) enhanced deodorization performance. These findings provide a theoretical basis for microbial community regulation and process optimization in food waste treatment facilities.

Loop-mediated isothermal amplification (LAMP), a rapid and sensitive method of nucleic acid isothermal amplification, is characterized by high specificity and sensitivity and holds broad application prospects in nucleic acid detection. However, the development of detection schemes based on LAMP encounters issues, such as non-specific and non-template amplification during the amplification process, which can affect the accuracy and specificity of the detection results. This article elaborates on the recent research progress in the strategies avoiding false positives and enhancing detection efficacy of LAMP, which include primer design, optimization of conditions, and introduction of special chemical substances. Finally, this article explores the breakthroughs and innovations of LAMP in the field of detection.