High-yield microcin MccJ25 antibiotic-free strain EY03 and application thereof

Abstract

The present application relates to the field of microbial technology, and particularly to a high-yield microcin MccJ25 antibiotic-free strain EY03 and application Escherichia coli EY03, which is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No. 38047. The above strain has the following advantages: no resistance marker: completely free of exogenous resistance genes, completely eliminating the risk of horizontal transfer of resistance genes from the root, high biological safety, and environmental friendliness; high genetic stability: the target gene is not lost and the expression level does not decrease after 50 generations of continuous passage, suitable for long-term preservation and industrial scale production; high yield and short cycle: MccJ25 yield > 10 g / L in a 5T fermenter for 24 h fermentation, and the production intensity and industrial adaptability are greatly improved; simple process: the fermentation process is easy to control, the fermentation level is stable, and the post-processing is simple.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to a high-yield microbialin-producing MccJ25 antibiotic-free strain EY03 and its applications. Background Technology

[0002] The antimicrobial peptide MccJ25 is a natural lasso peptide composed of 21 amino acids, microcin J25 (MccJ25), which primarily acts on Gram-negative bacteria, including pathogenic Escherichia coli, Salmonella, and Shigella. Its bactericidal mechanism differs from that of conventional antibiotics, making it less likely to induce drug resistance. Furthermore, MccJ25 peptides can also regulate the body's immunity, maintain intestinal flora balance, and improve nutrient absorption efficiency. Therefore, MccJ25 has broad application prospects in pharmaceutical antibacterial applications, food preservation, antibiotic-free feed additives, and livestock and poultry intestinal health.

[0003] However, the existing construction of McJ25 recombinant bacteria has the following technical defects: (i) Reliance on antibiotic labeling poses a biosafety risk. Most existing MccJ25 recombinant bacteria are plasmid recombinant bacteria carrying resistance genes. Fermentation requires the continuous addition of antibiotics to maintain plasmid stability, which increases production costs and poses serious biosafety risks: exogenous resistance genes may enter the environment or pathogenic microorganisms through horizontal transfer, exacerbating the spread of drug-resistant genes, increasing the risk of "superbug" growth, and threatening the ecological environment and public health safety.

[0004] (ii) Resistance genes can undergo horizontal transfer in the natural environment without human intervention. Studies have shown that plasmids can undergo horizontal transfer in the natural environment without artificial transformation. *E. coli* can undergo horizontal transfer in Ca²⁺. + / Mg² + In a divalent cation environment, naturally competent cells are formed, which directly take up naked plasmid DNA from the environment without artificial induction. Plasmids can also spread directly between bacteria through conjugation transfer. Pollutants such as polycyclic aromatic hydrocarbons, microplastics, and low concentrations of antibiotics in the environment can significantly promote this process.

[0005] In summary, once plasmids carrying resistance genes are released into the environment, they can undergo horizontal transfer without human intervention under the mediation of various environmental factors, and be captured and spread by indigenous bacteria (including potential pathogens).

[0006] (iii) Plasmids are easily lost, resulting in insufficient genetic stability. Traditional plasmid-recombinant bacteria are prone to plasmid loss in the absence of antibiotic selection pressure, leading to reduced yields and making them unsuitable for long-term stable industrial production.

[0007] In conclusion, developing a recombinant strain that is free of resistance markers, genetically stable, and produces high levels of McJ25 is key to solving the aforementioned industry pain points. Summary of the Invention

[0008] This invention provides a high-yield microbial inoculant MccJ25 antibiotic-free strain EY03 and its application, to solve the problems of existing strains having resistance markers, genetic instability, and low MccJ25 yield.

[0009] In a first aspect, the present invention provides an Escherichia coli strain. Escherichia coli EY03, the strain includes: Using Escherichia coli as the starting strain, the strain's genome integrates the mcjBC expression cassette and the mcjD expression cassette; the amino acid genes in the strain's genome are knocked out; the strain contains an antibiotic-free plasmid carrying the amino acid genes and the mcjA expression cassette; and the RBS sequences are modified accordingly.

[0010] In some implementations, the promoter of mcjA in the mcjA expression box is an optimized promoter; And / or, the promoter in the mcjBC expression box is an optimized promoter; And / or, the promoter in the mcjD expression box is an optimized promoter; And / or, the starting strain is Escherichia coli MC4100.

[0011] In some embodiments, the Escherichia coli EY03 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38047.

[0012] Secondly, the present invention provides a method for constructing the aforementioned Escherichia coli EY03, comprising: Using Escherichia coli as the starting strain, the mcjBC expression cassette and mcjD expression cassette were integrated into the strain's genome. The amino acid gene in the strain's genome was knocked out. Antibiotic-free plasmids carrying the amino acid gene and mcjA expression cassette were transformed into the strain, and the RBS sequence was modified accordingly.

[0013] Thirdly, the present invention provides a microbial inoculant containing the aforementioned Escherichia coli EY03, or its culture, or its fermentation product, or its metabolites.

