The widely used trypsin is mainly extracted from animal pancreas, which has the disadvantages of limited raw materials, high costs, and low purity. In addition, the autolysis of trypsin affects its stability in storage and application. [Objective] To obtain an anti-autolysis recombinant trypsin by heterologous expression and methylation. [Methods] We employed gene recombination to realize the heterologous expression of porcine trypsinogen in Pichia pastoris. Furthermore, we conducted single factor experiments to investigate and optimize the temperature, pH, and time of enzyme activation and improved the anti-autolysis performance of the recombinant trypsin by methylation. [Results] The engineered strain of P. pastoris expressing trypsinogen was successfully constructed. Under the trypsin concentration of 10 mg/mL, methylation reagent addition of 30 μL, and reaction time of 3 h, the methylated trypsin showed the activity loss of only 22% and the relative activity of 79% after autolysis for 6 h, which was about 3.4 times higher than that of the control, suggesting that the anti-autolysis performance of the recombinant trypsin was greatly improved. [Conclusion] This study successfully produced a novel anti-autolysis recombinant trypsin by heterologous expression and methylation, which can improve the production and application of trypsin in China.

Anaerobic digestion (AD) of biomass waste combined with waste activated sludge (WAS), which is characterized by great stability, low energy consumption, and biogas production, can effectively reduce the volume and improve the dehydration of WAS and thus has been widely applied in methane production. However, the AD process is susceptible to external factors such as microplastics (MPs) or nanoplastics (NPs), which can lead to reduced efficiency or even collapse of AD. The AD system needs the interdependence and interaction of the microbial community to keep stable operation in a dynamic equilibrium state, in which phages play a key role. Phages can not only regulate the structure of the microbial community in the sludge and direct the energy flow but also attach to MPs and NPs with bacteria and archaea for transmission. Nevertheless, the effects of MPs and NPs on such a process were underestimated in previous studies. In this paper, we summarize the research progress in the effects of different types and sizes of MPs and NPs on AD systems, with focus on the ecological connections among microbial communities, especially bacteria, archaea, and phages, in anaerobic systems. Furthermore, we put forward novel viewpoints about the effects of MPs and NPs on microbial communities and make an outlook on the future research directions in this field.

[Objective] To mine the key enzyme genes associated with spinosad synthesis and the biosynthetic gene clusters (BGCs) in Saccharopolyspora spinosa at different developmental stages by transcriptomics, thus laying the groundwork for the construction of high-yield strains. [Methods] The transcriptomes of S. spinosa during the logarithmic phase (T2-48 h) and the stationary phase (T6-144 h) were compared. The results from qRT-PCR and transcriptome sequencing were mutually validated. Gene ontology (GO) annotation and Kyoto encyclopedia of genes and genomes (KEGG) enrichment were performed for the differentially expressed genes (DEGs). Central carbon metabolism analysis was performed. [Results] The transcriptome sequencing of S. spinosa revealed 2 542 DEGs, including 1 188 genes with significantly up-regulated expression and 1 354 genes with significantly down-regulated expression. GO annotation indicated that the DEGs were primarily involved in carboxylic acid metabolic process, oxoacid metabolic process, organic acid metabolic process, and amino acid metabolic process. KEGG enrichment analysis demonstrated DEGs were mainly involved in pathways such as glycine, serine, and threonine metabolism, oxidative phosphorylation, and arginine biosynthesis. Further analysis identified seven genes related to spinosad biosynthesis. Among them, accB, Pfk, G6PD, and dsdA showed significantly up-regulated expression, while GAPDH, aceE, DLAT involved in the consumption of spinosad precursors, as well as genes in the TCA cycle and arginine biosynthesis, exhibited significantly down-regulated expression. The results of qRT-PCR were consistent with the trends observed in transcriptome sequencing, which revealed 12 upregulated BGCs: BGC2 (43 846 bp), BGC4 (18 330 bp), BGC9 (20 501 bp), BGC18 (62 621 bp), BGC22 (19 626 bp), BGC25 (42 896 bp), BGC26 (40 086 bp), BGC28 (39 392 bp), BGC30 (20 282 bp), BGC31 (53 657 bp), BGC34 (20 787 bp), and BGC35 (40 232 bp). [Conclusion] This study elucidated DEGs in S. spinosa at different developmental stages through transcriptome analysis, and analyzed the biosynthetic pathways and BGCs of spinosad. These findings pave the way for optimizing the spinosad biosynthetic pathways and genetically modifying S. spinosa to enhance the spinosad production in subsequent studies.

