A recombinant expression vector for producing antibacterial peptide microcin J25, a recombinant bacillus subtilis and application thereof

Abstract

The application belongs to the technical field of genetic engineering, and particularly relates to a recombinant expression vector for producing antibacterial peptide Microcin J25, a recombinant Bacillus subtilis and application. The recombinant expression vector comprises an antibacterial peptide MccJ25 gene cluster and a promoter, and the promoter comprises one or two of a P43 promoter, a Pveg promoter, a SPspovG promoter or a PHA-1 promoter. The application further provides a recombinant Bacillus subtilis comprising the recombinant expression vector, and systemically optimizes fermentation process parameters in a shake flask and a fermentation tank. The recombinant Bacillus subtilis constructed by the application can be applied to the prevention and control of bacterial diseases caused by Gram-negative bacteria such as Salmonella, Vibrio and Aeromonas in breeding, and provides a safe and efficient antibiotic replacement product for the breeding industry.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to a recombinant expression vector for producing the antimicrobial peptide Microcin J25, recombinant Bacillus subtilis, and its applications. Background Technology

[0002] Antimicrobial peptides (AMPs) are small molecule active polypeptides that are widely found in various organisms in nature, such as animals, plants, and microorganisms. As a core component of the innate immune system, they are the first line of defense against the invasion of external pathogens and play an irreplaceable role in the survival and evolution of organisms.

[0003] MccJ25 is a small bacteriocin produced by *Escherichia coli*. It is one of the few known cyclic antimicrobial peptides with a lasso structure, which endows it with excellent biological properties. Studies have shown that MccJ25 has potent inhibitory effects on a variety of pathogenic Gram-negative bacteria, especially maintaining high antibacterial activity against common multidrug-resistant Gram-negative bacteria. This characteristic makes it an important candidate molecule for addressing antibiotic resistance. Furthermore, MccJ25 exhibits excellent biocompatibility, extremely low toxicity to host cells, and good stability in complex physiological environments, laying the foundation for its application in medicine, animal husbandry, and food industries.

[0004] However, the research and industrialization of antimicrobial peptides still face numerous bottlenecks. Antimicrobial peptides generally suffer from low expression levels and complex separation and purification processes, resulting in high costs for large-scale production. Although existing studies often employ heterologous expression strategies to improve the expression and purification efficiency of antimicrobial peptides, factors such as differences in expression chassis strains and imperfect optimization of fermentation process parameters limit their large-scale preparation and industrial application. Therefore, constructing efficient heterologous expression chassis and optimizing fermentation processes are key challenges that urgently need to be overcome in the current field of antimicrobial peptide research. Summary of the Invention

[0005] To address the shortcomings of the existing technologies, the purpose of this invention is to provide a recombinant expression vector for producing the antimicrobial peptide Microcin J25, recombinant Bacillus subtilis, and its applications. This invention selects Bacillus subtilis 1A751 as the substrate strain and pMATE06 plasmid as the expression vector to conduct heterologous expression and fermentation process optimization studies of the antimicrobial peptide MccJ25. Ultimately, a highly efficient and stable expression system for the antimicrobial peptide MccJ25 is constructed, laying the theoretical and technical foundation for its large-scale production and practical application.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a recombinant expression vector for producing the antimicrobial peptide Microcin J25, the recombinant expression vector comprising an antimicrobial peptide MccJ25 gene cluster and a promoter, the promoter comprising P 43 promoter, P veg promoter, SP spovG promoter or P HA One or two of the -1 promoters; The nucleotide sequence of the antimicrobial peptide MccJ25 gene cluster is shown in SEQ ID NO:1; the P 43 The nucleotide sequence of the promoter is shown in SEQ ID NO:2, P veg The nucleotide sequence of the promoter is shown in SEQ ID NO:3, SP spovG The nucleotide sequence of the promoter is shown in SEQ ID NO:4, P HA The nucleotide sequence of the -1 promoter is shown in SEQ ID NO:5.

[0007] A second aspect of the present invention provides a method for constructing a recombinant expression vector as described in the first aspect, comprising the following steps: Construct carrying P 43 promoter, P veg promoter, mcjA Genes and mcjBCD The plasmid pMATE06-PAP containing the gene was double-digested, and the contents of the plasmid pMATE06-PAP were recovered. mcjBCD Linearized vector fragments of genes; PCR amplification of a fusion gene fragment, wherein the fusion gene fragment contains, from the 5' end to the 3' end, the target first promoter sequence, ... mcjA Gene, second promoter sequence; The fusion gene fragment and the linearized vector fragment are joined by a seamless cloning method to obtain a recombinant expression vector.

[0008] In a third aspect, the present invention provides a recombinant Bacillus subtilis that produces the antimicrobial peptide Microcin J25, wherein the recombinant Bacillus subtilis comprises the recombinant expression vector described in the first aspect.

[0009] A fourth aspect of the present invention provides a method for constructing recombinant Bacillus subtilis producing the antimicrobial peptide Microcin J25, comprising the following steps: The recombinant expression vector described in the first aspect was transferred into the competent Bacillus subtilis cells to obtain recombinant Bacillus subtilis producing the antimicrobial peptide Microcin J25.

[0010] A fifth aspect of the present invention provides a method for culturing recombinant Bacillus subtilis as described in the third aspect, comprising the following steps: The recombinant Bacillus subtilis producing the antimicrobial peptide Microcin J25 was revived and inoculated into a solid culture medium for culture; the recombinant Bacillus subtilis producing the antimicrobial peptide Microcin J25 after slant culture was inoculated into a primary liquid culture medium for primary seed culture; seed liquid was obtained; the seed liquid was inoculated into a secondary liquid culture medium for fermentation culture; when the cells grew to mid-log phase, an inducer was added to induce expression.

[0011] A sixth aspect of the present invention provides a fermentation method for recombinant Bacillus subtilis as described in the third aspect, comprising the following steps: The seed culture of recombinant Bacillus subtilis was inoculated into the fermentation medium and fermented in a fermenter. When the cells grew to the mid-log phase, an inducer was added to induce expression. The pH was maintained during the fermentation process, and the fermentation broth was collected.

[0012] A seventh aspect of the present invention provides the application of the recombinant expression vector described in the first aspect and the recombinant Bacillus subtilis described in the third aspect in the prevention and control of diseases in aquaculture.

[0013] Compared with the prior art, the technical solution of the present invention has the following beneficial effects: The recombinant expression vector provided by this invention carries the gene encoding the antimicrobial peptide MccJ25. mcjA Genes and mcjBCD The recombinant Bacillus subtilis strain containing the recombinant expression vector of this invention, along with its promoter sequence, was successfully introduced into Bacillus subtilis, enabling it to express and secrete the antimicrobial peptide MccJ25 with antibacterial activity. Antimicrobial activity assays showed that the recombinant Bacillus subtilis strain containing this invention exhibited significant antimicrobial activity against a variety of Gram-negative pathogens (including Salmonella, Vibrio, and Aeromonas), with inhibition zone diameters ranging from 33.90 to 39.61 mm and inhibition titers from 6.95 to 8.70 U / mL. This provides an important genetically engineered strain resource for the subsequent industrial production and application of the antimicrobial peptide MccJ25. Attached Figure Description

[0014] Figure 1 The diagram illustrates the construction of recombinant plasmids. In this diagram, A shows the fusion of the mcjA gene with different promoters, and B shows the construction of recombinant plasmids pMATE06-PAP, pMATE06-PA1, and pMATE06-1AP.

[0015] Figure 2The results show the identification of recombinant plasmids. In A: M is DL15000 DNA Ladder; 1 is recombinant plasmid pMATE06-PAP. In B: M is DL15000 DNA Ladder; 1 is recombinant plasmid pUC57-PA1; 2 is recombinant plasmid pUC57-1AP.

[0016] Figure 3 for mcjA -2 / 3 of the gene identification results, where A:M is the DL2000 DNA Ladder; 1-4 are the amplified genes. mcjA -2 gene, B:M is DL2000 DNA Ladder; 1-4 are amplified genes. mcjA -3 gene.

[0017] Figure 4 The results of PCR identification of recombinant plasmids pMATE06-PA1 and pMATE06-1AP are shown in Figures 1-3. In Figure 1, M represents the DL2000 DNA Ladder, and Figures 1-3 represent the PCR identification results of recombinant plasmid pMATE06-PA1. In Figure 2, M represents the DL2000 DNA Ladder, and Figures 1-3 represent the PCR identification results of recombinant plasmid pMATE06-1AP.

[0018] Figure 5 The PCR identification results of recombinant strains Bs-PAP, Bs-PA1, and Bs-1AP are shown below. In the figures, A: M is DL15000 DNA Ladder; 1 is recombinant plasmid pMATE06-PAP; 2 is recombinant plasmid pMATE06-PA1; 3 is recombinant plasmid pMATE06-1AP; B: M is DL2000 DNA Ladder; 1-4 are identification results of recombinant strain Bs-PAP; C: M is DL2000 DNA Ladder; 1-4 are identification results of recombinant strain Bs-PA1; D: M is DL2000 DNA Ladder; 1-4 are identification results of recombinant strain Bs-1AP.

[0019] Figure 6 This is the growth curve of the strain.

[0020] Figure 7The results of the fermentation broths of various strains against common pathogenic bacteria in livestock and poultry are shown. Among them, A is Salmonella enteritidis SE11; B is Salmonella H9825; C is Salmonella H9812; D is Salmonella pullorum SP12; E is Escherichia coli ATCC25922; F is Staphylococcus aureus SA25923; G is Staphylococcus aureus MASA21; H is Staphylococcus aureus ATCC6538; I is Pseudomonas aeruginosa CMCC10104; J is Clostridium perfringens CP24; K is Klebsiella pneumoniae KP17; L is Proteus mirabilis PM95; "+" is positive control chloramphenicol (25 μg / mL); "-" is blank control 2×SR medium; 1 is fermentation broth of strain Bs-PAP; 2 is fermentation broth of strain Bs-PA1; 3 is fermentation broth of strain Bs-1AP; 4 is fermentation broth of empty vector strain 1A751.

