[Objective] This study explored the application potential of symbiotic fungi and bacteria of Odontotermes formosanus in the degradation of wheat (Triticum aestivum L.) straw resources, aiming to provide a theoretical basis and supplement strain resources for the industrialization of straw biodegradation. [Methods] The lignocellulose-degrading fungi and bacteria were isolated from O. formosanus and screened by Congo red staining and the agar plate supplemented with carboxymethyl cellulose sodium (CMC-Na), and the enzyme production of the strains screened out was examined. Liquid fermentation at room temperature was carried out to evaluate the degradation effects of different strains and strain combinations on wheat straw. Fourier transform infrared spectroscopy (FTIR), X-ray crystal diffraction (XRD), and scanning electron microscopy (SEM) were employed to analyze the physical and chemical properties of wheat straw before and after degradation. [Results] Five species of fungi and three species of bacteria were isolated from the termite combs, including two species of Penicillium, one species of Purpureocillium, one species of Aspergillus, one species of Eutypella, one species of Bacillus, one species of Escherichia, and one species of Stenotrophomonas. Four efficient degrading strains were screened out and identified as Penicillium citrinum, Aspergillus nomiae, Bacillus subtilis, and Escherichia coli. In the liquid fermentation at room temperature, the combination of the four strains had the strongest comprehensive degradation performance, with the degradation rates of 24.35%, 47.24%, 35.75%, and 32.72% for dry matter, cellulose, hemicellulose, and lignin, respectively, in 12 days. The degradation destroyed the chemical bonds, the intermolecular force, and the composite structure of lignocellulose in wheat straw, and decreased the crystallinity of cellulose from 37.40% to 32.97%. [Conclusion] The combination of P. citrinum, A. nomiae, B. subtilis, and E. coli isolated from the combs of O. formosanus had a good degradation effect on wheat straw, demonstrating the application potential in the industrialization of straw biodegradation.

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] To homologously overexpress an alkaline fungal laccase PIE5 (CcPIE5) in Coprinopsis cinerea FA2222 under the control of the Agaricus bisporus gpdII promoter. [Methods] The laccase activity reached (24.2±1.1) U/mL in the supernatant after 7 days of cultivation at 37 ℃ in the mKjalke medium. The purified CcPIE5 showcased the best performance at pH 8.0 and 60 ℃. [Results] Unlike other characterized fungal laccases, CcPIE5 was tolerant to high concentrations of NaCl. Particularly, both k_cat and K_m decreased when the concentration of NaCl was increased from 0 to 1.5 mol/L, which indicated that CcPIE5 demonstrated application potential in the dye decoloring of texile finishing. In dye decolorization, CcPIE5 efficiently degraded (92.9±2.3)% indigo carmine at pH 8.5 and 60 ℃, with syringic acid as the mediator. Isatin 5-sulfonic acid (ISA) was identified by LC-MS as the primary byproduct of indigo carmine degradation. [Conclusion] CcPIE5 is best-suited in decolorizing dyes under high temperatures and alkaline and salty conditions. It serves as a good candidate for specific applications in the environment and industry.

Bacillus paralicheniformis, a Gram-positive, facultative anaerobic, motile rod-shaped endospore-forming bacterium, can be used as a species of potential plant growth-promoting rhizobacteria (PGPR). In this study, B. paralicheniformis HMPM220325 was isolated from the fruit fermented milk. This strain can form biofilms at the gas-liquid interface during static cultivation. [Objective] To study the effects of different environmental factors on the biofilm biomass of B. paralicheniformis HMPM220325 and provide data support for the later development and application of HMPM220325 as a PGPR strain. [Methods] Effects of different environmental factors and nutrients on the biofilm formation of B. paralicheniformis HMPM220325 were quantitatively detected by crystal violet staining, and the optimal conditions for the biofilm formation of the strain were optimized by orthogonal experiments. [Results] The optimal environmental conditions for the biofilm formation of B. paralicheniformis HMPM220325 were incubation at 50 ℃ and pH 9.0 for 36 h. The optimal medium was composed of maltose 15.0 g/L, urea 10.0 g/L, magnesium sulphate 20.0 mmol/L, disodium hydrogen phosphate 2.5 g/L, and bovine heart infusion 17.5 g/L. The optimized culture conditions increased the biofilm biomass by 58.28% compared with the original culture conditions. [Conclusion] This study explored the biofilm formation of B. paralicheniformis in a variety of environments and optimized the culture conditions for biofilm formation of this strain, providing an experimental basis for further development of PGPR.