[0014] In some embodiments, the metabolite includes MccJ25 antimicrobial peptide.

[0015] Fourthly, the present invention provides a method for preparing the aforementioned microbial inoculant, comprising the steps of culturing and fermenting the aforementioned Escherichia coli EY03.

[0016] Fifthly, the present invention provides that the Escherichia coli EY03 and the microbial fermentation agent have at least one of the following uses: 1) Its use in the preparation of the antimicrobial peptide MccJ25; 2) Application in the preparation of antibacterial growth-promoting additives for livestock and poultry feed; Optionally, the antibacterial growth-promoting additives for livestock and poultry feed include at least one of the following functions: inhibiting pathogenic Escherichia coli and / or Salmonella in livestock and poultry, reducing the diarrhea rate of weaned piglets and broilers, repairing the intestinal barrier, tolerating the high temperature of 121℃ feed pelleting, and / or replacing feed antibiotics. 3) Application in the preparation of food biological preservatives; Optionally, the food biological preservative includes at least one of the following functions: specifically inhibiting foodborne pathogens in fresh meat, eggs and dairy products and / or extending the shelf life of food; the foodborne pathogens include Escherichia coli O157:H7 and / or Salmonella; 4) Application in the preparation of anti-inflammatory drugs against multidrug-resistant Gram-negative bacteria and / or sepsis; Optionally, the anti-inflammatory drugs against multidrug-resistant Gram-negative bacteria and / or sepsis include targeting bacterial RNA polymerases for bactericidal activity, having no cross-resistance with clinical antibiotics, reducing inflammatory factors in the body, and / or having low cytotoxicity and no hemolysis.

[0017] In a sixth aspect, the present invention provides a method for producing the antimicrobial peptide MccJ25, comprising a fermentation step using the aforementioned Escherichia coli EY03.

[0018] In some embodiments, the method further includes the step of preparing the fermentation strain: activating and culturing Escherichia coli EY03, inoculating the activated culture into a seed culture medium for a first culture to obtain a primary seed liquid; inoculating the primary seed liquid into a seed culture medium for a second culture to obtain the fermentation strain; optionally, the seed culture medium is selected from LB medium; optionally, in the preparation of the primary seed liquid or fermentation strain, the inoculation amount is 1-5 v / v%; optionally, the conditions for the first culture are constant temperature shaking at 37±1℃ for 10-12 h at 100-300 rpm; optionally, the conditions for the second culture are constant temperature shaking at 37±1℃ for 10-12 h at 100-300 rpm, with an aeration ratio of 0.5-1 vvm. And / or, in the fermentation step, the seed culture of Escherichia coli EY03 is inoculated into the fermentation medium at an inoculation rate of 1-5 v / v%, and fermented and cultured at 36-38℃, aeration ratio of 0.5-1 vvm, tank pressure of 0.03-0.05 MPa, pH of 7.0-7.2, with 40-70% glucose added, and fermentation for 24-28 hours; And / or, during the fermentation process, the rotation speed is 550-450 rpm in the early stage of fermentation and 450-350 rpm in the later stage of fermentation; optionally, the early stage of fermentation is the fermentation time from 0 to 8 hours; optionally, the later stage of fermentation is the fermentation time from 8 hours to 24-28 hours. And / or, the fermentation medium comprises the following components at the following concentrations: yeast extract 10-70 g / L, magnesium sulfate 0.5-5 g / L, dipotassium hydrogen phosphate 0.5-5 g / L, citric acid 0-2 g / L, glucose 1-10 g / L, antifoaming agent 0.1-1 g / L, and initial pH 6.5-7.

[0019] The technical solution of this invention has the following advantages: This invention provides a strain of Escherichia coli. Escherichia coli EY03, deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38047, possesses the following advantages: No resistance markers: Completely free of exogenous resistance genes, eliminating the risk of horizontal transfer of resistance genes at the source, resulting in high biosafety and environmental friendliness; High genetic stability: The target gene is not lost or its expression level does not decrease after 50 consecutive generations, making it suitable for long-term preservation and industrial-scale production; High yield and short cycle: A 5T fermenter yields >10 g / L of MccJ25 after 24 hours of fermentation, significantly improving production intensity and industrial adaptability; Simple process: The fermentation process is easy to control, the fermentation level is stable, and post-processing is simple.