[0021] Figure 8 The results of the fermentation broths of each strain against common pathogenic bacteria in freshwater fish are shown. Among them, A is Providencia reticularis PR41; B is Aeromonas vesiculosus AV33; C is Aeromonas vesiculosus TH0426; D is Aeromonas hydrophila GDP34; E is Aeromonas guinea pig AC16; F is Aeromonas hydrophila TPS46; G is Nocardia ATCC43993; "+" is positive control chloramphenicol (25 μg / mL); "-" is blank control 2 × SR medium; 1 is fermentation broth of strain Bs-PAP; 2 is fermentation broth of strain Bs-PA1; 3 is fermentation broth of strain Bs-1AP; 4 is fermentation broth of empty vector strain 1A751.

[0022] Figure 9 The results of the fermentation broths of various strains against common pathogenic bacteria in marine fish are shown. Among them, A is Vibrio harveyi ATCC33868; B is Aeromonas salmonicidae AS14; C is Vibrio alfaquelae VA-1; D is Edwardsiella tarda ET47; E is Vibrio alginolyticus ATCC17749; F is Vibrio vulnificus ATCC27562; G is Vibrio anguillarum ATCC19264; H is Vibrio parahaemolyticus ATCC33847; "+" is the positive control chloramphenicol (25 μg / mL); "-" is the blank control 2 × SR medium; 1 is the fermentation broth of strain Bs-PAP; 2 is the fermentation broth of strain Bs-PA1; 3 is the fermentation broth of strain Bs-1AP; 4 is the fermentation broth of empty vector strain 1A751.

[0023] Figure 10The effect of different carbon sources on the antibacterial activity of strain Bs-PA1 was investigated. In this study, A represents starch; B represents mannose; C represents glycerol; D represents fructose; E represents trehalose; F represents dextrin; G represents xylose; H represents glucose; I represents sucrose; "+" represents the positive control chloramphenicol (25 μg / mL); "-" represents the blank control 2 × SR medium; P represents the fermentation broth of strain Bs-PA1; and J represents the bar graph of the antibacterial titers of different carbon sources. Different letters indicate significant differences between groups, and p < 0.05.

[0024] Figure 11 The effects of different organic nitrogen sources on the antibacterial activity of strain Bs-PA1 were investigated. A was soybean peptone; B was soybean meal; C was yeast extract HB8273; D was yeast extract LP0021B; E was beef extract; F was tryptone; G was tryptone:yeast extract (2:6); H was tryptone:yeast extract (4:4); I was tryptone:yeast extract (6:2); J was fish peptone; K was corn peptide; "+" was the positive control chloramphenicol (25 μg / mL); "-" was the blank control 2 × SR medium; and P was the fermentation broth of strain Bs-PA1.

[0025] Figure 12 The bar chart shows the antibacterial potency of different organic nitrogen sources; different letters indicate significant differences between groups, and p < 0.05.

[0026] Figure 13 The effect of different yeast species on the antibacterial activity of strain Bs-PA1 was investigated. In this study, A represents FM760; B represents FM802; C represents FM828; D represents FM902; E represents HB8273; F represents LM808; G represents LP0021B; "+" represents the positive control chloramphenicol (25 μg / mL); "-" represents the blank control 2 × SR medium; P represents the fermentation broth of strain Bs-PA1; H represents the bar graph of the antibacterial titers of different yeast species; different letters indicate significant differences between groups, and p < 0.05.

[0027] Figure 14 The effect of different inorganic nitrogen sources on the antibacterial activity of strain Bs-PA1 was investigated. In this study, A represents ammonium sulfate; B represents ammonium chloride; C represents urea; D represents ammonium nitrate; "+" represents the positive control chloramphenicol (25 μg / mL); "-" represents the blank control 2 × SR medium; P represents the fermentation broth of strain Bs-PA1; and E represents the bar graph of the antibacterial titers of different inorganic nitrogen sources. Different letters indicate significant differences between groups, and p < 0.05.

[0028] Figure 15The effect of different non-phosphorus nutrients on the antibacterial activity of strain Bs-PA1 was investigated. In this study, A represents calcium sulfate; B represents potassium sulfate; C represents magnesium sulfate; D represents calcium chloride; E represents potassium chloride; F represents magnesium chloride; G represents sodium chloride; "+" represents the positive control chloramphenicol (25 μg / mL); "-" represents the blank control 2 × SR medium; P represents the fermentation broth of strain Bs-PA1; and H represents the bar graph of the antibacterial titers of different non-phosphorus nutrients. Different letters indicate significant differences between groups, and p < 0.05.

[0029] Figure 16 The effect of different phosphates on the antibacterial activity of strain Bs-PA1 was investigated. In this study, A represents potassium dihydrogen phosphate; B represents sodium dihydrogen phosphate; C represents dipotassium hydrogen phosphate; D represents disodium hydrogen phosphate; E represents a 1:1 ratio of dipotassium hydrogen phosphate to potassium dihydrogen phosphate; F represents a 1:1 ratio of sodium dihydrogen phosphate to disodium hydrogen phosphate; "+" represents the positive control chloramphenicol (25 μg / mL); "-" represents the blank control 2 × SR medium; P represents the fermentation broth of strain Bs-PA1; and G represents a bar graph of the antibacterial titers of different phosphates. Different letters indicate significant differences between groups, with p < 0.05.

[0030] Figure 17 The effects of different trace elements on the antibacterial activity of strain Bs-PA1 were investigated. In this study, A represents manganese sulfate; B represents ferric sulfate; C represents copper sulfate; D represents zinc sulfate; E represents ferrous sulfate; F represents manganese chloride; G represents ferric chloride; "+" represents the positive control chloramphenicol (25 μg / mL); "-" represents the blank control 2 × SR medium; P represents the fermentation broth of strain Bs-PA1; and H represents the bar graph of the antibacterial titers of different trace elements. Different letters indicate significant differences between groups, and p < 0.05.

[0031] Figure 18 The results of the antibacterial activity analysis of strain Bs-PA1 in the PB experiment are shown. Among them, AL represents 1-12 different experimental groups designed in the PB experiment, "+" represents the positive control chloramphenicol (25 μg / mL), "-" represents the blank control 2 × SR medium, and P represents the fermentation broth of strain Bs-PA1.

[0032] Figure 19 The results of the antibacterial activity analysis of strain Bs-PA1 in the climbing experiment are shown. In the figure, A and G represent the 1-7 different gradient test groups set in the climbing experiment, respectively. "+" is the positive control chloramphenicol (25 μg / mL); "-" is the blank control 2 × SR medium; and P is the fermentation broth of strain Bs-PA1.

[0033] Figure 20The results of the antibacterial activity analysis of strain Bs-PA1 in the CCD experiment are shown. In the CCD experiment, AT represents 1-20 different experimental groups, "+" represents the positive control chloramphenicol (25 μg / mL), "-" represents the blank control 2 × SR medium, and P represents the fermentation broth of strain Bs-PA1.

[0034] Figure 21 The results of the antibacterial activity analysis of strain Bs-PA1 in the response surface methodology validation experiment are shown. In the figure, A is the antibacterial graph of the optimal culture medium test group, "+" is the positive control chloramphenicol (25 μg / mL), "-" is the blank control 2 × SR culture medium, P is the fermentation broth of strain Bs-PA1, B is the statistical bar graph of inhibition zone diameter, and ns indicates that there is no significant difference between the predicted value and the measured value group (p>0.05).

[0035] Figure 22 The effect of different maltose concentrations on the antibacterial activity of strain Bs-PA1 was investigated. A was 0.125%; B was 1%; C was 2%; D was 3%; E was 4%; F was 5%; "+" represented the positive control chloramphenicol (25 μg / mL); "-" represented the blank control 2 × SR medium; P represented the fermentation broth of strain Bs-PA1; and G represented the antibacterial titer bar graph. Different letters indicated significant differences between groups, with p < 0.05.

[0036] Figure 23 The effect of maltose addition on the antibacterial activity of strain Bs-PA1 is shown in Figure 1, where A represents OD200. 600 =0.5; B is OD 600 =0.6; C is OD 600 =0.7; D is OD 600 =0.8, "+" represents the positive control chloramphenicol (25 μg / mL); "-" represents the blank control 2× SR medium; P represents the fermentation broth of strain Bs-PA1; E represents the bar chart of antibacterial titer, different letters indicate significant differences between groups, and p<0.05.

[0037] Figure 24 The effect of different initial pH values ​​of the culture medium on the antibacterial activity of strain Bs-PA1 was investigated. In this study, A represents pH 5; B represents pH 6; C represents pH 7; D represents pH 8; "+" represents the positive control chloramphenicol (25 μg / mL); "-" represents the blank control 2 × SR medium; P represents the fermentation broth of strain Bs-PA1; and E represents the antibacterial titer bar graph. Different letters indicate significant differences between groups, and p < 0.05.

[0038] Figure 25The effect of different fermentation temperatures on the antibacterial activity of strain Bs-PA1 was investigated. In this study, A represents 30℃; B represents 35℃; C represents 37℃; D represents 40℃; "+" represents the positive control chloramphenicol (25 μg / mL); "-" represents the blank control 2 × SR medium; P represents the fermentation broth of strain Bs-PA1; and E represents the antibacterial titer bar graph. Different letters indicate significant differences between groups, and p < 0.05.

[0039] Figure 26 The effect of different shaking speeds on the antibacterial activity of strain Bs-PA1 was investigated. In this study, A was 100 rpm; B was 150 rpm; C was 220 rpm; D was 250 rpm; "+" represented the positive control chloramphenicol (25 μg / mL); "-" represented the blank control 2 × SR medium; P represented the fermentation broth of strain Bs-PA1; and E represented the antibacterial titer bar graph. Different letters indicated significant differences between groups, with p < 0.05.

[0040] Figure 27 To investigate the effect of the same liquid volume on the antibacterial activity of strain Bs-PA1, where A is 30 mL; B is 40 mL; C is 50 mL; D is 60 mL, "+" represents the positive control chloramphenicol (25 μg / mL); "-" represents the blank control 2 × SR medium; P is the fermentation broth of strain Bs-PA1; and E is the bar graph of antibacterial titer. Different letters indicate significant differences between groups, and p < 0.05.

[0041] Figure 28 The effect of different inoculum amounts on the antibacterial activity of strain Bs-PA1 was investigated. In this study, A was 2%; B was 4%; C was 6%; D was 8%. "+" represented the positive control chloramphenicol (25 μg / mL); "-" represented the blank control 2 × SR medium; P represented the fermentation broth of strain Bs-PA1; and E represented the antibacterial titer bar graph. Different letters indicated significant differences between groups, and p < 0.05.

