Lactobacillus johnsonii producing novel class iii bacteriocins, engineered bacteria and application thereof

Abstract

The present application relates to a lactobacillus johnsonii producing a novel class III bacteriocin, an engineered bacterium and application thereof, and belongs to the technical field of microorganisms. Lactobacillus johnsonii The JLJ071 strain of the lactobacillus johnsonii is preserved in the China General Microbiological Culture Collection Center, and the preservation number is CGMCC No.34541. The bacteriocin amino acid sequence is shown in Seq ID No.1. The expressed novel class III bacteriocin has good bactericidal activity on listeria monocytogenes and salmonella pullorum, and has good characteristics such as acid resistance, high temperature resistance and protease resistance, and has a good application prospect in feed additives.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to Lactobacillus johnsonii, a type III bacteriocinogen-producing bacterium, engineered bacteria, construction methods, and applications. Background Technology

[0002] The overuse of antibiotics in animal husbandry has led to the emergence of drug-resistant bacteria and increasingly serious antibiotic problems. Many countries and regions have explicitly banned the use of antibiotics in animal feed. Since 2020, my country's Ministry of Agriculture and Rural Affairs has also comprehensively banned the addition of growth-promoting antibiotics to feed to reduce the harm caused by antibiotic overuse and safeguard animal food safety and public health. Developing safe and effective new antibiotic alternatives has become an urgent need in the industry.

[0003] Bacteriocins are a class of low-molecular-weight proteins or polypeptides produced by bacteria during their reproductive metabolism. They possess advantages such as high antibacterial activity, high safety, and no drug resistance, and are considered promising alternatives to antibiotics. Since the US FDA approved nisin produced by lactic acid bacteria as a biological preservative in 1988, bacteriocins produced by lactic acid bacteria have become a research hotspot in food biological preservation and biopharmaceutical fields, demonstrating enormous potential and value. However, research and application as an animal antibiotic alternative in the livestock sector are limited. Furthermore, the yield of wild-type lactic acid bacteria bacteriocins is low, their isolation and purification are complex, and the cost is too high, making industrial-scale production difficult.

[0004] Bacteriocins are classified into three main categories—Class I, Class II, and Class III—based on their structural characteristics, molecular size, modification methods, and mechanisms of action. Class III bacteriocins are high molecular weight bacteriocins with a molecular weight greater than 30 kDa, mostly produced by *Lactobacillus* bacteria, and exert their antibacterial effects by disrupting the cell membrane or lysing the cell wall. Class III bacteriocins are further classified according to their mechanism of action into IIIa (lysed) and IIIb (non-lysed). Class IIIa bacteriocins achieve their bactericidal effect by lysing the bacterial cell wall, while Class IIIb bacteriocins have a gentler mechanism of action and do not cause cell lysis. Compared to Class I and Class II bacteriocins, although Class III bacteriocins have lower thermal stability, their larger molecular structure gives them a broader antibacterial spectrum and greater potential for industrial applications.

[0005] Patent application CN202510451449.8 discloses a Pediococcus pentosaceus strain producing novel class IIa bacteriocins, a genetically engineered strain, and its applications. The sequence encoding the bacteriocin gene was designed with primers, cloned by PCR, purified, inserted into an expression vector, and transformed into yeast to construct a heterologous expression strain capable of producing class IIa bacteriocins. Currently, there are no reports of engineered lactic acid bacteria expressing class III bacteriocins. The bacteriocin produced by *Lactobacillus johnsonii* has a structure and function similar to class III bacteriocins, exhibiting particularly strong activity against *Listeria* and *Salmonella*. Therefore, using genetic engineering techniques to construct engineered strains for heterologous expression of bacteriocins can improve their yield and application potential. Summary of the Invention

[0006] The purpose of this invention is to provide a novel Lactobacillus johnsonii producing a type III bacteriocin, an engineered strain, a construction method therefor, and its application, in order to solve the problems existing in the prior art.

[0007] This invention screened a strain of *Lactobacillus johnsonii* capable of producing class III bacteriocins from the feces of healthy broiler chickens. Lactobacillus johnsonii JLJ071 is a bacterium with excellent growth performance and acid and bile salt tolerance. It contains the amino acid sequence of a bacteriocin as shown in SEQ ID No. 1. This bacteriocin retains its antibacterial activity after treatment with proteases (trypsin, pepsin, papain, and proteinase K), high temperatures (40–100°C), and acid / alkali conditions (pH 3, 4, 5, 6, 7, 8). This bacterium was deposited on May 14, 2025, at the China General Microbiological Culture Collection Center (CGMCC) with accession number CGMCC No. 34541.

[0008] Based on this, the technical solution provided by the present invention is as follows: First, this invention provides a novel type III bacteriocin-producing Lactobacillus johnsonii (Lactobacillus johnsonii). Lactobacillus johnsonii )JLJ071.

[0009] Furthermore, the *Lactobacillus johnsonii* JLJ071 strain, isolated from the feces of healthy broiler chickens, exhibits excellent growth performance and resistance to acid and bile salts. This strain was deposited on May 14, 2025, at the China General Microbiological Culture Collection Center (CGMCC), with accession number CGMCC No. 34541.

[0010] The *Lactobacillus johnsonii* JLJ071 produces a class III bacteriocin, the amino acid sequence of which is shown in SEQ ID No. 1. This bacteriocin retains its antibacterial activity after treatment with proteases, high temperatures, and acids / bases; wherein the proteases include trypsin, pepsin, papain, and proteinase K; the high temperature is 40–100°C; and the pH of the acid / base is selected from 3, 4, 5, 6, 7, or 8.