[0020] Biological Preservation Escherichia coli EY03 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38047. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China. The deposit date is April 3, 2026. Attached Figure Description

[0021] To more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0022] Figure 1 The above are the electrophoresis results of the EY03 resistance fragment in Example 2 of this invention; wherein: M: nucleic acid marker; 1: ampicillin positive control; 2: kanamycin positive control; 3: EY03 (ampicillin primer amplification); 4: EY03 (kanamycin primer amplification); Figure 2 The growth of strain EY03 in Example 2 of this invention is shown in tubes with no antibiotics, kanamycin, and ampicillin; wherein: 1: tube without antibiotics; 2: kanamycin resistant tube; 3: ampicillin tube; Figure 3 The electrophoresis results of the genetic stability experiment of the recombinant strain EY03 in Example 3 of this invention are shown below. From left to right, the markers are shown, and lanes 1-16 are shown. Lane 1 is the positive control of the first generation of bacteria, and lanes 2-4 are the 10th generation, with 3 colonies selected from each generation. Similarly, lanes 5-16 are the 20th, 30th, 40th, and 50th generations for verification, with 3 colonies selected from each generation. Figure 4 The following are images showing the antibacterial effect of the fermentation broth of the genetically engineered strain EY03: (a) inhibition zones at 0, 6, 12, and 24 hours of fermentation (indicator bacteria: Escherichia coli 25922); (b) inhibition zones at 10, 100, and 1000 times dilution of the sample after 28 hours of fermentation (indicator bacteria: Escherichia coli 25922); (c) inhibition zones at 0, 6, 12, and 24 hours of fermentation (indicator bacteria: Bacillus subtilis); (d) inhibition zones at 0, 6, 12, and 24 hours of fermentation (indicator bacteria: Lactobacillus plantarum). Detailed Implementation The following embodiments are provided to better understand the present invention, but the following embodiments do not constitute a limitation on the content and scope of protection of the present invention. Any product that is the same as or similar to the present invention, derived by any person under the guidance of the present invention or by combining the features of the present invention with other prior art, falls within the scope of protection of the present invention.

[0023] Unless otherwise specified, all experimental steps or conditions in the examples were performed according to conventional experimental procedures and conditions in the art. Reagents or instruments whose manufacturers are not specified are all commercially available products.

[0024] The following embodiments involve: The method for determining the content of McJ25 is as follows: Under ultraviolet light at 33℃ and 280 nm wavelength, the product is separated by gradient elution using a Shim-pack Scepter C18 column at a flow rate of 1 mL / min. The specific operation steps are as follows.

[0025] Mobile phase A: Measure 1000 mL of ultrapure water, add 1 mL of TFA, filter with a 0.22 μm filter membrane, and degas by sonication for 3 min.

[0026] Mobile phase B: Measure 900 mL of acetonitrile, add 100 mL of ultrapure water, and use a pipette to transfer 0.85 mL of TFA into the acetonitrile-water solution. Filter through a 0.22 μm organic filter membrane and degas by sonication for 3 min.

[0027] Preparation of standard solution: Weigh 0.1 g of pure antimicrobial peptide MccJ25 into a 100 mL volumetric flask, add purified water to make up to the mark, shake to mix, and then filter through a 0.22 μm filter membrane.

[0028] After the column was equilibrated, the following gradient elution program was set up. Standard sample solutions of 0.8 g / L, 0.4 g / L, 0.2 g / L, 0.1 g / L, 0.05 g / L, and 0.025 g / L were prepared by adding purified water. After passing the solutions through a 0.22 μm filter membrane, the solutions were injected for detection at a volume of 10 μL. The gradient elution program shown in Table 1 was then started, and the peak areas at each concentration were plotted as external standard curves.

[0029] Table 1 Gradient elution program

[0030] LB plate formulation: 5 g / L yeast extract, 10 g / L peptone, 10 g / L sodium chloride, and 20 g / L agar powder.

[0031] LB seed culture medium formula: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L.

[0032] LB shake flask fermentation medium formula: yeast extract 5 g / L, peptone 10 g / L, sodium chloride 10 g / L.

[0033] Example 1: Construction method of recombinant strain EY03: 1. Expression element screening and strain construction The recombinant strain described in this invention modifies the mcjA, mcjBC, and mcjD promoters and the RBS sequence in MccJ25, and inserts the optimized mcjBC and mcjD expression cassettes into the expression cassettes using CRISPR-Cas9. Escherichia coli In the MC4100 genome, amino acid genes were simultaneously knocked out to obtain an amino acid-deficient recombinant chassis. High-level expression of MccJ25 was achieved by introducing the constructed mcjA expression plasmid (containing the complemented amino acid genes but without resistance genes) into the chassis strain. Specifically, the steps included were: (1) Integrating the optimized mcjBC and mcjD expression cassettes into the genome using CRISPR-Cas9. Escherichia coliIn the MC4100 genome; (2) knock out amino acid genes in the genome; (3) construct an mcjA expression plasmid with the amino acid gene complementation and without carrying the resistance gene, using the plasmid carrying the amino acid gene complementation expression cassette as the resistance-free plasmid, while also carrying the mcjA expression cassette; (4) transform the resistance-free plasmid, transforming the resistance-free plasmid in step (3) into the gene knockout strain in step (2); (5) modify the RBS sequence respectively. Finally, strain B was obtained.