[0042] Figure 29 This is the result of natural fermentation, where A represents the OD during the fermentation of the empty vector strain 1A751. 600 Changes in pH, glucose concentration, and maltose concentration; B represents the determination of antibacterial activity of the fermentation broth of the empty vector strain 1A751 at different time points; 0: initial fermentation broth; 1: OD at 1 h of fermentation. 600 = 0.7 Fermentation broth after maltose supplementation, and then the fermentation broth was collected every 3 h. C is the OD during the fermentation of recombinant strain Bs-PA1. 600 Changes in pH, glucose concentration, and maltose concentration; D represents the determination of the antibacterial activity of the fermentation broth of recombinant strain Bs-PA1 at different time points; 0: initial fermentation broth; 2: OD at 2 h of fermentation. 600= 0.7 Fermentation broth after maltose supplementation. Fermentation broth was collected every 3 h thereafter. Different letters in the bar chart indicate significant differences between groups, and p < 0.05.

[0043] Figure 30 The results show the fermentation of strain Bs-PA1 under constant pH conditions. A, C, E, and G represent the OD values ​​of recombinant strain Bs-PA1 during fermentation at constant pH 7.5, 7.0, 6.5, and 6.0, respectively. 600 The changes in pH, glucose concentration, and maltose concentration were used to determine the antibacterial activity of the recombinant strain Bs-PA1 at different time points under corresponding pH conditions. 0 represents the initial fermentation broth; 4, 2, 1.5, and 3 represent the fermentation time points to OD. 600 = 0.7 Fermentation broth after maltose supplementation. Fermentation broth was collected every 3 h thereafter. Different letters in the bar chart indicate significant differences between groups, and p < 0.05.

[0044] Figure 31 The results of fed-batch fermentation of strain Bs-PA1 are shown. A and B represent the antibacterial activity assays of the fermentation broth at different stages of both intermittent and continuous feeding fermentation. C and E represent the OD values ​​of the fermentation broth in both intermittent and continuous feeding fermentation. 600 The changes in pH, glucose concentration, and maltose concentration were shown. D and F represent the antibacterial titers of the fermentation broth at different time points during intermittent and continuous feeding fermentation. 0: initial fermentation broth; 1.5: OD at 1.5 h of fermentation. 600 = 0.7 Fermentation broth after maltose supplementation. Fermentation broth was collected every 3 h thereafter. Different letters in the bar chart indicate significant differences between groups, and p < 0.05. Detailed Implementation

[0045] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0046] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, and / or combinations thereof.

[0047] A typical embodiment of the present invention provides a recombinant expression vector for producing the antimicrobial peptide Microcin J25, the recombinant expression vector comprising the antimicrobial peptide MccJ25 gene cluster and a promoter, the promoter comprising P 43 promoter, P veg promoter, SP spovG promoter or P HA One or two of the -1 promoters; The nucleotide sequence of the antimicrobial peptide MccJ25 gene cluster is shown in SEQ ID NO:1; the P 43 The nucleotide sequence of the promoter is shown in SEQ ID NO:2, P veg The nucleotide sequence of the promoter is shown in SEQ ID NO:3, SP spovG The nucleotide sequence of the promoter is shown in SEQ ID NO:4, P HA The nucleotide sequence of the -1 promoter is shown in SEQ ID NO:5.

[0048] In some embodiments, the recombinant expression vector is constructed using pMATE06 as a backbone, and the recombinant expression vector sequentially contains a first promoter, mcjA Gene, second promoter, mcjBCD Gene.

[0049] In some implementations, the promoter includes P 43 / P veg SP spovG / P HA -1 or P HA -1 / SP spovG .

[0050] In some embodiments, the recombinant expression vector sequentially contains P 43 promoter, mcjA Gene, P veg promoter, mcjBCD Gene.

[0051] In some embodiments, the recombinant expression vector sequentially contains SP spovG promoter, mcjA Gene, P HA -1 promoter, mcjBCD Gene.

[0052] In some embodiments, the recombinant expression vector sequentially contains P HA -1 promoter, mcjA Genes, SP spovG promoter, mcjBCD Gene.

[0053] Another typical embodiment of the present invention provides a method for constructing a recombinant expression vector as described in the present invention, comprising the following steps: Construct carrying P 43 promoter, mcjA Gene 、 P veg promoters and mcjBCD The plasmid pMATE06-PAP containing the gene was double-digested, and the contents of the plasmid pMATE06-PAP were recovered. mcjBCD Linearized vector fragments of genes; PCR amplification of a fusion gene fragment, wherein the fusion gene fragment contains, from the 5' end to the 3' end, the target first promoter sequence, ... mcjA Gene, second promoter sequence; The fusion gene fragment and the linearized vector fragment are joined by a seamless cloning method to obtain a recombinant expression vector.

[0054] In some implementations, restriction endonucleases are used. Bst AP I and Eco NI was used to double-digest the pMATE06-PAP plasmid.

[0055] In some embodiments, the fusion gene fragment contains SP from the 5' end to the 3' end. spovG , mcjA Gene, P HA -1, the sequence of the fusion gene is shown in SEQ ID NO:6.

[0056] In some embodiments, the fusion gene fragment contains P from the 5' end to the 3' end. HA -1、 mcjA Genes, SP spovG The sequence of the fusion gene is shown in SEQ ID NO:7.

[0057] A third embodiment of the present invention provides a recombinant Bacillus subtilis that produces the antimicrobial peptide Microcin J25, wherein the recombinant Bacillus subtilis comprises the recombinant expression vector described in the present invention.

[0058] In some embodiments, the recombinant Bacillus subtilis uses Bacillus subtilis 1A751 as the chassis strain.

[0059] In some embodiments, the sensitive targets of the recombinant Bacillus subtilis are mainly distributed in the genera *Salmonella*, *Vibrio*, and *Aeromonas*, including *Salmonella enteritidis* SE11, *Salmonella* H9825, *Salmonella* H9812, *Salmonella pullorum* SP12, *Providencera retinoi* PR41, *Aeromonas vesiculosus* AV33, *Vibrio harveyi* ATCC33868, and *Vibrio parahaemolyticus* ATCC33847.

[0060] A fourth embodiment of the present invention provides a method for constructing recombinant Bacillus subtilis producing the antimicrobial peptide Microcin J25, comprising the following steps: The recombinant expression vector described in this invention was transferred into the competent cells of Bacillus subtilis to obtain recombinant Bacillus subtilis producing the antimicrobial peptide Microcin J25.

[0061] In some embodiments, the present invention does not specifically limit the transfer method, and conventional transfer methods in the art can be used, such as chemical conversion or electroconversion.

[0062] A fifth embodiment of the present invention provides a method for culturing recombinant Bacillus subtilis as described in the present invention, comprising the following steps: The recombinant Bacillus subtilis producing the antimicrobial peptide Microcin J25 was revived and inoculated into a solid culture medium for culture; the recombinant Bacillus subtilis producing the antimicrobial peptide Microcin J25 after slant culture was inoculated into a primary liquid culture medium for primary seed culture; seed liquid was obtained; the seed liquid was inoculated into a secondary liquid culture medium for fermentation culture; when the cells grew to mid-log phase, an inducer was added to induce expression.

[0063] In some embodiments, the secondary liquid culture medium comprises 30-60 g / L glucose, 6-16 g / L yeast extract, 4-10 g / L ammonium chloride, 1-4 g / L magnesium sulfate, 1-4 g / L dipotassium hydrogen phosphate, and 0.5-2 g / L potassium dihydrogen phosphate; preferably, the secondary liquid culture medium comprises 40-60 g / L glucose, 10-13 g / L yeast extract, 4-8 g / L ammonium chloride, 2-3 g / L magnesium sulfate, 2-3 g / L dipotassium hydrogen phosphate, and 0.5-1.5 g / L potassium dihydrogen phosphate; more preferably, the secondary liquid culture medium comprises 50 g / L glucose, 11.64 g / L yeast extract, 5 g / L ammonium chloride, 2.51 g / L magnesium sulfate, 1 g / L potassium dihydrogen phosphate, and 2.54 g / L dipotassium hydrogen phosphate.

[0064] In some embodiments, the yeast powder includes HB8273 and FM760, preferably FM760.

[0065] In some implementations, the OD in the logarithmic mid-term 600 It is 0.6-0.8.

[0066] In some embodiments, the inducer comprises maltose at a concentration of 1-3% (w / v).

[0067] In some embodiments, the culture conditions include an initial pH of 5.0-8.0, a temperature of 30-40°C, a shaking speed of 100-250 rpm, a liquid volume of 30-60 / 250 mL, and an inoculum size of 2%-8% (v / v).

[0068] A sixth embodiment of the present invention provides a fermentation method for recombinant Bacillus subtilis as described in the present invention, comprising the following steps: The fermentation seed liquid was inoculated into the fermentation medium and fermented in a fermenter. When the cells grew to the mid-log phase, an inducer was added to induce expression. The pH was maintained during the fermentation process, and the fermentation broth was collected.

[0069] In some embodiments, the fermentation medium comprises 50-65 g / L glucose monohydrate, 10-16 g / L yeast extract, 2.5-3.5 g / L anhydrous magnesium sulfate, 2.5-3.5 g / L dipotassium hydrogen phosphate, 1-1.5 g / L potassium dihydrogen phosphate, and 4-6 g / L ammonium chloride.

[0070] In some embodiments, the fermentation medium comprises 45-50 g / L glucose monohydrate, 10-12 g / L yeast extract, 2.3-2.5 g / L anhydrous magnesium sulfate, 2.3-2.5 g / L dipotassium hydrogen phosphate, 0.9-1.0 g / L potassium dihydrogen phosphate, and 4.5-5.0 g / L ammonium chloride.

[0071] In some embodiments, the inducer is maltose, and the final concentration of the maltose is 1-3% (w / v).

[0072] In some implementations, the logarithmic midpoint is OD. 600 It is 0.5-0.8.

[0073] In some implementations, the constant pH is 6.0-7.5.

[0074] In some embodiments, the method further includes feeding during fermentation, wherein the feeding method is intermittent or continuous.

[0075] In some implementations, the feed carbon source can be glucose or maltose, and the feed nitrogen source can be yeast powder or ammonium chloride, in order to extend the production cycle of the antimicrobial peptide.