[Objective] To obtain a yeast strain efficiently producing the acidic protease PrA for applications in food processing, feed additives, and other related industries. [Methods] We constructed a recombinant strain of Pichia pastoris expressing PrA by fermentation in shake flasks and measured the enzymatic properties of the expressed PrA. Several strategies, such as signal peptide modification, gene dosage optimization, and co-expression with molecular chaperones, were employed to enhance the production of PrA. Additionally, high-density fermentation was employed to further improve the expression level. [Results] The expressed enzyme PrA showcased the specific activity of 3 974.00 U/mg, with the optimum performance at pH 3.0 and 45 ℃. The production of PrA by the parental strain was 738.03 U/mL. The modification of the MF4I signal peptide increased the production of PrA to 1 206.52 U/mL. Moreover, an increase in the copy number of prA further increased the PrA production to 2 406.47 U/mL. Additionally, co-expression with single or combined molecular chaperones increased the PrA production to 4 091.27 U/mL. After undergoing high-density fermentation, the enzyme activity reached 43 088.00 U/mL within 168 h, representing a 58.4-fold increase compared with the initial production. [Conclusion] High-level expression of PrA was achieved in P. pastoris, which laid a foundation for the future industrial applications. The results provide valuable insights into the research and development of PrA for applications in food processing and feed additives.

Levilactobacillus brevis is a common species of lactic acid bacteria mainly detected on the surface of plant stems and leaves and in pickles, dairy products, and intestines. With excellent physiological functions, L. brevis is a potential probiotic species. With the rise of genomics, it is of great significance to reveal the genetic characteristics and functional gene properties of L. brevis at the gene level for application of this bacterium. This paper reviews the genetic background and major functional genes of L. brevis, aiming to lay a theoretical foundation for the application of L. brevis.

[Objective] Outer membrane vesicles (OMVs) are spherical bilayer membrane structures secreted by Gram-negative and some Gram-positive bacteria. OMVs contain abundant surface antigens and are of great research significance in vaccine development. However, the presence of lipopolysaccharides (LPS), which is the primary component of OMVs, arouses safety concern. Therefore, genetically modifying bacterial LPS to produce safe and efficient OMVs is a viable approach to enhance the production and application of OMVs. [Methods] We modified Salmonella minnesota Re595 with O antigen and most core antigen deletions by deleting the acyl chain coding gene msbB and inserting the phosphatase coding gene lpxE from Francisella novicida to reduce acyl chains and phosphate groups on lipid A, thus obtaining less toxic LPS. LPS and OMVs were extracted from the starting strain and modified strain, and their pro-inflammatory activities were compared between the two strains. In addition, inactivated foot-and-mouth disease virus vaccines were prepared with OMVs to assess the immune adjuvant activity of OMVs. [Results] The modification of LPS reduced the endotoxin activity and pro-inflammatory responses while significantly increasing the immune adjuvant activity of OMVs. [Conclusion] This study demonstrates that the modification of LPS can attenuate the toxicity and enhance the immune adjuvant activity of bacterial OMVs. These findings provide a theoretical foundation for utilizing OMVs as immune adjuvants in the future.

[Objective] To investigate the effects of a metabolite cocktail composed of indole-3-propionic acid (IPA), sodium butyrate (SB), and valeric acid (VA) of gut microbiota on the proliferation of hepatocellular carcinoma cells. [Methods] The human hepatocellular carcinoma HepG2 cells were cultured in vitro and treated with the cocktail at different concentrations (1×, 2×, 3×, 4×, and 5×). The total cholesterol (TC) and triglyceride (TG) levels in the cells were determined by the total cholesterol and triglyceride assay kits. The Cell Counting Kit-8 (CCK-8) and colony formation assays were employed to examine the cell proliferation. Twelve BALB/c athymic nude mice were randomized into a control (Ctrl) group and a treatment (Treat) group and then subjected to subcutaneous injections of HepG2 cells. The tumor size was measured every three days, and the tumor volume and tumor inhibition rate were calculated. When the tumor volume reached 100 mm³, the mice in the Ctrl group were administered with sterile water by gavage daily, while those in the Treat group received the cocktail via gavage until euthanized under anesthesia. After 27 days of treatment, the body weights of mice in both groups were measured, and tumors were excised and weighed, with the tumor weight/body weight ratio calculated. The content of Ki-67 protein in the tumors was determined by immunohistochemical (IHC) staining, and the lipid accumulation within tumor cells was assessed by Oil Red O staining. [Results] The cocktail of IPA, SB, and VA lowered the levels of TC and TG in hepatocellular carcinoma HepG2 cells and exerted an inhibitory effect on the proliferation of HepG2 cells. Both CCK-8 and colony formation assays indicated that the cocktail inhibited the proliferation of HepG2 cells in a dose-dependent manner. The oral administration of the cocktail inhibited the growth of hepatocellular carcinoma cells, as evidenced by smaller and lighter tumors and lower tumor weight/body weight ratios in the Treat group than in the Ctrl group (Ctrl: 723 mm³, 0.47 g, 22.23%; Treat: 526 mm³, 0.32 g, 16.65%). IHC and Oil Red O staining further demonstrated reductions in Ki-67 expression and lipid accumulation in the mice administered with the cocktail via gavage. [Conclusion] The cocktail of IPA, SB, and VA can inhibit the proliferation and suppress the lipid synthesis of hepatocellular carcinoma cells.