[0034] 2. Strains optimization and screening This embodiment also examines the effects of promoters in the mcjA expression box, mcjBC expression box, and mcjD expression box: During the construction of recombinant E. coli EY03, multiple expression protocols were designed and evaluated for the mcjA, mcjBC, and mcjD genes. For the mcjD gene expression, a total of 10 protocols were designed and evaluated. Of these, only 3 could normally express the MccJ25 peptide, with the highest expression level being 16 times that of the lowest. For the mcjA gene expression, a total of 9 protocols were designed and evaluated. Of these, 8 could achieve peptide expression, with the highest expression level being 23 times that of the lowest. For the mcjBC gene, a total of 10 protocols were designed and evaluated. Of these, only 2 protocols could achieve normal peptide expression. Of these 2 protocols, the optimal expression level was twice that of the lowest.

[0035] Multiple expression strains were obtained by arranging and combining three expression elements, and the fermentation content of different combinations of strains was evaluated using a 3L fermenter.

[0036] (1) Escherichia coli was inoculated into LB medium and cultured for 8 hours to obtain the fermentation strain; (2) Prepare fermentation medium: yeast extract 40g / L, magnesium sulfate 2g / L, dipotassium hydrogen phosphate 2g / L, citric acid 4g / L, glucose 5g / L, defoamer (defoamer BK-8, purchased from Beijing Zhubao New Technology Co., Ltd.) 1g / L, adjust pH to 7.0-7.2 with NaOH, prepare 50% glucose as feed, and sterilize at 121℃ for 20min; (3) Inoculate the cultured seed liquid into the fermentation medium at an inoculation rate of 1% (v / v) and carry out fermentation culture.

[0037] (4) During the fermentation process, the temperature is maintained at 36-38℃, the rotation speed is 450rpm, the aeration ratio is 0.5-1vvm, the tank pressure is 0.03-0.05Mpa, and the pH is 7.0-7.1.

[0038] Table 2. Content of different element combinations in strains at different time periods

[0039] The obtained strain B was named Escherichia coli EY03 and deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38047. The deposit address is No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, 100101, China. The deposit date is April 3, 2026.

[0040] Example 2: Verification of the lack of resistance in recombinant strain EY03 To verify whether the recombinant strain EY03 constructed in this invention carries an exogenous resistance gene, the ampicillin resistance gene (Amp) used in the modification process was used. + ) and kanamycin resistance gene (Kan + Specific primers (see Amp-U, Amp-D, KanU, KanD, as shown in SEQ ID NO.1~4) were used to amplify the EY03 genomic DNA by PCR, and a recombinant bacterium carrying the corresponding resistance gene (the control strain is Escherichia coli F-750, see patent document CN119709572A, which is deposited at the China General Microbiological Culture Collection Center, accession number CGMCCNo.32623) was used as a positive control.

[0041] PCR amplification system: Table 3 PCR amplification system

[0042] PCR amplification program: Pre-denaturation: 95℃ 5min; Denaturation: 95℃ 15s; Annealing: 56℃ 45s; Extension: 72℃ 15s; 30 cycles; Extension: 72℃ 10min.

[0043] The results are attached. Figure 1 As shown, the positive control lanes (lane 1 corresponding to ampicillin and lane 2 corresponding to kanamycin) amplified the expected size of the resistance gene fragment, while the EY03 strain (lanes 3 and 4 corresponding to ampicillin and kanamycin, respectively) showed no amplification bands. This result indicates that the EY03 strain completely lacks both ampicillin and kanamycin resistance genes.

[0044] Strawberry strain EY03 was streaked onto LB agar plates. Single colonies were inoculated into antibiotic-free LB tubes, LB tubes containing 50 μg / mL kanamycin, and LB tubes containing 100 μg / mL ampicillin. The plates were incubated at 37°C with shaking at 200 rpm for 24 hours. Results are attached. Figure 2 As shown, EY03 can grow normally in test tube 1, but not in test tubes 2 and 3, confirming that EY03 is a strain without resistance markers and has good biosafety.

[0045] Example 3: Verification of the genetic stability of recombinant strain EY03 To investigate the genetic stability of the non-resistant recombinant strain EY03 constructed in this invention during the production of microinfectin MccJ25, the EY03 strain was continuously passaged for 50 generations.

[0046] The EY03 strain was streaked from a glycerol storage tube onto LB agar plates and incubated upside down at 37°C for 16–20 h. Once single colonies appeared, they were picked and inoculated into 10 mL of LB liquid medium and incubated at 37°C with shaking at 220 rpm for 12 h; this was the first generation culture. Then, 100 μL of this culture was transferred to 10 mL of fresh LB liquid medium and incubated at 37°C with shaking at 220 rpm for 12 h; this was the second generation. This process was repeated until the 50th generation. The strains from generations 0, 10, 20, 30, 40, and 50 were then tested as follows: (1) Verification of gene stability: Genomic DNA was extracted from each generation of strains, and specific primers (BC-F, BC-R, as shown in SEQ ID NO.5~6) were used to amplify the MccJ25 synthesis-related genes (PCR amplification of the MccJ25BC gene, since the fermentation broth of the strain contains it, it can be proven that mcjABCD are present) by PCR.

[0047] PCR amplification system: Table 4 PCR amplification system

[0048] PCR amplification program: Pre-denaturation: 95℃ 5min; Denaturation: 95℃ 15s; Annealing: 56℃ 45s; Extension: 72℃ 15s; 30 cycles; Extension: 72℃ 10min.