[0076] In some embodiments, the method for preparing the fermentation seed liquid includes: reviving the recombinant Bacillus subtilis producing the antimicrobial peptide Microcin J25 and inoculating it in a solid culture medium for culture; inoculating the recombinant Bacillus subtilis producing the antimicrobial peptide Microcin J25 after slant culture into a primary liquid culture medium for primary seed culture; obtaining seed liquid; and inoculating the seed liquid into a secondary liquid culture medium for culture to obtain fermentation seed liquid.

[0077] In some embodiments, the secondary liquid culture medium comprises 30-60 g / L glucose, 6-16 g / L yeast extract, 4-10 g / L ammonium chloride, 1-4 g / L magnesium sulfate, 1-4 g / L dipotassium hydrogen phosphate, and 0.5-2 g / L potassium dihydrogen phosphate; preferably, the secondary liquid culture medium comprises 40-60 g / L glucose, 10-13 g / L yeast extract, 4-8 g / L ammonium chloride, 2-3 g / L magnesium sulfate, 2-3 g / L dipotassium hydrogen phosphate, and 0.5-1.5 g / L potassium dihydrogen phosphate; more preferably, the secondary liquid culture medium comprises 50 g / L glucose, 11.64 g / L yeast extract, 5 g / L ammonium chloride, 2.51 g / L magnesium sulfate, 1 g / L potassium dihydrogen phosphate, and 2.54 g / L dipotassium hydrogen phosphate.

[0078] In some embodiments, the yeast powder includes HB8273 and FM760, preferably FM760.

[0079] In some implementations, the OD in the logarithmic mid-term 600 It is 0.4-0.6.

[0080] In some embodiments, the culture conditions include an initial pH of 5.0-8.0, a temperature of 30-40°C, a shaking speed of 100-250 rpm, a liquid volume of 30-60 / 250 mL, and an inoculum size of 2%-8% (v / v).

[0081] The seventh embodiment of the present invention provides the application of the recombinant expression vector and recombinant Bacillus subtilis described in the present invention in the prevention and control of diseases in aquaculture.

[0082] In some implementations, the aquaculture includes aquaculture and livestock and poultry farming.

[0083] In some embodiments, the aquaculture includes freshwater aquaculture and marine aquaculture.

[0084] In some implementations, aquaculture includes, but is not limited to, fish (such as grass carp, common carp, tilapia, large yellow croaker, grouper, and perch), shrimp (such as Litopenaeus vannamei and Penaeus monodon), crabs (such as Chinese mitten crab and swimming crab), and shellfish farming.

[0085] In some embodiments, the livestock and poultry farming includes, but is not limited to, pigs, chickens, ducks, geese, pigeons, cattle, and sheep.

[0086] In some embodiments, the disease includes diseases of farmed animals caused by pathogens.

[0087] In some embodiments, the pathogens include Gram-negative pathogens and Gram-positive pathogens; preferably, they are Gram-negative pathogens.

[0088] In some embodiments, the pathogenic bacteria include pathogenic Escherichia coli, Salmonella, Staphylococcus aureus, Klebsiella pneumoniae, Proteus mirabilis, Providencia retinae, Aeromonas verrucosa, Aeromonas guinea pig, Nocardia, Vibrio harveyi, Aeromonas salmonicidae, Vibrio alfakella, and Vibrio alginolyticus, etc. In some embodiments, the application methods of the recombinant Bacillus subtilis include, but are not limited to: directly sprinkling the fermentation liquid or powder of the recombinant Bacillus subtilis into the aquaculture water, mixing it into the feed or feed and feeding it, adding it to drinking water, or directly administering it orally.

[0089] In some embodiments, the recombinant expression vector or recombinant Bacillus subtilis may also be used in combination with other probiotics (such as lactic acid bacteria and yeast) or herbal extracts to synergistically enhance the disease control effect.

[0090] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0091] Example 1: Construction and Activity Analysis of Recombinant Bacillus subtilis Expressing Antimicrobial Peptide McJ25 1. Test strains and plasmids Table 1. Strains used

[0092] Table 2. Plasmids used

[0093] 2. Carrier Construction The gene sequence of the antimicrobial peptide MccJ25 was retrieved from the GenBank database (GenBank accession number: AF061787.1), enabling... mcjA and mcjBCD Genes maintain the same direction; screen and obtain P 43 P veg SP spovG and P HA -1 promoter sequence. The project was commissioned to Sangon Biotech (Shanghai) Co., Ltd. mcjA and mcjBCD Gene sequence and P 43 P veg Promoter fusion, according to P 43 , mcjA P veg , mcjB , mcjC , mcjD The sequence (nucleotide sequence as shown in SEQ ID NO:8) was used to construct and synthesize the secretory expression vector pMATE06, named pMATE06-PAP; Wuhan Jinkairui Biotechnology Co., Ltd. was commissioned to convert the two promoters (P) of the recombinant plasmid pMATE06-PAP into a single gene. 43 P veg The sum of the sequences at both ends mcjA Genes and SP respectively spovG / P HA -1, P HA -1 / SP spovG Promoter fusion was performed, and the resulting plasmid pUC57 was constructed and synthesized. Finally, the plasmid was constructed into the secretory expression vector pMATE06, named pMATE06-PA1 and pMATE06-1AP. Figure 1 ).

[0094] like Figure 1 As shown, according to promoter P 43 and P veg Select the sequences at both ends Bst AP I and Eco Using NI as the restriction site, specific amplification primers BE-F / BE-R were designed to amplify... mcjA -2 gene and mcjA -3 gene. Using F- mcjA -1 / R- mcjA -1 Primer verification mcjA -1 gene (sequencing results show the nucleotide sequence as shown in SEQ ID NO:9).

[0095] Table 3 Primer sequences used

[0096] pMATE06-PAP, pUC57-PA1, and pUC57-1AP plasmids were extracted and detected by agarose gel electrophoresis, as follows: Figure 2 As shown in A and B, the plasmid integrity is good. Restriction endonuclease was used. Bst AP I and Eco NI performed double enzyme digestion on the pMATE06-PAP plasmid and recovered the linearized vector fragment pMATE06- mcjBCDUsing the extracted pUC57-PA1 and pUC57-1AP plasmids as templates, primers BE-F / BE-R were used to... mcjA -2、 mcjA -3 gene amplification. The amplification products were detected by agarose gel electrophoresis, and the results are as follows: Figure 3 As shown, a single, bright target band appeared at approximately 495 bp, with no non-specific amplification bands, indicating successful amplification of the target gene, consistent fragment size with expectations, and complete alignment of the sequencing results with the expected gene sequence. mcjA-2 , mcjA-3 The nucleotide sequences are shown in SEQ ID NO:10 and SEQ ID NO:11, respectively. The target gene fragment was recovered using a gel. Following the instructions of the ready-to-use seamless cloning kit, the recovered target gene fragment (…) mcjA -2、 mcjA -3 gene) and linearized vector fragment pMATE06- mcjBCD Perform seamless cloning ligation. Recombinant plasmid heat shock method is used for transfer. E. coli In DH5α competent cells, positive clones were selected for PCR identification and sequencing. PCR identification was performed using primers BE-F / BE-R, and the results are as follows. Figure 4 As shown, the expected target band appeared at 495 bp, with no extraneous bands; the sequencing results were completely consistent with the expected gene sequence. The correctly identified recombinant plasmids were named pMATE06-PA1 and pMATE06-1AP, respectively, and stored at -20°C for later use.

[0097] 3. Construction and identification of recombinant Bacillus subtilis Preparation of Bacillus subtilis 1A751 competent cells: 1A751 cells were inoculated into GM I medium and cultured overnight, then transferred at 10% (v / v) to fresh GM I medium and cultured until OD. 600 The concentration was approximately 0.7, and then transferred to GM II medium at 10% (v / v) for 1.5 h to induce competent cell formation.

[0098] 1 μg each of pMATE06-PAP, pMATE06-PA1, and pMATE06-1AP plasmids were transformed into 100 μL of Bacillus subtilis 1A751 competent cells. After incubation at 37°C for 1 h, GM II medium was added for recovery for 3 h. The cells were then plated on LB agar plates containing kanamycin to screen for positive transformants. Plasmids from each strain were extracted and identified by PCR using BE-F / BE-R. The results are shown in Table 5. The PCR amplification product of strain Bs-PAP showed the expected band at 612 bp (…). Figure 5 In strain B), the amplification products of strains Bs-PA1 and Bs-1AP showed the expected band at 495 bp. Figure 5 C and Figure 5 (D) Sequencing results showed that the target gene sequence of the plasmid in the strain was completely consistent with the expected sequence, indicating that the recombinant plasmid was successfully transformed into Bacillus subtilis 1A751, and the recombinant Bacillus subtilis was successfully constructed. The correctly identified recombinant strains were named Bs-pMATE06-PAP (Bs-PAP), Bs-pMATE06-PA1 (Bs-PA1), and Bs-pMATE06-1AP (Bs-1AP), respectively. 4. Determination of the growth curve of recombinant Bacillus subtilis Strains Bs-PAP, Bs-PA1, Bs-1AP, and Bacillus subtilis 1A751 containing the empty vector pMATE06 were inoculated into LB medium containing kanamycin and cultured at 37°C with shaking at 220 rpm for 12 h to obtain seed culture. The seed culture was then transferred to 100 mL LB medium at a 1% (v / v) inoculation rate and cultured at 37°C with shaking at 220 rpm. Oddi concentration (OD) was measured every 3 h starting from 0 h. 600 Growth curves of the strains were plotted, and the differences in growth rate and growth cycle between each recombinant strain and the empty vector strain 1A751 were analyzed and compared. The results are as follows: Figure 6 As shown, during the lag phase (0-6 h), the recombinant bacteria were basically the same as the empty vector strain 1A751; during the logarithmic phase (6-14 h), the growth rate of Bs-PA1 was better than that of Bs-PAP and Bs-1AP; after 14 h, the bacteria entered the stationary phase, and the cell density of Bs-PA1 was not significantly different from that of the empty vector, and its stability was better than the other two strains.

