[Objective] To use microorganisms to mobilize unavailable nutrients in soil for the utilization by leguminous crops and the sustainable and long-term utilization of soil resources. [Methods] Microbial culture and micro-plot experiments were carried out with Ceriporia lacerata HG2011, a new isolate of white-rot fungus, to investigate the fungal secretion, soil nitrogen (N) and phosphorus (P) mobilization, and influences on the nutrient uptake, growth, and yields of Vigna radiata and Vicia villosa. [Results] C. lacerata released cellulase, chitinase, β-l,3-glucanase, protease, phosphatase, and siderophore, and dissolved Ca₃(PO₄)₂ in pure culture. After being inoculated on the soil surface, this fungus formed colonies, with some mycelia extending into the soil, which decrease soil pH but increase the content of NH₄⁺-N, alkali-hydrolyzed N, water-soluble P and Olsen P, and the activities of protease and phosphatase. In general, C. lacerata inoculation improved soil N and P supplies, enhanced root activity, and promoted root growth, nodule formation and development, thus increasing the nutrient uptake, grain yield of V. radiata, and biomass of V. villosa. [Conclusion] C. lacerata dwelling in soil mobilized soil N and P to increase fertilizer use efficiency and promote crop growth. C. lacerata can be cultured with sawdust, straw, husk and other organic agricultural and forestry wastes, with low production costs. This study provides a new strategy for mobilizing soil nutrients, promoting the growth of leguminous crops such as V. radiata and V. villosa, and benefiting the conservation and sustainable use of cultivated lands.

[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.

[Objective] To investigate the biological role played by the glucoside transporter Lmo0738 in the virulence of Listeria monocytogenes. [Methods] The lmo0738-deleted mutant (Δlmo0738) and complementation mutant (CΔlmo0738) were constructed by homologous recombination. The growth, hemolytic activity, cellular adhesion and invasion, and intracellular migration of the wild type strain and the mutants were assessed by the growth curves, sheep red blood cell hemolysis assay, infection of human epithelial cells (Caco-2), and infection of mouse fibroblastic cells (L929), respectively. Additionally, the mRNA and protein levels of the virulence factor listeriolysin O (LLO) were determined by real-time quantitative reverse transcription PCR (RT-qPCR) and Western blotting, respectively. [Results] The L. monocytogenes strain with the deletion of lmo0738 demonstrated weakened growth and diminished hemolytic activity. Notably, Δlmo0738 exhibited significantly reduced cell adhesion, invasion, and intracellular migration compared with the wild type strain. In addition, the mRNA and protein levels of LLO were significantly down-regulated in Δlmo0738. [Conclusion] This study provides the evidence that the absence of lmo0738 attenuates the virulence of L. monocytogenes, laying a crucial foundation to illustrate the mechanism of the phosphotransferase system (PTS) in regulating the sugar catabolism and the infection mechanism of major food-borne pathogens including L. monocytogenes.

[Objective] To investigate the role of WekM, the O-antigen glycosyltransferase of avian pathogenic Escherichia coli (APEC) O₁, in lipopolysaccharide biosynthesis and environmental adaptation. [Methods] The wekM-deleted strain ΔwekM of APEC O₁ was constructed by Red homologous recombination, and then the complementary strain CΔwekM was constructed. The impacts of wekM on bacterial growth and motility were examined. The lipopolysaccharide (LPS) profile and reactivity with rabbit anti-O₁ serum of each strain were identified by silver staining and Western blotting. Real-time fluorescence quantitative PCR was conducted to determine the transcriptional levels of flagellum-related genes, and ethidium bromide was used to measure the bacterial cell membrane permeability. Finally, the drug sensitivity test was carried out to identify the bacterial susceptibility to antibiotics such as ciprofloxacin. [Results] The constructed ΔwekM and CΔwekM were verified by PCR amplification and DNA sequencing. Compared with the wild type, ΔwekM showed incomplete LPS profile and absence of some O-antigen bands. Western blotting results showed that ΔwekM did not react with the anti-O₁ serum, suggesting that the loss of WekM impaired the LPS production. The deletion of wekM reduced the swimming motility and did not impact the bacterial growth rate compared with the wild type. The transcription levels of flagellum-related genes such as flgC were down-regulated in ΔwekM. The results implied that the reduced motility of ΔwekM was caused by the decrease in flagellar production. In addition, ΔwekM demonstrated increased cell membrane permeability compared with the wild type (P < 0.01), and ΔwekM improved bacterial sensitivity to 7 antibiotics including polymyxin. This result suggested that the adaptability of ΔwekM to the environment was inhibited due to the increased cell membrane permeability. [Conclusion] The deletion of wekM in APEC results in diminished swimming motility, increased antibiotic resistance, improved cell membrane permeability, and damaged LPS integrity. The findings lay a foundation for mining the role of wekM and enrich our understanding of the stress resistance mechanism of APEC.