[0049] The results are attached. Figure 3 As shown, each generation of strains can amplify the target band of the expected size, and the band brightness is consistent, indicating that the target gene has not been lost or mutated after 50 consecutive generations.

[0050] Yield stability verification: Each generation of strains was cultured in a 30L fermenter. *E. coli* (EY03) was inoculated into liquid LB seed medium and cultured at 37℃ and 200 rpm for 8 hours with shaking to obtain the fermentation strain. The prepared seed culture was then inoculated into the fermentation medium at a rate of 1% (v / v) for fermentation. Throughout the fermentation process, the temperature was maintained at 36-38℃, the aeration ratio at 0.5-1 vvm, the tank pressure at 0.03-0.05 MPa, the pH at 7.0-7.1, the fermentation speed at 450 rpm, and the fermentation time at 28 hours. After fermentation, the MccJ25 content in the fermentation broth was measured.

[0051] The results showed that the MccJ25 yields of the 1st, 10th, 20th, 30th, 40th and 50th generation strains were 9.38 g / L, 9.07 g / L, 9.49 g / L, 9.96 g / L, 9.24 g / L and 9.73 g / L, respectively, with no significant differences between generations, indicating that the recombinant strain maintained a stable high yield after 50 consecutive generations.

[0052] The above results indicate that the non-resistant recombinant strain EY03 described in this invention has good genetic stability and is suitable for long-term preservation and industrial-scale production.

[0053] Example 4: Environmental Simulation Validation Experiment for Horizontal Transfer of Resistance Genes 4.1 Ca² + / Mg² + Induced natural transformation experiment Experimental materials: The recipient bacterium was Escherichia coli CVCC 233, and the donor plasmid was pUC18 (Amp... + ) and pACYC177 (Kan + The main reagent is a 0.1 mol / L CaCl2 solution (containing 10 mmol / L MgCl2).

[0054] Experimental Methods: *E. coli* CVCC 233 was cultured to the logarithmic growth phase, and bacterial cells were collected. In the treatment group, the bacterial cells (80 μL) were resuspended in pre-cooled CaCl2-MgCl2 solution (11.098 g of anhydrous CaCl2 and 0.9521 g of anhydrous MgCl2, diluted to 1 L of deionized water). After standing on ice for 30 minutes, 1 μg of plasmid DNA was added, and the mixture was incubated on ice for another 45 minutes. After the reaction, without heat shock, the cells were directly plated onto LB agar plates containing the appropriate antibiotics and incubated at 37°C for 16–24 hours.

[0055] Table 5 Ca² + / Mg² + Effects on plasmid transfer

[0056] Results analysis: Relying solely on Ca² + / Mg² + The presence of divalent cations allows E. coli to take up naked plasmid DNA from the environment and acquire corresponding antibiotic resistance without the need for artificial induction steps such as heat shock or electroporation, demonstrating the significant risk of gene transfer under natural conditions.

[0057] 4.2 Experiment on plasmid transformation mediated by biofilm on microplastic surface Experimental materials: The recipient bacterium was Escherichia coli CVCC 233, and the donor plasmid was pUC18 (Amp... + The microplastics were polypropylene (PP), polyethylene (PE), and polyvinyl chloride (PVC) fragments (particle size 1-5 mm), and some microplastics were aged by UV irradiation for 7 days.

[0058] Experimental method: Add 50 mL of LB medium diluted 10 times to a 250 mL Erlenmeyer flask, and add 1 g of microplastic fragments and 1 mL of bacterial suspension (approximately 10 g of LB medium). 8 Add 5 μg pUC18 plasmid DNA to the microplastic and incubate at 25°C for 48 hours. After incubation, wash off the biofilm bacteria from the microplastic surface, spread the microplastics on LB agar plates containing 100 μg / ml ampicillin, count the transformants, and calculate the transformation frequency as the number of transformants (cfu) / the total number of viable bacteria in the biofilm (cfu).

[0059] Table 6. Effects of microplastics on plasmid transfer

[0060] Results analysis: In the presence of microplastic fragments, wild-type strains can efficiently take up plasmid DNA, and the "plastic ring" biofilm formed on the surface of microplastics is a "hotspot" for bacteria to acquire exogenous resistance genes.

[0061] 4.3 Intercellular co-culture plasmid transformation experiment To verify that plasmids carrying resistance genes can undergo horizontal transfer without artificial intervention under direct intercellular contact conditions, thereby demonstrating the biosafety advantages of the recombinant strain without resistance markers of the present invention, the present invention references and reproduces the intercellular plasmid transformation (Cell-to-Cell Transformation) experimental system reported in the literature.

[0062] Experimental materials: The donor bacteria were threonine-deficient Escherichia coli MC4100 (Escherichia coli MC4100 was purchased from Zhili Zhongte Biotechnology Co., Ltd., and the ThrBC gene was knocked out of Escherichia coli MC4100 using conventional CRISPR-Cas9 methods), carrying the pHSG299 plasmid (kanamycin resistant, non-conjugating plasmid); the recipient bacteria were two wild-type Escherichia coli strains isolated from the environment.