[0099] 5. Analysis of the antibacterial activity of recombinant Bacillus subtilis secreted expression McJ25 Single colonies of each strain were picked and inoculated into LB medium containing kanamycin, and cultured at 37°C with shaking at 220 rpm for 12 h. The inoculum was then transferred to 100 mL of 2×SR medium at a 1% (v / v) inoculation rate and cultured at 37°C until OD500. 600 When the concentration of maltose was approximately 0.7, 3% maltose was added to induce expression. After culturing for another 48 hours, the supernatant was collected by centrifugation and became the fermentation broth.

[0100] Using 27 common livestock, poultry, and aquatic pathogens as indicator bacteria, the Oxford cup method was used to detect their antibacterial activity: the indicator bacteria were mixed with 50°C semi-solid LB medium and poured into a petri dish containing an Oxford cup. After cooling, the Oxford cup was removed, and 200 μL of the sample to be tested (containing 25 μg / mL of positive control chloramphenicol, 2×SR medium for blank control, fermentation broth of negative control 1A751, and fermentation broth of each recombinant bacteria) was added to the well. After incubation at 37°C for 16-24 h, the diameter of the inhibition zone was measured.

[0101] Analysis results showed that all three recombinant Bacillus subtilis strains constructed exhibited significant Gram-negative bacteria-targeting antibacterial properties. Figure 7 , 8 (See Table 4). The antibacterial activity of the three recombinant strains was mainly concentrated on Gram-negative pathogens, with weak inhibitory ability against Gram-positive bacteria. Among them, only Staphylococcus aureus ATCC6538 showed a weak inhibitory effect. The sensitive targets of the three recombinant strains were mainly distributed in Salmonella, Vibrio, and Aeromonas.

[0102] The three recombinant strains showed significant differences in their antibacterial spectrum and antibacterial level. Bs-PA1 exhibited moderate antibacterial spectrum coverage, Bs-PAP's antibacterial spectrum was essentially the same as Bs-PA1's, and Bs-1AP had the narrowest antibacterial spectrum. Bs-PA1 showed the strongest antibacterial activity, Bs-PAP's overall antibacterial activity was relatively stable, and Bs-1AP's overall antibacterial activity was relatively weak and limited to only a few tested strains. Considering both the antibacterial spectrum and antibacterial activity, strain Bs-PA1 demonstrated the best antibacterial activity and was determined to be the preferred strain for subsequent research and fermentation optimization.

[0103] Table 4. Antibacterial spectrum analysis of Bs-PAP, Bs-PA1, and Bs-1AP

[0104] Note: No inhibition zone is marked as "-", inhibition zone diameter <10 mm is marked as "+" (weak activity), and inhibition zone diameter 10 mm ≤ <15 mm is marked as "++" (moderate activity).

[0105] Example 2: Optimization of culture medium and culture conditions for recombinant Bacillus subtilis Bs-PA1 1. Basal culture medium The basal culture medium was formulated with yeast extract LP0021B 5 g / L, tryptone 3 g / L and dipotassium hydrogen phosphate 0.6 g / L.

[0106] 2. An Attempt at Quantitative Methods for McJ25 MccJ25 standard was custom-synthesized by MedChemExpress (MCE), the only company on the market that sells this product.

[0107] (2) Expression and purification of MccJ25 Based on the constructed pMATE06-PAP plasmid, specific primers were designed to amplify... mcjABCD Gene sequence; selection Hin d III and Eco RI serves as the restriction site, allowing... mcjABCD The gene fragment was ligated with the linearized pET28a vector fragment and transformed into... E. coliBL21(DE3) was induced to express.

[0108] The MccJ25 standard provided by MCE showed a single peak in chromatographic analysis, but this standard lacked antibacterial activity and was a linear peptide, making it unsuitable as a MccJ25 standard. Furthermore, chromatographic analysis of the fermentation broth of strain Bs-PA1 revealed numerous overlapping peaks, making it impossible to distinguish the peak corresponding to MccJ25. In addition, antibacterial tests on samples collected after induction of the constructed *E. coli* expressing MccJ25 (BL21-MccJ25) showed no antibacterial activity; purification of the samples using Ni-NTA and SDS-PAGE did not detect the target protein. Therefore, subsequent studies will use the diameter of the inhibition zone and the antibacterial titer as evaluation indicators to indirectly quantify the antibacterial activity of MccJ25.

[0109] Methods for determining potency: The Oxford cup inhibition test was used, with chloramphenicol as a positive control, to determine the volumetric activity (U / mL) of the fermentation supernatant of strain Bs-PA1. Vibrio parahaemolyticus ATCC33847 was used as an indicator bacterium (1 × 10⁻⁶). 6 The diameter of the inhibition zone produced by 200 μL of 25 μg / mL chloramphenicol solution is defined as 1 U. The formula for calculating the volumetric activity of the fermentation supernatant is:

[0110] Where: Diameter AMP Diameter is the diameter of the inhibition zone produced by the fermentation broth of the test strain. Cm V represents the diameter of the inhibition zone produced by the chloramphenicol solution; V is the sample volume (mL, 0.2 mL in this experiment).

[0111] 3. Seed culture Strawberry strain Bs-PA1 was streaked onto solid LB medium and incubated upside down at 37°C for 24 h. Single colonies were then picked and inoculated into liquid LB medium and cultured at 37°C with shaking at 220 rpm for 12 h to allow the cells to grow to the mid-logarithmic growth phase (OD). 600 (Reaching 0.6-0.8), to be used as seed liquid for later use.

[0112] 4. Screening of carbon source types The optimal carbon source was screened using single-factor experiments. The fermentation medium formulation consisted of yeast extract LP0021B 5 g / L, tryptone 3 g / L, and dipotassium hydrogen phosphate 0.6 g / L, with all carbon source concentrations at 30 g / L (carbon source types included starch, mannitol, glycerol, fructose, trehalose, dextrin, xylose, glucose, and sucrose). Initial liquid volumes were 50 / 250 mL. The initial pH of the medium was adjusted to 7.0 with 1 mol / L HCl or NaOH, and the medium was autoclaved at 115°C for 15 min, then cooled to room temperature. Seed culture was inoculated into each treatment group at a 4% (v / v) inoculum rate and cultured at 37°C with constant temperature shaking at 220 rpm. When OD... 600 When the concentration reached 0.7, maltose at a final concentration of 3% (w / v) was added to induce expression. After culturing for another 48 h, the fermentation supernatant was collected, and the antibacterial activity of the fermentation broth was determined by the Oxford cup antibacterial test, with the diameter of the inhibition zone as the evaluation index. The method was the same as in Example 1.

[0113] The results showed that the carbon source had a significant effect on the ability of strain Bs-PA1 to synthesize MccJ25. Figure 10 Using antibacterial potency as the evaluation index, the antibacterial potency of the nine carbon sources, from highest to lowest, was: glucose > starch > mannitol > dextrin > xylose > trehalose > sucrose > fructose > glycerol. Among them, when glucose was used as the carbon source, the diameter of the inhibition zone in the fermentation broth reached its maximum (23.01 mm), with an antibacterial potency of 3.91 U / mL, demonstrating significantly better antibacterial activity than other carbon sources. Considering both antibacterial activity and cost-effectiveness, 30 g / L glucose was selected as the carbon source for subsequent experiments.

[0114] 5. Screening of nitrogen source types The optimal nitrogen source was screened using single-factor experiments. Three types of nitrogen sources were screened: organic nitrogen source, yeast extract, and inorganic nitrogen source. The fermentation system and culture conditions were the same as those in section 4, screening of carbon source types, only the nitrogen source composition and concentration were changed.

[0115] (1) Organic nitrogen sources: The concentration of all organic nitrogen sources was 8 g / L. The organic nitrogen sources included soybean peptone, soybean meal, yeast extract HB8273, yeast extract LP0021B, beef extract, tryptone, fish peptone, and corn peptides, as well as different ratios of tryptone and yeast extract LP0021B complexes (2:6, 4:4, and 6:2, w / w). The screening results of organic nitrogen sources showed that ( Figure 11 and 12 When yeast extract HB8273 was used as a single organic nitrogen source, the fermentation broth exhibited the largest inhibition zone diameter (23.52 mm) and an inhibition titer of 4.71 U / mL, demonstrating a significantly better antibacterial effect than other single organic nitrogen sources. The antibacterial effects of the combined nitrogen sources were all lower than those of the single yeast extract HB8273, and no synergistic effect was observed.

[0116] (2) Yeast extracts: The concentration of all yeast extracts was 8 g / L. The types of yeast extracts included yeast powder FM760, FM802, FM828, FM902, HB8273, LM808, and LP0021B. The screening results of the seven yeasts showed that when yeast powder FM760 was used as a nitrogen source, the diameter of the inhibition zone (26.07 mm) of the fermentation broth of strain Bs-PA1 was significantly larger than that of other yeast extracts, and the antibacterial activity was the best, with an antibacterial titer of 5.29 U / mL; the antibacterial activity of HB8273 was second, and the antibacterial activity of other yeast nitrogen source groups was weak. Figure 13 Meanwhile, yeast powder FM760 has the advantages of stable supply and low price. Therefore, yeast powder FM760 was selected as the organic nitrogen source for subsequent experiments.

[0117] (3) Inorganic nitrogen source: The concentration of inorganic nitrogen sources was 5 g / L, and the types of inorganic nitrogen sources included ammonium sulfate, ammonium chloride, urea, and ammonium nitrate. Adding different inorganic nitrogen sources to yeast powder FM760 as an organic nitrogen source resulted in significant differences in the antibacterial activity of the fermentation broth. Figure 14 Among them, when 5 g / L ammonium chloride was added, the diameter of the inhibition zone in the fermentation broth increased significantly (27.41 mm), and the inhibition titer reached 5.38 U / mL. Therefore, 5 g / L ammonium chloride was selected as the inorganic nitrogen source.

[0118] 6. Screening of nutrients The optimal nutrient salts were screened using single-factor experiments. Three types of nutrients were screened: non-phosphorus source nutrients, phosphates, and trace elements. The fermentation system and culture conditions were the same as those in section 4, screening of carbon source types, only the nutrient salt composition was changed.

[0119] (1) Non-phosphorus nutrients: glucose 30 g / L, yeast powder FM760 8 g / L, ammonium chloride 5 g / L, dipotassium hydrogen phosphate 0.6 g / L, the concentration of non-phosphorus nutrients was 1 g / L. The types of non-phosphorus nutrients included calcium sulfate, potassium sulfate, magnesium sulfate, calcium chloride, potassium chloride, magnesium chloride, and sodium chloride. The screening results of the 7 non-phosphorus nutrients showed that ( Figure 15 When magnesium sulfate was used as an inorganic salt component, strain Bs-PA1 exhibited the largest inhibition zone diameter (27.99 mm) and an inhibition titer of 5.52 U / mL. Therefore, 1 g / L magnesium sulfate was selected as a non-phosphorus nutrient salt for subsequent experiments.