Pullulan is an exopolysaccharide produced by Aureobasidium spp. Despite its widespread biotechnological applications, the mechanisms underlying the biosynthesis and regulation of pullulan remain to be studied. In recent years, researchers have employed molecular biological techniques to elucidate the molecular mechanisms of pullulan synthesis and regulation. The transmembrane protein AmAgs2 is identified as a key enzyme for the synthesis of pullulan, and the cAMP-protein kinase A (cAMP-PKA), target of rapamycin 1 (TORC1), high osmotic glycerol 1 (HOG1), and sucrose nonfermentable 1 (Snf1) signaling pathways are involved in the regulation of pullulan synthesis. We reviewed the research progress in this field, aiming to give insights into the research on the synthesis and regulation mechanisms of fungal extracellular polysaccharide and provide theoretical support for building cell factories with high yields of pullulan.

[Objective] Long-term and excessive application of chemical fertilizers leads to soil degradation and an imbalanced microbial community structure in soil. The combination of organic active substances with chemical fertilizers is considered an important approach for controlling soil degradation and maintaining microbial community stability. [Methods] Metagenomics was employed to study the effects of combining the bioactive substance γ-polyglutamic acid (γ-PGA) with chemical fertilizer on soil microbial community and function in cotton fields. Four fertilization groups were designed: chemical fertilizer (NK), chemical fertilizer combined with γ-PGA aqueous solution (YT), chemical fertilizer combined with γ-PGA granules (GT), and no fertilizer (CK). [Results] GT and YT groups outperformed NK and CK groups in terms of cotton growth and soil nutrient content. The combination of γ-PGA with chemical fertilizer significantly increased the microbial abundance and diversity in soil, while chemical fertilizer alone did not improve soil microbial diversity. In addition, the application of γ-PGA changed soil microbial community composition. Compared with the NK group, YT and GT groups showed a 9.70%–12.72% decrease in the relative abundance of Proteobacteria and 13.33%–20.90% and 8.09%–13.01% increases in the relative abundance of Bacteroidetes and Actinobacteria, respectively. In addition, the relative abundance of Rhizophagus (a genus of mycorrhiza fungi) increased by 19.71% in the YT group. The functional gene analysis showed that GT and YT significantly increased the abundance of functional genes related to amino acid biosynthesis, secondary metabolite biosynthesis, and ABC transporters. [Conclusion] The application of γ-PGA has the potential of improving soil microbial diversity and ecosystem stability in Xinjiang cotton fields.

[Objective] Certain strains of Ligilactobacillus salivarius possess excellent probiotic properties and strong potential for application. This study characterized strain ATCC 11741 in terms of the growth characteristics, stress tolerance, and adhesion capacity, aiming to provide a theoretical basis for the utilization of L. salivarius. [Methods] L. salivarius ATCC 11741's accuracy was verified through morphological observations and 16S rRNA gene sequencing; its growth characteristics were investigated by tracking growth and acid production curves; additionally, a range of tolerance tests, including those with acid, alkali, bile salts, hyperosmolarity and temperature, were conducted to evaluate its tolerance; lastly, self-aggregation and hydrophobicity experiments were used to indirectly determine its adhesion. [Results] The growth curve of L. salivarius ATCC 11741 was formed like a "S". The strain reached its highest acid production between 2–7 h, stabilizing at pH 4.3 after 14 h. After being cultured in the MRS medium at pH 2.0 for 4 h, the strain had a survival rate of 50.48% and grew well in the pH 4.0–11.0 range. Following 2 h of incubation in the MRS medium with 0.10% bile salt, the strain's survival rate was 94.440 0%. In the MRS medium with 6% NaCl, the strain continued to proliferate. The growth of strain ATCC 11741 was significantly affected by temperature, being promoted at 30–42 ℃ and inhibited at 20 ℃ and 50 ℃. At the 5 h mark, the strain's hydrophobicity (measured by the hydrocarbon compound adhesion method) was 44.5% and its self-aggregation rate was 41.4%. [Conclusion] L. salivarius ATCC 11741 is a strain with fast growth, strong tolerance to acids, alkali, and salts, moderate tolerance to bile salt, a wide range of suitable temperatures, and strong adhesion capacity.