[0063] Experimental method: Donor bacteria and recipient bacteria were mixed at a volume ratio of 1:1 and cultured at 37°C with shaking for 32 hours without the addition of any antibiotics. Threonine-deficient Escherichia coli MC4100 could not grow on inorganic salt plates without threonine. Transformants were plated on these selection plates and counted. Transformation frequency = number of transformants (cfu) / total recipient bacteria (cfu).

[0064] Table 7. Effects of bacterial co-culture on plasmid transfer

[0065] Results analysis: Direct co-culturing of donor and recipient bacteria can achieve highly efficient plasmid transfer, with a frequency as high as 1.5 × 10⁻⁶. -6 / cell, no thermal shock, electroporation or Ca² + deal with.

[0066] 4.4 Experimental Conclusions Through the above environmental simulation verification experiments, this invention confirms that: (1) In Ca² + / Mg² + Under conditions of divalent cations that are common in the natural environment, wild Escherichia coli can directly take up naked plasmid DNA from the environment without heat shock or electroporation and acquire antibiotic resistance. (2) Biofilms on microplastic surfaces can significantly promote the transformation of plasmid DNA into environmental bacteria; (3) Highly efficient plasmid transfer can be achieved through direct intercellular contact (frequency up to 10). -6 / cell), if traditional recombinant bacteria carrying resistance genes are used, during the production and processing, if problems such as strain leakage or plasmid gene residue occur, the resistance genes can be released under various natural environmental factors (Ca²). + / Mg² + Horizontal transfer without human intervention can occur through the mediation of ions, microplastic biofilms, and intercellular contact, leading to risks such as the spread of drug resistance genes. However, the plasmid in Escherichia coli EY03 of this invention does not possess resistance and therefore poses no environmental safety risk.

[0067] Example 5: Fermentation optimization of strain EY03 A 30L fermenter was used to evaluate the effect of segmented regulation on the fermentation content of strain EY03.

[0068] Escherichia coli (EY03) was inoculated into liquid LB seed medium and cultured with shaking at 37℃ and 200 rpm for 8 hours to obtain the fermentation strain. The cultured seed liquid was then inoculated into the fermentation medium at a rate of 1% (v / v) for fermentation. During the fermentation process, the temperature was maintained at 36-38℃, the aeration ratio at 0.5-1 vvm, the tank pressure at 0.03-0.05 MPa, and the pH at 7.0-7.1. A segmented speed control strategy was adopted during the fermentation process: a higher speed of 550-450 rpm was used in the early stage (0-8h), and the speed was reduced to 450-350 rpm in the later stage (8-28h).

[0069] The effects of segmented rotation speed on McJ25 content and biomass are shown in Table 8.

[0070] Table 8 Effect of rotational speed on McJ25 content

[0071] Example 6: Scale-up stability verification of recombinant strain EY03 6.1 Seed liquid preparation Escherichia coli with accession number CGMCC No. 38047 was used. Escherichia coli The EY03 strain was isolated by streak plating on LB agar plates and incubated statically at 37°C for 12-16 hours until single colonies appeared. Morphologically typical single colonies were picked and inoculated into test tubes containing 5 mL of LB liquid medium, and activated by shaking at 37°C and 200 rpm for 12 hours. The activated culture was then transferred at a 1% (v / v) inoculation rate to Erlenmeyer flasks containing 300 mL of LB liquid medium and incubated at 37°C and 200 rpm for another 12 hours to obtain the primary seed culture. The primary seed culture was then inoculated at a 1% (v / v) inoculation rate into a seed tank containing 300 L of LB liquid medium and incubated at 37°C, 200 rpm, and an aeration ratio of 1:1 (vvm) for 12 hours to obtain the secondary seed culture (fermentation strain).

[0072] 6.2 Fermentation tank cultivation Fermentation medium formula: yeast extract 40g / L, magnesium sulfate 2g / L, dipotassium hydrogen phosphate 2g / L, citric acid 4g / L, glucose 5g / L, defoamer 1g / L, pH adjusted to 7.2 with NaOH, and autoclaved at 121℃ for 20 minutes for later use.

[0073] The prepared secondary seed culture was inoculated at a rate of 2% (v / v) into a 5-ton fermenter containing 3 tons of fermentation medium for fed-batch fermentation. The fermentation process control parameters were: temperature 37℃, aeration ratio 1:1 (vvm), turbine rotation speed 130 rpm in the early stage (0-8h), and adjusted to 110 rpm in the middle and late stages (8-28h). Carbon source supply was maintained by feeding a 60% (w / v) glucose solution, and the pH of the fermentation broth was controlled within the range of 7.0-7.2. The total fermentation cycle was 28 hours.

[0074] Samples were taken at 24 and 28 hours of fermentation, and the fermentation supernatant was collected by centrifugation. The content of MccJ25 was determined by high-performance liquid chromatography (HPLC). The results showed that the yield of MccJ25 was 10.17 g / L at 24 hours and 11.26 g / L at 28 hours.