[0120] (2) Phosphate: Glucose 30 g / L, yeast powder FM760 8 g / L, ammonium chloride 5 g / L, magnesium sulfate 1 g / L, phosphate concentration of 2 g / L for all. Phosphate types included dipotassium hydrogen phosphate, potassium dihydrogen phosphate, a complex of dipotassium hydrogen phosphate and potassium dihydrogen phosphate (1:1, w / w), disodium hydrogen phosphate, sodium dihydrogen phosphate, and a complex of disodium hydrogen phosphate and sodium dihydrogen phosphate (1:1, w / w). The results showed that ( Figure 16 The combined phosphate treatment group, consisting of 1 g / L dipotassium hydrogen phosphate and 1 g / L potassium dihydrogen phosphate, exhibited the largest inhibition zone diameter (29.79 mm) in the fermentation broth, with an inhibition titer of 5.87 U / mL, demonstrating significantly superior antibacterial effect compared to other groups. Therefore, 1 g / L dipotassium hydrogen phosphate and 1 g / L potassium dihydrogen phosphate were selected as the phosphates for subsequent experiments.

[0121] (3) Trace elements: glucose 30 g / L, yeast powder FM760 8 g / L, ammonium chloride 5 g / L, magnesium sulfate 1 g / L, dipotassium hydrogen phosphate 1 g / L, potassium dihydrogen phosphate 1 g / L, the concentration of each trace element is 1 mg / L, and the trace elements include manganese sulfate, ferric sulfate, copper sulfate, zinc sulfate, ferrous sulfate, manganese chloride and ferric chloride. Compared with the phosphate screening results, the trace element screening results show that ( Figure 17 The addition of trace elements did not significantly enhance the antibacterial activity of strain Bs-PA1. Among them, manganese sulfate, which showed good antibacterial effect, had an inhibition zone of only 25.95 mm and an inhibition titer of 5.46 U / mL. Therefore, the aforementioned exogenous trace elements were not added to the culture medium formulation determined in this experiment.

[0122] 7. Plackett-Burman method for screening key influencing factors Six components—glucose, yeast powder FM760, ammonium chloride, magnesium sulfate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate—were selected as factors for investigation. A PB experimental design was used to screen key influencing factors. Each factor was set to two levels: low (-1) and high (1). The factor codes and level settings are shown in Table 5. A PB experimental design matrix with N = 12 trials was used, with three parallel trials per group. The experimental design is shown in Table 6.

[0123] Table 5. Factor coding and levels in the Plackett-Burman experimental design.

[0124] Table 6 Plackett-Burman Experimental Design Matrix

[0125] The optimal culture medium composition for strain Bs-PA1 was ultimately determined through single-factor experiments to be: glucose, yeast extract FM760, ammonium chloride, magnesium sulfate, dipotassium hydrogen phosphate, and potassium dihydrogen phosphate. The PB experiment, with six culture medium components as the investigative factors and the diameter of the inhibition zone as the response value, showed that ( Figure 18 (See Table 7). The constructed regression model has a P = 0.0146, indicating that the model fits well and has significant statistical significance, which can effectively explain the influence of each factor on the response value.

[0126] Table 7. Results of the Plackett-Burman experiment

[0127] The experimental results are shown in Table 8. The significance analysis of each factor showed that magnesium sulfate, dipotassium hydrogen phosphate, and yeast extract FM760 all had p-values ​​less than 0.05, making them key factors affecting the antibacterial activity of strain Bs-PA1. Their order of influence was D > E > B. Factors A, C, and F all had p-values ​​greater than 0.05, indicating they were not significant factors and had no significant impact on the antibacterial activity of the strain. These results provide a basis for the selection of factors in subsequent steepest climb experiments and central composite designs.

[0128] Table 8. Significance Test of Regression Equations for Factors in the Plackett-Burman Experimental Design

[0129] Note: R 2 = 0.9152, Adi R 2 = 0.8135; p <0.01 indicates a highly significant difference. p <0.05 indicates a significant difference. p >0.05 indicates no significant difference.

[0130] 8. Steepest Climb Test Three key components—yeast extract FM760, magnesium sulfate, and dipotassium hydrogen phosphate—were selected as critical factors. A steepest ramp-up experiment was used to determine the center value of the optimal response region. In the experiment, the ramp-up direction of each key factor was determined based on the regression equation coefficients obtained from the PB experiment: factors with positive coefficients showed an increase in concentration from low to high levels; factors with negative coefficients showed a decrease in concentration from high to low levels. Three parallel experiments were set up for each group.

[0131] Based on the PB experiment results, the three non-significant factors—glucose, ammonium chloride, and potassium dihydrogen phosphate—were fixed at 50 g / L, 5 g / L, and 1 g / L, respectively. A steepest climbing experiment was designed targeting the three key factors: yeast powder FM760, magnesium sulfate, and potassium dihydrogen phosphate. The experimental results showed ( Figure 19 (Table 9) As the concentrations of yeast powder FM760, magnesium sulfate, and dipotassium hydrogen phosphate increased in a gradient, the diameter of the inhibition zone in the fermentation broth of strain Bs-PA1 showed a trend of first increasing and then decreasing: group 4 exhibited the best antibacterial activity. Based on this, yeast powder FM760 11.5 g / L, magnesium sulfate 2.5 g / L, and dipotassium hydrogen phosphate 2.5 g / L were determined to be the central values ​​for subsequent central combination experiments.

[0132] Table 9 Results of the steepest climb test

[0133] 9. Central composite experiment Based on the optimal range obtained from the steepest ascent experiment, five levels (-1.682, -1, 0, 1, and 1.682) were set for the three key factors: yeast powder FM760, magnesium sulfate, and dipotassium hydrogen phosphate. These levels were coded, and Design-Expert 12.0 software was used to design 20 groups, with three parallel experiments per group. The experimental data were fitted and analyzed using the software, constructing a quadratic polynomial regression equation for the inhibition zone diameter on the three key factors. Response surface 3D plots and contour plots were used to visually demonstrate the intensity and trend of interactions among the factors.

[0134] CCD test results show ( Figure 20 (Table 10) The actual values ​​of the inhibition zone diameters of the 20 groups of experiments were basically consistent with the model predictions, indicating that the model has good predictive ability; among them, the central group (11.5 g / L yeast powder FM760, 2.5 g / L magnesium sulfate, and 2.5 g / L dipotassium hydrogen phosphate) showed the best antibacterial activity.

[0135] Software was used to fit the experimental data to construct a quadratic polynomial regression equation between the diameter of the inhibition zone and three key factors: Y = 33.01 + 1.08A + 1.83B + 0.0.3887C - 2.87AB + 0.9096AC - 0.0946BC - 2.39A 2 -2.71B 2 -3.570C 2 In the formula, Y is the predicted diameter of the inhibition zone (mm), and A, B, and C are the coding values ​​for yeast powder FM760, dipotassium hydrogen phosphate, and magnesium sulfate, respectively. The model fit analysis results show that the coefficient of determination of this regression model is... R 2 = 0.9615, indicating that the model fits the experimental data well and can effectively predict the effects of key factors on the antibacterial activity of strain Bs-PA1.

[0136] Table 10. Centralized Combined Experimental Design and Results

[0137] The regression equation was solved using software, and the optimal solution was obtained as follows: yeast extract FM760 = 11.64 g / L, dipotassium hydrogen phosphate = 2.54 g / L, magnesium sulfate = 2.51 g / L. At this point, the predicted inhibition zone diameter Y = 33.42 mm. Combining this with the fixed concentrations of non-significant factors, the optimal culture medium formulation and concentration for strain Bs-PA1 were determined to be: glucose 50 g / L, yeast extract FM760 11.64 g / L, ammonium chloride 5 g / L, magnesium sulfate 2.51 g / L, potassium dihydrogen phosphate 1 g / L, and dipotassium hydrogen phosphate 2.54 g / L.

[0138] To verify the reliability of the optimization results, a validation experiment was conducted using the above-mentioned optimal culture medium formulation. Figure 21 The validation experiment was conducted in triplicate. The measured average diameter of the inhibition zone in the fermentation broth was 33.77 mm (inhibition titer reached 6.46 U / mL), with a relative error of only 0.148% compared to the model prediction of 33.72 mm. These results demonstrate that the established response surface methodology has good predictive accuracy and practical application value, and can provide effective guidance for optimizing the fermentation process of strain Bs-PA1.

[0139] 10. Optimization of cultivation conditions (1) Maltose concentration Based on the optimal culture medium obtained in the previous screening, the initial pH of the medium was adjusted to 7.0, and the liquid volume was 50 / 250mL Erlenmeyer flasks. Six maltose concentration gradients were set at 0.125%, 1%, 2%, 3%, 4%, and 5% (w / v), and the medium was cultured at 220 rpm with shaking until OD. 600 When the concentration of maltose was 0.7, different concentrations of maltose were added, and the mixture was incubated for another 48 hours. All experiments were conducted according to the standard culture conditions established in Section 3.3.3, and antibacterial assays were performed to determine the optimal maltose concentration. The results showed that ( Figure 22 As the maltose concentration increased, the diameter of the inhibition zone in the fermentation broth showed a trend of first increasing and then stabilizing: when the maltose concentration was 2%, the antibacterial activity was the strongest, and the inhibition diameter was 33.79 mm. Therefore, the optimal maltose induction concentration for strain Bs-PA1 to synthesize MccJ25 was determined to be 2% (w / v).

[0140] (2) Maltose induction node time Based on the optimal culture medium obtained in the previous screening, the initial pH of the culture medium was adjusted to 7.0, and the liquid volume was set in 50 / 250mL Erlenmeyer flasks with four different OD values ​​of 0.5, 0.6, 0.7, and 0.8. 600 Gradient culture, incubated at 220 rpm with oscillation until OD. 600At different gradients, 2% (w / v) maltose was added, and the culture was continued for 48 h. All experiments were conducted according to the standard culture conditions established in Section 3.3.3, and antibacterial assays were performed to determine the optimal point for adding maltose. The results showed ( Figure 23 When OD 600 At a concentration of 0.7, the antibacterial activity was optimal, with an inhibition diameter of 33.82 mm and an antibacterial titer of 6.49 U / mL. Therefore, the optimal induction time for adding maltose is at OD... 600 = 0.7.