[Objective] A facultative anaerobic bacterium Klebsiella sp. CW-D3T utilizing sulfate as the terminal electron acceptor for anaerobic respiration was used for degradation of target pollutants in the system with phenanthrene (PHE)-Cd²⁺ co-contamination. The response mechanism of the strain to different Cd²⁺ concentrations in the sulfate reduction system and the anaerobic metabolic pathways of the strain for degrading PHE were studied. [Methods] A sulfate reduction system with an initial sulfate concentration of 20 mmol/L was developed to enhance the growth and metabolic activity of functional bacteria and improve the bacterial performance for remediating PHE-Cd²⁺ co-contamination. The changes in extracellular polymer secretion and the vibrational characteristics of characteristic peaks were analyzed to explore the cellular responses to different Cd²⁺ concentrations. Furthermore, qualitative and quantitative analyses of the metabolic products of PHE in the sulfate reduction system were conducted by GC-MS and HPLC. [Results] In the presence of 0.5–50.0 mg/L Cd²⁺, the sulfate reduction system of Klebsiella sp. CW-D3T enhanced the remediation efficiency of target compounds, with the PHE and Cd²⁺ removal rates above 70.00% when the initial Cd²⁺ concentration was below 10 mg/L. As the concentration of Cd²⁺ increased, the secretion of extracellular polysaccharides in extracellular polymeric substances (EPS) was more than that of extracellular proteins, and the intensity of characteristic peaks of polysaccharides and protein functional groups on the surface of bacterial cells was enhanced. The initial activation of PHE in the sulfate reduction system tended to favor carboxylation to produce 2-phenanthroic acid under Cd²⁺ stress. When the initial Cd²⁺ concentrations were 10 mg/L and 50 mg/L, the content of 2-phenanthroic acid peaked at 15.56 μg/L and 10.23 μg/L on day 5, respectively, which decreased by 27.56% and 52.37% compared with that of the control group without the addition of Cd²⁺. Cd²⁺ stress significantly affected the 2-phenanthroic acid content within the cycle and at the end of the cycle. [Conclusion] The biodegradation efficiency of PHE by Klebsiella sp. CW-D3T was significantly improved when sulfate was used as an electron acceptor in the presence of Cd²⁺. The extracellular polysaccharides and proteins played a positive role in enhancing the microbial tolerance to Cd²⁺ stress by regulating the detoxification process.

[Objective] We compared the microbial communities in the rhizosphere of plants with different genotypes and explored the relationship between microbial community and soil-borne disease occurrence, aiming to reveal the underlying mechanisms by which rhizosphere microorganisms assist plants in defending against pathogen invasion. [Methods] A pot experiment was conducted with the soil experiencing severe continuous cropping obstacles to compare the microbial communities in the rhizosphere of a susceptible watermelon variety 'zaojia 8424' and a resistant variety 'xinong 8' to Fusarium wilt. Furthermore, the relationship between microbial community and the occurrence of Fusarium wilt was explored. [Results] The resistant watermelon variety exhibited significantly lower disease index and pathogen (Fusarium oxysporum f. sp. niveum, FON) abundance than the susceptible watermelon variety. Although no significant difference was observed in the bacterial and fungal alpha diversity in the rhizosphere between resistant and susceptible varieties, the microbial beta diversity showcased significant difference between the two varieties. Moreover, both bacterial and fungal community composition was significantly correlated with pathogen abundance. Linear discriminant analysis effect size (LEfSe) further revealed that the resistant watermelon variety enriched more potential antagonistic or plant growth-promoting taxa represented by Actinobacteria and Rhizobiaceae in the rhizosphere. Interestingly, Fusarium was also enriched in the rhizosphere of the resistant variety, mainly composed of unclassified Fusarium and F. solani. Notably, the co-occurrence network of microorganisms in the rhizosphere of the resistant variety exhibited higher complexity and stability than that of the susceptible variety, with an increase of 18.18% in average degree and the nodes and edges dominated by Actinobacteria. [Conclusion] The watermelon varieties resistant and susceptible to Fusarium wilt demonstrate different microbial community composition in the rhizosphere. The enrichment of beneficial microbial taxa and interconnected co-occurrence network of the resistant variety contribute to plant defense against the pathogen invasion. This study disentangles the relationship between rhizosphere microbial community and soil-borne disease occurrence, providing important information and a theoretical basis for preventing and managing soil-borne diseases.