[Objective] Multipurpose bioorganic fertilizers contribute to the sustainable development of the Carya cathayensis Sarg. industry. This work aims to explore the resources of phosphorus (P)-mobilizing bacteria (PMBs) with plant growth-promoting effects from the rhizosphere soil of C. cathayensis Sarg. [Methods] PMBs were isolated with the dilution-plate coating method and identified based on 16S rRNA gene homology. Moreover, plate and liquid culture tests were conducted to determine their biological functions. [Results] A total of 34 strains of PMBs were isolated from the rhizosphere soil of C. cathayensis Sarg. These PMBs belonged to 10 genera of four phyla: Bacillota, Proteobacteria, Actinobacteriota, and Gracilicutes. Among them, Bacillus (12 strains), Burkholderia (9 strains), and Pseudomonas (5 strains) were the dominant genera, with the strains accounting for 76.47% of the total isolated PMBs. After inoculation of PMBs, the content of soluble P produced by PMBs was 7.01–49.97, 3.61–27.11, 4.56–342.82, 27.71–544.53, and 3.28–27.17 mg/L in the culture media with AlPO₄, FePO₄, Ca₃(PO₄)₂, sodium phytate, and lecithin as the sole P source, respectively. Twenty-three strains were capable of simultaneously mobilize insoluble inorganic and organic P components. Additionally, 20, 7, 23, 12, 10, 14, and 13 strains of PMBs could produce indole-3-acetic acid (IAA), siderophores, extracellular protease, β-1, 3-glucanase, cellulase, phosphatase, and lipase, respectively, among which strains S3-6L and S3-22L exhibited five biological functions. [Conclusion] The PMBs identified in this study possess high P mobilization capability and multiple biological functions, enriching the resources of PMB strains and laying a foundation for the development of efficient, green, and composite microbial fertilizers for C. cathayensis Sarg.

[Objective] To investigate the enzymatic properties and straw-degrading effect of a recombinant xylanase rRuXyn024. [Methods] We cloned RuXyn024 from the rumen of beef cattle and used bioinformatics tools for detailed sequence analysis. The expression vector pET-RuXyn024 was constructed and transformed into Escherichia coli BL21(DE3) for heterologous expression of RuXyn024. Furthermore, the enzymatic properties and straw-degrading effect of rRuXyn024 were examined. [Results] RuXyn024 was composed of 358 amino acid residues and had a molecular weight of approximately 40 kDa, belonging to the GH 10 family. The optimal pH and temperature of rRuXyn024 were pH 7.0 and 40 ℃, respectively. The relative activity of RuXyn024 at pH 6.0−9.0 and 30−70 ℃ remained above 60% and 70%, respectively. With xylan from wheat straw as the substrate, rRuXyn024 showcased the K_m and V_max of 18.8 g/L and 82.6 µg/min, respectively. The activity of rRuXyn024 was inhibited by Mg²⁺, Zn²⁺, Cu²⁺, Ni²⁺, EDTA, and SDS at 1 mmol/L and 5 mmol/L, as well as 5 mmol/L Ca²⁺ and β-mercaptoethanol. Cu²⁺ and β-mercaptoethanol at 5 mmol/L nearly inactivated the enzyme. Mn²⁺ at 1 mmol/L and 5 mmol/L increased the activity of rRuXyn024 by 46.9% and 35.8%, respectively. The degradation of xylan from wheat straw by rRuXyn024 produced oligosaccharides, including xylotriose and xylobiose. rRuXyn024 could degrade maize straw, rice straw, soybean straw, and rapeseed straw, with the strongest degrading effect on maize straw. [Conclusion] rRuXyn024 exhibits tolerance to broad scopes of pH and temperature and significant potential for improving the utilization of straw by ruminants.