[0141] (3) Initial pH of the culture medium Based on the optimal culture medium obtained in the previous screening, four different initial pH values ​​(5, 6, 7, and 8) were set in 50 / 250 mL Erlenmeyer flasks and cultured at 220 rpm until OD. 600 When pH = 0.7, 2% (w / v) maltose was added, and the culture was continued for 48 h. All experiments were conducted according to the standard culture conditions established in Section 3.3.3, and antibacterial assays were performed to determine the optimal initial pH of the culture medium. The results showed ( Figure 24 The antibacterial activity was optimal at an initial pH of 7.0, with an inhibition diameter of 33.84 mm and an antibacterial titer of 6.56 U / mL.

[0142] (4) Fermentation temperature Based on the optimal culture medium obtained in the previous screening, the initial pH of the culture medium was adjusted to 7.0, and the liquid volume was 50 / 250mL Erlenmeyer flasks. Four different temperature gradients were set at 30, 35, 37 and 40°C, and the medium was cultured at 220 rpm with shaking until OD. 600 When the temperature reached 0.7, 2% (w / v) maltose was added, and the mixture was cultured for another 48 h. All experiments were conducted according to the standard culture conditions established in Section 3.3.3, and antibacterial assays were performed to determine the optimal fermentation temperature. The results showed that ( Figure 25 37°C is the optimal fermentation temperature for strain Bs-PA1, at which the diameter of the inhibition zone in the fermentation broth is the largest (33.85 mm), and the inhibition titer reaches 6.59 U / mL.

[0143] (5) Shaking speed Based on the optimal culture medium obtained in the previous screening, the initial pH of the culture medium was adjusted to 7.0, and the liquid volume was 50 / 250mL Erlenmeyer flasks. Four different shaking speed gradients of 100, 150, 220, and 250 rpm were set, and the medium was cultured under different shaking conditions until OD. 600 At a concentration of 0.7, 2% (w / v) maltose was added, and the mixture was incubated for another 48 h. All experiments were conducted according to the standard culture conditions established in Section 3.3.3, and antibacterial assays were performed to determine the optimal shaking speed. The results showed ( Figure 26 220 rpm is the optimal shaking speed for strain Bs-PA1. At this speed, the antibacterial activity is optimal, with an inhibition diameter of 33.86 mm and an antibacterial titer of 6.67 U / mL.

[0144] (6) Liquid volume Based on the optimal culture medium obtained in the previous screening, the initial pH of the culture medium was adjusted to 7.0, and four different liquid volumes of 30, 40, 50, and 60 mL were set. The medium was cultured at 220 rpm until OD. 600 When the concentration of maltose reached 0.7, 2% (w / v) maltose was added, and the culture was continued for 48 h. All experiments were conducted according to the standard culture conditions established in Section 3.3.3, and antibacterial assays were performed to determine the optimal liquid volume. The results showed that ( Figure 27 The antibacterial effects of 40, 50, and 60 mL of liquid volume were better than those of 30 mL, but there was no significant difference among the three. Taking into account cell growth, dissolved oxygen conditions, and fermentation broth yield, 60 / 250 mL was selected as the optimal liquid volume (inhibition diameter of 33.88 mm and antibacterial titer of 6.71 U / mL).

[0145] (7) Inoculation volume Based on the optimal culture medium obtained in the previous screening, the initial pH of the medium was adjusted to 7.0, and the liquid volume was 60 / 250mL Erlenmeyer flasks. Four inoculum sizes were set: 2%, 4%, 6%, and 8% (v / v). The culture was carried out under shaking at 220 rpm until OD. 600 When the inoculum concentration reached 0.7, 2% (w / v) maltose was added, and the culture was continued for 48 h. All experiments were conducted according to the standard culture conditions established in Section 3.3.3, and antibacterial assays were performed to determine the optimal inoculum size. The results showed ( Figure 28 The antibacterial activity of 4%, 6%, and 8% inoculum was better than that of 2%, but there was no significant difference among the three. Taking into account both the amount of seed liquid and the antibacterial potency, 4% (v / v) was finally determined to be the optimal inoculum for strain Bs-PA1 (the diameter of the inhibition zone was 33.90 mm, and the antibacterial potency reached 6.95 U / mL).

[0146] In summary, the optimal culture medium formulation and concentration for laboratory shake-flask fermentation are: glucose 50 g / L, yeast extract FM7601 1.64 g / L, ammonium chloride 5 g / L, magnesium sulfate 2.51 g / L, potassium dihydrogen phosphate 1 g / L, and dipotassium hydrogen phosphate 2.54 g / L. Optimal culture conditions: at OD... 600The optimal induction conditions were achieved by adding 2% (w / v) maltose at pH 0.7; the optimal culture conditions were an initial pH of 7.0, a fermentation temperature of 37℃, a shaking speed of 220 rpm, a liquid volume of 60 / 250 mL, and an inoculum size of 4% (v / v). Under these optimal conditions, the Bs-PA1 fermentation broth exhibited significant antibacterial activity against Vibrio parahaemolyticus ATCC33847, with an inhibition zone diameter of 33.90 mm and an inhibition titer of 6.95 U / mL.

[0147] Example 3: Optimization of Fermentation Process for Recombinant Bacillus subtilis Bs-PA1 1. Seed culture medium and fermentation culture medium Seed culture medium: glucose 50 g / L, yeast powder FM760 11.64 g / L, ammonium chloride 5 g / L, magnesium sulfate 2.51 g / L, potassium dihydrogen phosphate 1 g / L, dipotassium hydrogen phosphate 2.54 g / L, pH adjusted to 7.0, sterilized at 115°C for 15 min.

[0148] Fermentation medium: 175 g glucose monohydrate, 40.74 g yeast powder, 8.785 g anhydrous magnesium sulfate, 8.89 g dipotassium hydrogen phosphate, and 3.5 g potassium dihydrogen phosphate were dissolved in 3 L of pure water to form the main fermentation medium solution; 17.5 g ammonium chloride was weighed and dissolved in 100 mL of pure water to prepare an ammonium chloride solution; 70 g maltose was weighed and dissolved in 100 mL of pure water to prepare a maltose solution; 500 mL of pure water was prepared as sterilization water. The main fermentation medium solution, ammonium chloride solution, maltose solution, and sterilization water were then placed together in an automatic high-pressure steam sterilizer and sterilized at 115°C for 15 min.

[0149] Bs-PA1 strain, preserved at -80°C in glycerol, was activated and cultured on solid LB medium at 37°C for 12 h. A single colony was picked and inoculated into 50 mL of kanamycin-resistant liquid LB medium, and cultured at 37°C and 220 rpm for 12 h until the strain entered the logarithmic growth phase (OD2). 600 The inoculum was 0.8%. At an inoculum rate of 4% (v / v), the culture was transferred to the sterilized seed culture medium and incubated at 37°C with shaking at 220 rpm until mid-log (OD2). 600 Within the range of 0.4-0.6, a seed liquid for fermentation was obtained for subsequent fermentation experiments in fermenters.

[0150] 2. Natural fermentation The initial fermentation conditions for strain Bs-PA1 were set as follows: pH 7.0, stirring rate 400 rpm, temperature 37°C, and aeration rate 6 L / min. No pH adjustment or feeding was performed during fermentation. Cell growth was continuously monitored after the start of natural fermentation, and the OD (Organic Degradation) was measured. 600 When the concentration reaches 0.7, maltose with a final concentration of 2% (w / v) is added. 10 mL of fermentation broth is collected every 3 hours via a sterile sampling valve, and the cell growth (OD) is monitored in real time. 600 The changes in antibacterial activity of the fermentation broth, maltose concentration (Fehling's reagent titration method), and glucose concentration (measured using an SBA-40D biosensor analyzer) were recorded until the pH of the fermentation broth reached its lowest point, indicating the end of natural fermentation. The dynamic changes of each indicator were analyzed. Simultaneously, an empty vector strain 1A751 was used as a control, and natural fermentation experiments were conducted under the same conditions to compare the differences in fermentation characteristics between the two strains. The Oxford cup antibacterial method was the same as in Example 1.

[0151] A natural fermentation experiment was conducted in a 5 L fermenter. The dynamic changes of various indicators during natural fermentation are as follows: Figure 29 As shown. OD of empty vector strain 1A751 600 The value reached 0.7 at 1 hour and peaked at 19 hours (OD). 600 (2.47); OD of strain Bs-PA1 600 The value reached 0.7 at 2 hours and peaked at 20 hours (OD). 600 The lowest pH value of the fermentation broth for the empty vector strain 1A751 reached 5.48, while the lowest pH value of the fermentation broth for strain Bs-PA1 only dropped to 5.92. When the OD in the fermentation broth... 600 After the concentration reached 0.7 and maltose was added, the antibacterial activity of the fermentation broth of the empty vector strain 1A751 did not increase significantly during the entire fermentation process, and the overall antibacterial activity was weak; while the antibacterial activity of the fermentation broth of strain Bs-PA1 increased significantly after the addition of maltose inducer, and the antibacterial activity reached its maximum at 17 h (the diameter of the inhibition zone was 36.15 mm, and the antibacterial titer was 7.39 U / mL).

[0152] 3. Analysis of the effects of different pH values ​​on the growth and antibacterial activity of strain Bs-PA1 Four gradient pH-controlled fermentation experiments were designed to explore the optimal pH conditions by setting different fermentation pH environments. Group 1 was set at pH 7.5, with 2 mol / L NaOH continuously added via an automatic feed pump to maintain pH stability; Group 2 was set at pH 7.0, implementing bidirectional dynamic acid-base control; Group 3 was set at pH 6.5; and Group 4 was set at pH 6.0. All experimental groups used a 5 L fermentation system, a fermentation temperature of 37°C, a stirring rate of 200 rpm, and an aeration rate of 6 L / min. Complete glucose consumption was used as the fermentation endpoint. Samples were taken every 3 hours to measure cell growth (OD). 600 The concentrations of glucose and maltose, as well as the antibacterial activity, were measured using the same Oxford cup method as in Example 1. By comparing the changes in various indicators across the four groups of experiments, the effects of different pH control strategies on the growth and metabolism of strain Bs-PA1 were evaluated, thereby identifying the optimal fermentation pH. The results are as follows: Figure 30 As shown.