[Objective] To investigate the changes of pathogenicity and DNA methylation levels and patterns of Ralstonia solanacearum strains with different pathogenicity during consecutive subculture. [Methods] R. solanacearum strains with different pathogenicity were consecutively subcultured for 50 passages. The pathogenicity of different strains was determined by the attenuated index (AI) method and the pot experiments. Methylation sensitive amplification polymorphism (MSAP) analysis was performed to profile the DNA methylation levels of different strains. Moreover, the relative expression levels of genes related to methylases and demethylases were determined by real-time fluorescent quantitative PCR (qRT-PCR). [Results] After 50 passages, both of the virulent strain FJAT15304 and the intermediate strain FJAT445 evolved into avirulent strains, while the avirulent strain FJAT15249 remained to be avirulent. Compared with F₁ strains, FJAT15304.F₅₀ and FJAT445.F₅₀ showed the total methylation rates increasing by 7.82% and 38.22%, respectively. However, both of FJAT15249.F₁ and FJAT15249.F₅₀ had the total methylation rate of 33.33%. Full methylation was the main pattern in the virulent and intermediate strains, while hemi-methylation was the main pattern in all the avirulent strains. Compared with F₁ strains, strains FJAT15304.F₅₀ and FJAT445.F₅₀ showed up-regulated expression of three methylase-related genes dam, dcm, and ftsZ and down-regulated expression of demethylase-related gene alkB, which suggested that the change of DNA methylation might play a key role in the debilitation of pathogenicity. [Conclusion] The pathogenicity of R. solanacearum attenuates during the consecutive subculture, which might be related to the level of DNA methylation. The findings provide a scientific basis for the application of avirulent strains in the biocontrol of bacterial wilt.

[Objective] The plasmid interference system of CRISPR/LshCas13a was constructed in Escherichia coli MG1655-ΔrecA and Escherichia coli DH10B to analyze the escape phenomenon in RNA editing experiments by targeting the non-essential gene lacZ and the essential gene polA. [Methods] An inducible CRISPR/LshCas13a RNA editing system- associated plasmid was designed with LshCas13a from Leptotrichia shahii. MG1655-ΔrecA and DH10B were selected as the research objects. The Crisporo algorithm was employed to design the CRISPR RNA (crRNA) sequences targeting lacZ and polA, and the LshCas13a plasmid interference experiment was carried out to study the escape phenomena targeting lacZ and polA. The escape phenomenon of the LshCas13a system was evaluated based on the number and sequences of escaped colonies. PCR and Sanger sequencing were conducted to explore the escape events of the LshCas13a system. The escaped colonies carrying the LshCas13a system disrupted by the insertion sequence (IS) were selected, and OD₆₀₀ was measured to evaluate the growth recovery of the strains. [Results] The LshCas13a system was used to target lacZ and polA in MG1655-ΔrecA and DH10B. MG1655-ΔrecA escaped through point mutation of LshCas13a and IS-mediated transposition when lacZ was targeted. When polA was targeted, MG1655-ΔrecA and DH10B escaped by point mutation of LshCas13a, IS-mediated transposition, and mutation of the direct repeat (DR) sequence of crRNA. The mutation of LshCas13a promoted the recovery of strain growth. [Conclusion] The LshCas13a plasmid interference system successfully revealed the diversified escape phenomena during the RNA editing of E. coli, including IS-mediated the transposition of LshCas13a, point mutation of LshCas13a, and DR sequence mutation or recombination of crRNA. The results laid a foundation for optimization of the CRISPR/LshCas13a gene editing system.

Microalgae are rich in lipids, proteins, and exopolysaccharides, serving as potential producers of high-value by-products. Compared with monoculture, microalgal co-culture offers advantages such as fast growth rates and strong resistance, increasing the microalgal biomass and lipid production. Biomass production by microalgae co-culture is influenced by environmental conditions, nutrient composition, and external stress, and the produced biomass can be utilized for biofuel production and food processing. This article introduces the types of microalgal co-culture systems and reviews the related studies on the production of high-value by-products. It summarizes the factors influencing biomass production in microalgal co-culture systems and highlights the potential of microalgal co-culture for resource utilization. Furthermore, this article discusses the prospects and challenges of microalgal co-culture.