[0075] After fermentation, the fermentation broth was plated and gene stability was assessed. PCR amplification (BC-F, BC-R, as shown in SEQ ID NO. 5~6) was performed on the MccJ25 synthesis-related genes (PCR amplification of the MccJ25BC gene; since the fermentation broth contained the gene, it can be confirmed that mcjABCD was present). The results showed that mcjABCD was 100% intact.

[0076] PCR amplification system: Table 9 PCR amplification system

[0077] PCR amplification program: Pre-denaturation: 95℃ 5min; Denaturation: 95℃ 15s; Annealing: 56℃ 45s; Extension: 72℃ 15s; 30 cycles; Extension: 72℃ 10min.

[0078] 6.3 Control recombinant strain F-750 cultured in a fermenter The recombinant strain F-750 integrates the mcjD gene into the host bacterium's chromosomal genome, while the mcjA and mcjBC genes responsible for MccJ25 synthesis are expressed using conventional plasmids with resistance selection markers (such as kanamycin resistance), see CN119709572A.

[0079] The method for preparing the seed liquid is the same as in Example 6.1 of this invention.

[0080] The fermenter culture medium formula, sterilization conditions, and fermentation control parameters (temperature, aeration ratio, dissolved oxygen, addition of 70% glucose, etc.) are completely consistent with those in Example 6.2 of this invention.

[0081] Using the same HPLC method as in the embodiments of the present invention, samples were taken at 24 hours and 28 hours of fermentation to detect the MccJ25 content. The results showed that the MccJ25 yield was 5.1 g / L at 24 hours and 6.75 g / L at 28 hours.

[0082] After fermentation, the fermentation broth was plated and compared with LB agar plates containing 100 μg / mL ampicillin and 50 μg / mL kanamycin. Gene stability was then assessed. 28% of colonies failed to grow on the antibiotic-free plates, indicating that at least 28% of colonies had lost a plasmid. With further expansion of fermentation, it is reasonable to predict that the proportion of strains without plasmid burden will increase significantly, affecting production stability and yield.

[0083] Example 7: Validation of the antibacterial activity of MccJ25 This embodiment is based on the antibacterial experiment of the fermentation broth of the recombinant strain EY03 in the aforementioned embodiment 6. Specifically, the antibacterial activity of the antimicrobial peptide MccJ25 was determined by the agar diffusion method.

[0084] Using Escherichia coli 25922 (from the American Center for Type Culture Collection) as the indicator bacterium, a layer of 2% LB agar medium was first poured into a sterilized 90 mm Petri dish. After solidification, 0.1 mL of diluted 25922 bacterial suspension (OD200) was added. 600 =0.1-0.2), spread evenly with a sterile spreader. Punch holes in the culture dish using a sterilized punch, ensuring a distance of at least 25 mm between the centers of each hole. Using a micropipette, add an appropriate amount of the antibacterial agent from the EY03 fermentation broth (after centrifugation, retain the supernatant, and then pass it through a 0.22 μm membrane) to the agar wells, cover the dish, and incubate upright at 37℃ for 18 hours. Observe the inhibition zone as shown in the attached figure. Figure 4 (a) shows the inhibition zones at 6, 12, 24, and 28 hours of fermentation, which are 7 mm, 13 mm, 18 mm, and 21 mm, respectively. (b) shows the inhibition zone diameters of the spray-dried samples of the fermentation broth after 28 hours of fermentation at 10x, 100x, 1000x, and physiological saline, which are 21 mm, 17 mm, 9 mm, and 0 mm, respectively.

[0085] It is evident that the fermentation product of the recombinant bacteria EY03 described in this invention has a strong inhibitory effect on Escherichia coli 25922, but does not inhibit beneficial bacteria such as Lactobacillus plantarum and Bacillus subtilis. The antibacterial experimental method using commercially available Lactobacillus plantarum and Bacillus subtilis to replace Escherichia coli 25922 was described, referring to... Figure 4 The results of the test in (a) are as follows: Figure 4 In the middle (c) and (d), (c) is Bacillus subtilis and (d) is Lactobacillus plantarum, which can be used for antibacterial, preservative and immune-regulating purposes in food or aquaculture.

[0086] In summary, the high-yield MccJ25 recombinant strain described in this invention is non-resistant, green and safe, has a short fermentation cycle, is easy to control, and exhibits stable fermentation scale-up, with a scale-up content reaching over 11 g / L, far exceeding the levels reported in other studies to date.

[0087] Obviously, the above embodiments are merely illustrative examples for clear explanation and are not intended to limit the implementation. Those skilled in the art will recognize that other variations or modifications can be made based on the above description. It is neither necessary nor possible to exhaustively list all possible implementations here. However, obvious variations or modifications derived therefrom are still within the scope of protection of this invention.