[0153] (1) Fermentation at constant pH 7.5: The results showed that the cell growth OD 600 The value was lower than that of the naturally fermented group (OD). 600 The antibacterial activity of the fermentation broth was 2.38, and the antibacterial activity of the fermentation broth was significantly lower than that of the natural fermentation group (the diameter of the inhibition zone was 36.15 mm, and the antibacterial titer was 7.39 U / mL).

[0154] (2) Constant pH 7.0 fermentation: The glucose consumption rate of strain Bs-PA1 was faster than that of the pH 7.5 group, and the cell growth was slightly increased, but the antibacterial activity of the fermentation broth was still lower than that of the natural fermentation group.

[0155] (3) Constant pH 6.5 fermentation: Under pH 6.5 conditions, the OD of strain Bs-PA1 600 The value reached 0.7 at 1.5 h, and OD was at 17 h. 600 The pH value began to decline after reaching 2.35, and glucose was completely consumed at 16.5 h. The inhibition zone diameter reached its maximum of 36.86 mm at 16.5 h of fermentation, with an inhibition titer of 7.84 U / mL. The strain exhibited the most vigorous growth and metabolism under this pH condition. Simultaneously, the antibacterial activity of the fermentation broth was significantly higher than that of the natural fermentation group and other pH gradient groups, indicating that the weakly acidic pH 6.5 environment is not only suitable for the growth of strain Bs-PA1 but also significantly promotes the expression of the antibacterial active substance MccJ25, making it the optimal pH condition for the fermentation of this strain.

[0156] (4) Constant pH 6.0 fermentation: Under this pH condition, the glucose consumption rate of strain Bs-PA1 slows down, and OD 600 The value decreased, and the growth of the bacteria was significantly inhibited.

[0157] 4. Fed-feed fermentation At pH 6.5, OD 600 When the ratio is 0.7, add 2% (w / v) maltose to initiate induction and compare the two feeding strategies: (1) Intermittent fed-batch fermentation: Maltose was added every 3 hours, with each addition being 2% (w / v) of the final concentration, until the fermentation endpoint was reached. The Oxford cup antibacterial method was the same as in Example 1. The results are as follows: Figure 31 As shown, the OD of strain Bs-PA1 600 The value continued to rise, reaching 2.44 at 22.5 h of fermentation. The antibacterial activity did not decrease significantly. The diameter of the inhibition zone reached 39.53 mm at 22.5 h of fermentation, and the antibacterial titer was 8.56 U / mL.

[0158] (2) Continuous feeding fermentation: A high-concentration maltose (75%) solution was dynamically added to the fermentation system via an automatic feed pump to maintain the maltose concentration in the fermentation broth until the fermentation endpoint. The Oxford cup antibacterial method was the same as in Example 1. Results are as follows: Figure 31 As shown, the OD of strain Bs-PA1 600 The antibacterial activity continuously increased, reaching 2.45 after 22.5 h of fermentation, and did not show a significant decrease after 17 h of induction. The antibacterial titer at 16.5 h of continuous fed-batch fermentation (8.59 U / mL) was basically the same as that at 22.5 h of intermittent fed-batch fermentation (8.56 U / mL). At 22.5 h of continuous fermentation, the inhibition zone diameter reached 39.61 mm, with an antibacterial titer of 8.70 U / mL. Both feeding methods effectively enhanced the antibacterial activity of strain Bs-PA1, with continuous fed-batch fermentation showing better results and representing the optimal feeding strategy for this strain.

[0159] 5. Determination of fermentation organic acid products An Agilent 1260 Infinity II high-performance liquid chromatography system was used. Chromatographic separation was performed using a C18 reversed-phase column (5 μm particle size, 4.6 × 250 mm) with the column temperature maintained at 35°C. The mobile phase consisted of phase A (0.1% phosphoric acid) and phase B (methanol) in gradient elution mode at a flow rate of 0.6 mL / min. The detection wavelength was 210 nm and the injection volume was 10 μL.

[0160] When the pH of strain Bs-PA1 dropped to 5.92 during natural fermentation, the composition was measured as shown in Table 11. Three major organic acids were detected: lactic acid, acetic acid, and citric acid. Lactic acid and acetic acid are the most important acidic metabolites during the fermentation process of strain Bs-PA1.

[0161] Table 11 Results of organic acid detection in fermentation broth of strain Bs-PA1

[0162] 6. Data Analysis SPSS 27.0 statistical software was used to perform one-way ANOVA on the experimental data, and Duncan's multiple comparison method was used to test the significance of differences between groups. Different letters indicate significant differences (p<0.05).

[0163] In summary, (1) the results of the natural fermentation experiment showed that the minimum pH of recombinant Bacillus subtilis Bs-PA1 fermentation could be reduced to 5.92, and the acidic substances were mainly lactic acid, acetic acid and citric acid. The results of the constant pH gradient fermentation experiment showed that recombinant Bacillus subtilis Bs-PA1 had the best growth rate and MccJ25 expression effect at pH 6.5, and the inhibition diameter against Vibrio parahaemolyticus ATCC33847 reached 36.86 mm with an inhibition titer of 7.84 U / mL.

[0164] (2) The results of the fed-bacterial fermentation experiment showed that when the feeding was intermittent, the diameter of the inhibition zone of recombinant Bacillus subtilis Bs-PA1 against Vibrio parahaemolyticus ATCC33847 reached 39.53 mm and the inhibition titer was 8.56 U / mL; when the feeding was continuous, the diameter of the inhibition zone reached 39.61 mm and the inhibition titer was 8.70 U / mL.

[0165] Finally, it should be noted that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions described in the foregoing embodiments or make equivalent substitutions for some of them. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A recombinant expression vector for producing the antimicrobial peptide Microcin J25, characterized in that, The recombinant expression vector contains the antimicrobial peptide MccJ25 gene cluster and a promoter, the promoter including P 43 promoter, P veg promoter, SP spovG promoter or P HA One or two of the -1 promoters; The nucleotide sequence of the antimicrobial peptide MccJ25 gene cluster is shown in SEQ ID NO:1; the P 43 The nucleotide sequence of the promoter is shown in SEQ ID NO:2, P veg The nucleotide sequence of the promoter is shown in SEQ ID NO:3, SP spovG The nucleotide sequence of the promoter is shown in SEQ ID NO:4, P HA The nucleotide sequence of the -1 promoter is shown in SEQ ID NO:

5.

2. The recombinant expression vector as described in claim 1, characterized in that, The recombinant expression vector is constructed using pMATE06 as a backbone, and the recombinant expression vector contains, in sequence, a first promoter, and... mcjA Gene, second promoter, mcjBCD Gene.

3. A method for constructing a recombinant expression vector as described in claim 1 or 2, characterized in that, Includes the following steps: Construct carrying P 43 promoter, P veg promoter, mcjA Genes and mcjBCD The plasmid pMATE06-PAP containing the gene was double-digested, and the contents of the plasmid pMATE06-PAP were recovered. mcjBCD Linearized vector fragments of genes; PCR amplification of a fusion gene fragment, wherein the fusion gene fragment contains, from the 5' end to the 3' end, the target first promoter sequence, ... mcjA Gene, second promoter sequence; The fusion gene fragment and the linearized vector fragment are joined by a seamless cloning method to obtain a recombinant expression vector.

4. A recombinant Bacillus subtilis producing the antimicrobial peptide Microcin J25, characterized in that, The recombinant Bacillus subtilis comprises the recombinant expression vector as described in claim 1 or 2.

5. A method for constructing recombinant Bacillus subtilis producing the antimicrobial peptide Microcin J25, characterized in that, Includes the following steps: The recombinant expression vector according to claim 1 or 2 is transferred into the competent Bacillus subtilis cells to obtain recombinant Bacillus subtilis producing the antimicrobial peptide Microcin J25.

6. A method for culturing recombinant Bacillus subtilis as described in claim 4, characterized in that, Includes the following steps: The recombinant Bacillus subtilis producing the antimicrobial peptide Microcin J25 was revived and inoculated into a solid culture medium for culture; the recombinant Bacillus subtilis producing the antimicrobial peptide Microcin J25 after slant culture was inoculated into a primary liquid culture medium for primary seed culture. Seed liquid was obtained; The seed culture was inoculated into a secondary liquid culture medium for fermentation; when the cells grew to the mid-log phase, an inducer was added to induce expression.

7. The cultivation method as described in claim 6, characterized in that, The secondary liquid culture medium comprises 30-60 g / L glucose, 6-16 g / L yeast extract, 4-10 g / L ammonium chloride, 1-4 g / L magnesium sulfate, 1-4 g / L dipotassium hydrogen phosphate, and 0.5-2 g / L potassium dihydrogen phosphate. The culture conditions include an initial pH of 5.0-8.0, a temperature of 30-40°C, a shaking speed of 100-250 rpm, a liquid volume of 30-60 / 250 mL, and an inoculum volume of 2%-8% (v / v).

8. A fermentation method for recombinant Bacillus subtilis as described in claim 4, characterized in that, Includes the following steps: The seed culture of recombinant Bacillus subtilis was inoculated into the fermentation medium and fermented in a fermenter. When the cells grew to the mid-log phase, an inducer was added to induce expression. The pH was maintained during the fermentation process, and the fermentation broth was collected.

9. The fermentation method as described in claim 8, characterized in that, The fermentation medium comprises 50-65 g / L glucose monohydrate, 10-16 g / L yeast extract, 2.5-3.5 g / L anhydrous magnesium sulfate, 2.5-3.5 g / L dipotassium hydrogen phosphate, 1-1.5 g / L potassium dihydrogen phosphate, and 4-6 g / L ammonium chloride; or, The inducing agent is maltose, and the final concentration of maltose is 1-3% (w / v); the logarithmic midline is OD. 600 It is 0.5-0.8; or, The constant pH is 6.0-7.5; or, The method also includes feeding during the fermentation process, wherein the feeding method is intermittent feeding or continuous feeding.

10. The application of the recombinant Bacillus subtilis according to claim 4 or the fermentation broth prepared by the fermentation method according to claim 8 or 9 in the prevention and control of diseases in aquaculture.

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