[Objective] Pseudomonas as one of the dominant spoilage bacteria highly form biofilms in chilled meat products and processing environment when contaminating single or mixed with other species. This study aims to investigate the antibiofilm properties of the cell-free supernatants (CFSs) of three Bacillus species isolated from fermented food and rice seeds on Pseudomonas lundensis (PL) or and Acinetobacter johnsonii (AJ) as mono- or dual-species. [Methods] Biofilm biomass, extracellular polymeric substances (EPSs), and biofilm structure were measured by crystal violet staining, spectrophotometry, confocal laser scanning microscopy (CLSM), respectively, as well as transcription of biofilm-related genes determined by qPCR. [Results] The CFSs of Bacillus amyloliquefaciens ZG08, B. velezensis B5, and B. subtilis YB11 inhibited the biofilm formation of PL and AJ without affecting their growth. The treatment with 50% CFSs of ZG08 and B5 decreased the cell viability of two biofilms by 12.73%–21.04%, which was higher than that of YB11 (0.15%–4.38%). The inhibition rates of 50% CFSs of the three strains were 59.75%–79.59% against the PL biofilm and 63.62%–78.57% against the biofilm of PL+AJ, in which the CFS of YB11 had weaker activity. The content of exopolysaccharides and exoprotein in the two biofilms treated with these CFSs were reduced by 53.77%–73.30% and 54.84%–62.38%, respectively. The treatment with the three CFSs also reduced the adhesive cells, loosened biofilm structures, and thinned their thickness by 57.63%–74.49% and 60.43%–64.64%, respectively. Moreover, the CFSs of ZG08 and B5 effectively eradicated by 41.77%–69.79% against the mature biofilms of PL and PL+AJ, compared to weak activity of YB11. In addition, the antibiofilm activities of the three CFSs were stable under four enzyme digestion and heating conditions. Compared with the control, the CFSs of ZG08 and B5 significantly down-regulated the expression of six biofilm-related genes, lapA, alg44, pelG, luxR, wspR, and rpoS. [Conclusion] The CFSs of ZG08 and B5 have strong antibiofilm activities against PL and AJ as mono- or dual-species.

[Objective] To systematically analyze the enzymatic properties of transglutaminase (TGase) from Streptomyces mobaraensis CGMCC 4.1851 (strain XM4) and subsequently develop a high-yielding strain by engineering for achieving efficient expression of TGase in Streptomyces with reduced fermentation duration and enhanced production efficiency. [Methods] The pH of the fermentation broth and TGase activity were measured to assess the fermentation characteristics of strain XM4. TGase from XM4 was purified by alcohol precipitation combined with ion-exchange chromatography. The reaction conditions (pH, temperature, metal ions) were optimized for the enzyme, and the enzymatic kinetics were tested. The catalytic efficiency was evaluated by casein cross-linking experiments. Subsequently, genetic engineering was employed to enhance the modified strain through heterologous expression and replacement of the ribosome-binding site (RBS), followed by measurement of TGase production. [Results] TGase from strain XM4 exhibited good activity and stability within the range of pH 4.0–11.0, with the highest activity at 50 ℃ and pH 10.0. The modification realized efficient expression of TGase in S. mobaraensis, inceasing the production by 103.3% compared with the original strain and reducing the fermentation time to 24 h. [Conclusion] TGase from strain XM4 demonstrates excellent acid-base tolerance and thermal stability, demonstrating broad application prospects in the food industry, especially dairy processing. Additionally, the engineered strain enables efficient production of TGase, providing new options for the industrial production and application of TGase.