Claims

1. A type of Escherichia coli Escherichia coli EY03 is characterized by, The strains include: Using Escherichia coli as the starting strain, the strain's genome integrates the mcjBC expression cassette and the mcjD expression cassette; the amino acid genes in the strain's genome are knocked out; the strain contains an antibiotic-free plasmid carrying the amino acid genes and the mcjA expression cassette; and the RBS sequences are modified accordingly.

2. The *Escherichia coli* EY03 according to claim 1, characterized in that, The promoter for mcjA in the mcjA expression box is an optimized promoter; And / or, the promoter in the mcjBC expression box is an optimized promoter; And / or, the promoter in the mcjD expression box is an optimized promoter; And / or, the starting strain is Escherichia coli. Escherichia coli MC4100.

3. The *Escherichia coli* EY03 according to claim 1 or 2, characterized in that, The Escherichia coli EY03 is deposited at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 38047.

4. A method for constructing Escherichia coli EY03 as described in any one of claims 1-3, characterized in that, include: Using Escherichia coli as the starting strain, the mcjBC expression cassette and mcjD expression cassette were integrated into the strain's genome. The amino acid gene in the strain's genome was knocked out. Antibiotic-free plasmids carrying the amino acid gene and mcjA expression cassette were transformed into the strain, and the RBS sequence was modified accordingly.

5. A microbial inoculant, characterized in that, Contains Escherichia coli EY03 as described in claim 1, or its culture, fermentation product, or metabolite.

6. The microbial agent according to claim 5, characterized in that, The metabolites include MccJ25 antimicrobial peptide.

7. A method for preparing a microbial inoculant as described in claim 5 or 6, characterized in that, The method includes the steps of culturing and fermenting the Escherichia coli EY03 as described in any one of claims 1-3.

8. The *Escherichia coli* EY03 according to claim 1, and the microbial fermentation agent according to claim 5 or 6, have at least one of the following uses: 1) Its use in the preparation of the antimicrobial peptide MccJ25; 2) Application in the preparation of antibacterial growth-promoting additives for livestock and poultry feed; Optionally, the antibacterial growth-promoting additives for livestock and poultry feed include at least one of the following functions: inhibiting pathogenic Escherichia coli and / or Salmonella in livestock and poultry, reducing the diarrhea rate of weaned piglets and broilers, repairing the intestinal barrier, tolerating the high temperature of 121℃ feed pelleting, and / or replacing feed antibiotics. 3) Application in the preparation of food biological preservatives; Optionally, the food biological preservative includes at least one of the following functions: specifically inhibiting foodborne pathogens in fresh meat, eggs and dairy products and / or extending the shelf life of food; the foodborne pathogens include Escherichia coli O157:H7 and / or Salmonella; 4) Application in the preparation of anti-inflammatory drugs against multidrug-resistant Gram-negative bacteria and / or sepsis; Optionally, the anti-inflammatory drugs against multidrug-resistant Gram-negative bacteria and / or sepsis include targeting bacterial RNA polymerases for bactericidal activity, having no cross-resistance with clinical antibiotics, reducing inflammatory factors in the body, and / or having low cytotoxicity and no hemolysis.

9. A method for producing the antimicrobial peptide MccJ25, characterized in that, The method includes the step of fermentation using Escherichia coli EY03 as described in any one of claims 1-3.

10. The method according to claim 9, characterized in that, The method also includes the following steps for preparing the fermentation strain: activating and culturing Escherichia coli EY03, inoculating the activated culture into a seed culture medium for a first culture to obtain a primary seed liquid; inoculating the primary seed liquid into a seed culture medium for a second culture to obtain the fermentation strain; optionally, the seed culture medium is selected from LB medium; optionally, in the preparation of the primary seed liquid or fermentation strain, the inoculation amount is 1-5 v / v%; optionally, the conditions for the first culture are constant temperature shaking at 37±1℃ for 10-12 h at 100-300 rpm; optionally, the conditions for the second culture are constant temperature shaking at 37±1℃ for 10-12 h at 100-300 rpm, with an aeration ratio of 0.5-1 vvm. And / or, in the fermentation step, the seed culture of Escherichia coli EY03 is inoculated into the fermentation medium at an inoculation rate of 1-5 v / v%, and fermented and cultured at 36-38℃, aeration ratio of 0.5-1 vvm, tank pressure of 0.03-0.05 MPa, pH of 7.0-7.2, with 40-70% glucose added, and fermentation for 24-28 hours; And / or, during the fermentation process, the rotation speed is 550-450 rpm in the early stage of fermentation and 450-350 rpm in the later stage of fermentation; optionally, the early stage of fermentation is the fermentation time from 0 to 8 hours; optionally, the later stage of fermentation is the fermentation time from 8 hours to 24-28 hours. And / or, the fermentation medium comprises the following components at the following concentrations: yeast extract 10-70 g / L, magnesium sulfate 0.5-5 g / L, dipotassium hydrogen phosphate 0.5-5 g / L, citric acid 0-2 g / L, glucose 1-10 g / L, antifoaming agent 0.1-1 g / L, and initial pH 6.5-7.

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