[Objective] Considering the important role of signal peptides in the secretory expression of heterologous proteins, we devised an automated high-throughput platform for the automatic screening of signal peptides, aiming to explore the effects of different signal peptides in Bacillus subtilis on the expression of heterologous proteins. [Methods] First, using the Escherichia coli-B. subtilis shuttle vectors pHP13 and pMA5 as the skeleton, we amplified the cell division B lethal gene (ccdB) and then ligated it to the middle of the promoter and the target gene to build the signal peptide screening vector. With the genomic DNA of B. subtilis 168 as the template, 173 signal peptides were amplified. An automated platform was established for the expression and screening of heterologous proteins in B. subtilis. Furthermore, the recombinant strains of heterologous proteins containing different signal peptides were constructed, and the effects of different signal peptides on the secretory expression of heterologous proteins were investigated. [Results] Five signal peptides (RpmG, AspB, CitH, LytF, and YkwD) showed strong abilities to induce the export of GFP from B. subtilis. Among them, RpmG had the strongest ability to induce the export of GFP, and the extracellular GFP fluorescence of the recombinant strain increased by 236% compared with that of the control strain. In addition, 41 signal peptides were not compatible with pullulanase (PulA), while the two signal peptides RpmG and AspB showed strong abilities to export PulA. The highest PulA activity of 116 U/mL was detected from the recombinant strain carrying the signal peptide RpmG, and the extracellular enzyme activity was 52 U/mL. The secretion rate of the PulA recombinant strain carrying the signal peptide AspB reached 74%, which was 68% higher than that of the control strain. [Conclusion] We developed an automated platform for high-throughput screening of the heterologous protein signal peptides in B. subtilis and obtained the signal peptides capable of improving the secretory expression of GFP and PulA. This automated platform allowed the parallel processing of a considerable number of samples, which simplified the repetitive manual laboratory work. This platform outperformed manual operation in terms of both time consumption and cost. The advantage of the automated high-throughput platform will be more significant with the increase in sample size. In summary, the established automatic high-throughput screening platform not only accelerates the screening process of signal peptides of heterologous proteins, but also provides new technical support for the modification and iteration of industrial strains of other value-added proteases.

[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.

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 develop a rapid nucleic acid detection method for Marburg virus based on clustered regularly interspaced short palindromic repeats/associated protein 13a (CRISPR/Cas13a). [Methods] According to the conserved region of Marburg virus nucleoprotein (NP) gene, specific primers for reverse transcription recombinase-aided amplification (RT-RAA) and CRISPR RNA (crRNA) were designed and synthesized. RT-RAA was employed to amplify the target sequence. The amplification products were detected by the CRISPR-Cas13a system, and the results were interpreted by easy-readout and sensitive enhanced (ERASE) lateral flow test strips. Finally, the national reference panel was used to evaluate the sensitivity and specificity of the new method. [Results] A set of high-efficiency RT-RAA primers and crRNA targeting Marburg virus NP gene was screened, on the basis of which a CRISPR-ERASE method for the detection of Marburg virus was developed. The target nucleic acid with a concentration of 1 copy/μL could be detected within 1 h, and there was no cross-reaction with other several pathogens. [Conclusion] In this study, a rapid, simple, highly sensitive, and specific nucleic acid detection method for Marburg virus was developed based on CRISPR/Cas13a.

[Objective] To study the physicochemical properties and active components of extracellular antibacterial substances from Bacillus subtilis subsp. spizizenii Bspi2104. [Methods] The Oxford cup method was employed to determine the antibacterial activities and physicochemical properties of B. subtilis subsp. spizizenii Bspi2104 with broad-spectrum antibacterial activity and B. subtilis subsp. spizizenii BspiL6 without antibacterial activity. Metabolomics was employed to detect the antibacterial components of the extracellular products of the strain Bspi2104. [Results] The treatment with trypsin, papain, protease K, pepsin, and lipase had no significant effect on the antibacterial activity of extracellular products. Extracellular products lost the antibacterial activity after treatment at 80 ℃ and 100 ℃ and presented decreased antibacterial activity at pH 9.0 and pH 11.0, especially at higher pH values. There was no significant difference in the antibacterial activity between the extracellular products treated with and without UV (P>0.05). The ammonium sulfate precipitates at 60%, 70%, and 80% saturation exerted antibacterial activities, which was the strongest at the saturation of 70%. The extracellular products of the two strains were extracted by hydrochloric acid precipitation, combined with methanol extraction, ethyl acetate extraction, and chloroform extraction, and all the extracts showed antibacterial activities. The ethyl acetate extract had the strongest antibacterial activity. LC-MS/MS was employed to analyze the composition of extracellular products of Bspi2104 and BspiL6 extracted with different methods. There were 35 common differential metabolites in the extracellular products of the two strains extracted with different methods. The differential metabolites belonged to 37 categories of compounds, including carboxylic acids and derivatives, fatty acids, organic oxygen-containing compounds, organic nitrogen-containing compounds, steroids and derivatives, pregnenolone lipids, phenols, alkaloids and derivatives, glycerol phosphates, isoflavonoids, and benzene and substituted derivatives. Some of these compounds, such as kurarinone, and surfactin B, had antibacterial activities. [Conclusion] The extracellular products of B. subtilis subsp. spizizenii Bspi2104 had good physicochemical stability and maintained high antibacterial activity after treatment with various proteases and lipases, and at −20 ℃ to 60 ℃, pH 1.0–11.0, and UV irradiation for 3 h. The ammonium sulfate precipitates and the extracts from hydrochloric acid precipitation combined with methanol extraction, ethyl acetate extraction, and chloroform extraction of the extracellular products of B. subtilis subsp. spizizenii Bspi2104 had antibacterial activities. Among them, the 70% ammonium sulfate precipitate and ethyl acetate extract had the best antibacterial effects. The extracellular products of the strain contained diverse categories of antibacterial compounds. The findings provide theoretical reference for the discovery and screening of antibacterial components of Bacillus, and the related metabolites have research prospects.

[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] 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] 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] 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.