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.

[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.

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] Human norovirus (HuNoV) is one of the most common pathogens causing acute gastroenteritis in humans worldwide. Currently, there are no approved vaccines to prevent this disease. This study aimed to prepare virus-like particles (VLPs) of HuNoV in the flashBAC baculovirus expression system, laying a foundation for the development of vaccines against HuNoV. [Methods] After codon optimization, the full-length gene sequence of the VP1 protein of HuNoV GⅡ.4 was synthesized and cloned into the baculovirus pBacPAK9 transfer vector to obtain the recombinant plasmid pBacPAK9-8his-VP1. After enzyme digestion and sequencing, the recombinant plasmid was co-transfected with the linear baculovirus plasmid (Bacmid) into SF9 cells to obtain a recombinant baculovirus carrying the VP1 gene. Hi-Five (HF) cells were infected by the recombinant baculovirus for protein expression, and the expression of VP1 was analyzed by SDS-PAGE and Western blotting. VP1 was purified by Ni-NTA affinity chromatography and identified by SDS-PAGE and Western blotting. The purity of VP1 was examined by high performance liquid chromatography (HPLC). The VLPs were observed by transmission electron microscopy. [Results] A transfer plasmid pBacPAK9-8his-VP1 was constructed, and the VP1 protein, with a molecular weight of approximately 58 kDa, was mainly expressed in the cytoplasm of HF cells. The HPLC results showed that the purity of VP1 was over 99%. The VLPs with a regular shape, uniform sizes, and diameters of 30–40 nm were observed by transmission electron microscopy. [Conclusion] The VLPs of HuNoV GⅡ.4 were prepared with a baculovirus expression system, laying a foundation for the development of HuNoV vaccines.

[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] 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] To screen microbial strains with high production of exopolysaccharides (EPS) and provide strain resources for the development of soil improvement agents. [Methods] The string test with a LB-aniline blue plate was employed to qualitatively screen the strains of plant growth-promoting rhizobacteria (PGPR) with EPS production. After fermentation with each strain in four media, the EPS content in the fermentation liquid was determined by low temperature alcohol precipitation and the sulfate-anthranone colorimetric method, on the basis of which the PGPR strain with high EPS production was screened out. The fermentation conditions of the strain screened out were optimized by orthogonal test with EPS content in fermentation liquid as the indicator. The influence of the fermentation liquid of strain F1 on the content of macro-aggregates in sandy loam soil was analyzed by the petri dish culture experiment. [Results] Eight EPS-producing PGPR strains were primarily screened out, among which strain F1 had the highest EPS production. PDA was the best medium for F1 to produce EPS, with the EPS content of 867.54 μg/mL. Based on morphological, physiological and biochemical characteristics, phylogenetic analysis based on 16S rRNA gene sequence, and also average nucleotide identity analysis, F1 was identified as a strain of Bacillus megaterium. The optimal culture conditions for F1 to produce EPS were 28 ℃ and 180 r/min for 24 h, under which the EPS yield reached 1 123.39 μg/mL. After F1 was incubated in sandy loam soil for 40 days, the content of water-stable macro-aggregates with the grain diameter > 0.25 mm in the soil increased by 4.44 times compared with that of the control. [Conclusion] Strain F1 with high EPS production can promote the formation of water-stable macro-aggregates in sandy loam soil. The optimal conditions for F1 to produce of EPS was incubation in PDA at 28 ℃ and 180 r/min for 24 h.

[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.