[Objective] The protein SakA encoded by sakA is a member of the mitogen-activated protein kinase (MAPK) family in Aspergillus niger. However, little is known about the roles of SakA in A. niger. In this study, we constructed the A. niger strains with knockout of sakA to investigate the roles of this gene. [Methods] The Agrobacterium-mediated method was utilized to construct ΔsakA strains from A. niger RAF106 (the wild type, WT). The growth and spore production of ΔsakA and WT were observed on three different media. The sensitivity of ΔsakA and WT to different stress conditions was studied. The intracellular and extracellular levels of amylase, pectinase, and cellulase were compared between ΔsakA and WT. Real-time quantitative polymerase chain reaction (qRT-PCR) was employed to determine the relative transcript levels of the genes associated with spore production, amylase, pectinase, cellulase, and hyperosmotic regulation. [Results] Three ΔsakA strains were successfully obtained and verified by PCR and qRT-PCR. The ΔsakA strains had slow growth, delayed spore production, and delayed conidiophore differentiation compared with WT. The ΔsakA strains showcased slower colony growth than WT under the stress conditions of 0.6 mol/L KCl, 0.8 mol/L NaCl, and 1.2 mol/L NaCl. Compared with WT, the knockout of sakA increased the extracellular amylase production by 20.68%–21.43% and decreased the intracellular amylase production by 19.18%–20.26%, decreased the extracellular pectinase production by 36.71%–38.30% and increased the intracellular pectinase production by 35.68%–36.53%, decreased the extracellular cellulase production by 28.04%–33.82% and increased the intracellular cellulase production by 15.28%–18.19%. Compared with WT, the knockout of sakA down-regulated the transcript levels of spore production-related genes (fluG, sfgA, flbA, flbB, flbD, laeA, brlA, abaA, vosA, stuA, and velB) by 8.53%–90.87%. Furthermore, it down-regulated the transcript levels of amylase-related genes (amyC, amyD, amyE, amyF, amyG, and amyH) and the transcription factor (amyR) by 8.87%–87.50%, the pectinase-related genes (aglB, lacA, pexB, pecA, pecC, pecB, endA, endC, and poly) by 23.23%–84.01%, the cellulase-related genes (xlnR, chbA, chbB, and eglB) by 3.75%–81.02%, and the hyperosmotic regulation-related genes (ena1, ena2, sho1, nik1, ypdl, pkA, and hAD) by 5.27%–94.36%. [Conclusion] The sakA gene of A. niger positively regulates spore production and is essential for spore production. The knockout of sakA affects the spore production of A. niger. Furthermore, SakA plays a crucial role in the synthesis and secretion of amylase, pectinase, and cellulase as well as osmotic stress response.

[Objective] Poly(butylene adipate-co-butylene 2,5-furandicarboxylate) (PBAF) is a biodegradable furan-based copolyester plastic. In the chemical synthesis of PBAF, the randomness of oligomer polymerization leads to the formation of complex polymers, such as block copolymers, random copolymers, and alternating copolymers. In this study, dimethyl furan-2,5-dicarboxylate (FDME) and 1,4-butanediol (BDO) were used as substrates to synthesize bis-BDO ester by enzymatic reaction to provide the bioplastic precursor for controllable polymerization and avoid complex by-products. [Methods] RgPETase from Rhizobacter gummiphilus was heterogeneously expressed in Escherichia coli BL21(DE3). RgPETase exhibited acyltransferase activity for FDME and BDO. The reaction conditions including pH, temperature, content of BDO (as both substrate and solvent), and amount of enzyme for the synthesis of bis-BDO ester were optimized. [Results] The optimum reaction conditions of RgPETase were pH 8.0, BDO content of 30%, and reaction temperature within the range of 25–30 ℃. Under the optimum conditions (30 ℃ and enzyme concentration of 6 μmol/L), RgPETase can catalyze 10 mmol/L FDME to produce (2.96±0.01) mmol/L bis-BDO ester. [Conclusion] RgPETase exhibits high acyltransferase activity and catalyzes the generation of bis-BDO ester from FDME via acyl transfer reaction under mild conditions, which provides a green and sustainable approach for synthesizing the precursor of PBAF.

[Objective] Ergothioneine, a rare natural amino acid, is a powerful antioxidant with important physiological functions in the body. It has been widely used in the fields of food, medicine, and cosmetics. However, extracting ergothioneine from mushrooms and chemical synthesis suffer from low yields and high costs. This study aims to use metabolic engineering approaches to improve the yield of ergothioneine in Actinoplanes sp. HS. [Methods] Firstly, we locked onto the genes potentially involved in ergothioneine synthesis in Actinoplanes sp. HS by bioinformatics analysis. Then, we identified the functions of these genes by heterologous expression in Escherichia coli BL21(DE3). Finally, the identified functional genes were combined and overexpressed in Actinoplanes sp. HS, and the yields of ergothioneine in the mutant strains were measured. By adding different concentrations of precursors to the fermentation medium, we investigated the impact of precursor concentration on the yield of ergothioneine. [Results] The enzymes encoded by BC03-04016, BC03-04015, BC03-04014, and BC03-04013 in Actinoplanes sp. HS could synthesize the ergothioneine precursor hercynine-cysteine-sulphoxide (HER-Cys-Sul). The enzymes encoded by BC03-04046 and BC03-04917 had the function of cleaving carbon-sulfur bonds, which could catalyze the formation of the final product ergothioneine from HER-Cys-Sul. The mutant strains YC313 and YC314 obtained through overexpression of genes in ergothioneine synthesis showed ergothioneine yields of 125 mg/L and 108 mg/L, respectively, which were 2.9 times and 2.5 times that of the wild-type strain Actinoplanes sp. HS. Additional supplementation of 0.35 g/L methionine in the fermentation medium increased the ergothioneine yield of YC313 by 24% compared with that in the original medium, and the addition of 10 g/L soybean meal resulted in a 19% increase in the ergothioneine yield. [Conclusion] In this study, we identified the genes and their functions in ergothioneine synthesis in Actinoplanes sp. HS and obtained two strains with high yields of ergothioneine through metabolic engineering. Furthermore, we investigated the impact of precursor supply on the yield of ergothioneine, which provided strategic support for the production of ergothioneine by Actinoplanes sp.