[0011] Secondly, the present invention provides a bacteriocin.

[0012] The amino acid sequence of the bacteriocin is: (a) the amino acid sequence shown in SEQ ID No. 1; or (b) A sequence with equivalent function formed by replacing, deleting or adding one or more amino acids to the sequence shown in SEQ ID No. 1.

[0013] Furthermore, the present invention provides a gene encoding the bacteriocin.

[0014] Furthermore, the nucleotide sequence encoding the bacteriocin is shown in SEQ ID No. 2. Those skilled in the art will understand that, considering codon degeneracy, the nucleotide sequence encoding the gene may also include a sequence capable of expressing an equivalent protein by substituting, deleting, or adding one or more nucleotides to the sequence shown in SEQ ID No. 2.

[0015] In addition, the present invention also includes complementary sequences of the above-mentioned coding genes, transcribed RNA, etc.

[0016] The present invention also provides an expression vector containing the said gene.

[0017] The vector can be a cloning vector or an expression vector.

[0018] The present invention also provides engineered bacteria for transforming the expression vector.

[0019] Furthermore, the present invention provides an engineered strain of Lactobacillus johnsonii that produces a novel type III bacteriocin, wherein the engineered strain is a genetically engineered strain capable of expressing the bacteriocin of claim 2.

[0020] As a preferred embodiment, the starting strain of the engineered bacteria is yeast, specifically Saccharomyces cerevisiae.

[0021] This invention also provides a method for preparing a novel type III bacteriocinogen-producing engineered bacterium, comprising the following steps: (1) Construction of bacteriocin expression vector Using Lactobacillus johnsonii JLJ071 genomic DNA as a template, bacteriocin upstream and downstream amplification primers were designed to amplify the bacteriocin gene fragment; the linear plasmid fragment pAM1 and the PCR product were ligated in one step, and the ligation product was transformed into Escherichia coli DH5α by heat shock transformation, and positive transformants were screened and verified. (2) Transplant yeast and verify its function. The recombinant expression vector plasmid verified in step (1) was introduced into YPG30 yeast by chemical transformation, transferred to SD medium plates without Ura3, cultured at 30℃ for 3 days, the target protein was extracted and identified by SDS-PAGE, and the recombinant Lactobacillus johnsonii engineered bacteria expressing the bacteriocin synthesis regulatory gene was obtained.

[0022] The present invention also provides a method for preparing bacteriocins, which are obtained by culturing the Lactobacillus johnsonii or the engineered bacteria.

[0023] In addition, the present invention also provides products containing the Lactobacillus johnsonii, the bacteriocin, or the engineered bacteria.

[0024] Furthermore, the products include, but are not limited to: feed additives, feed, food additives, food, preservatives, pharmaceuticals, or health products.

[0025] The present invention also provides the use of the Lactobacillus johnsonii, the bacteriocin, or the engineered bacteria in the preparation of products for the prevention and treatment of diseases caused by Gram-negative bacteria.

[0026] As a preferred embodiment, the Gram-negative bacteria include Listeria monocytogenes and Salmonella pullorum.

[0027] Based on the above technical solution, the present invention has the following technical effects: This invention screened and constructed a novel type III bacteriocin-producing Lactobacillus johnsonii and its engineered strain. This bacterium exhibits good acid and bile salt resistance, and the bacteriocin it secretes possesses good antibacterial activity, protease stability, high-temperature stability, and acid-base stability. The constructed engineered strain can efficiently express type III bacteriocin in a yeast expression system, and can be used to prevent and treat diseases caused by Gram-negative bacteria such as Listeria and Salmonella. This invention can provide technical support for the development of bacteriocins as novel alternative antibiotics and has certain application value. Attached Figure Description

[0028] Figure 1 This is the colony morphology of Lactobacillus johnsonii JLJ071; Figure 2 This is the cell morphology of Lactobacillus johnsonii JLJ071; Figure 3 The diagram shows the results of the organic acid and hydrogen peroxide exclusion test for the bacteriocins produced by the strains of this invention. Figure 4 The image shows the results of protease treatment experiments on the bacteriocins produced by the strains of this invention. Figure 5 The figure shows the results of temperature treatment tests on the bacteriocins produced by the strains of this invention. Figure 6 The figure shows the results of acid-base treatment tests on the bacteriocins produced by the strains of this invention. Figure 7 This is an electrophoresis diagram of the bacteriocin expressed by the engineered bacteria of this invention. In the diagram, M represents the marker, 1 and 2 represent two replicates of the engineered bacteria, G represents galactose induction, and R represents raffinose induction control. Detailed Implementation

[0029] The following detailed embodiments are used to further illustrate the present invention, but should not be construed as limiting the present invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the present invention.

[0030] Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0031] Example 1. Screening and identification of bacteriocinogenic strains First, a highly acidic culture medium with added bile salts is used as a screening tool to initially screen out lactic acid bacteria strains that have both acid and bile salt tolerance. Then, strains with high antibacterial activity are selected from the initially screened strains. This can greatly simplify the screening process and efficiently obtain target strains that have both environmental tolerance and bacteriocinogenic ability.

[0032] Weigh an appropriate amount of cecal contents from broiler chickens, place them in sterile physiological saline, shake thoroughly, and perform a tenfold serial dilution. For each dilution, take 0.1 mL of the bacterial solution and spread it onto MRS medium (purchased from Beijing Aoboxing). Incubate under anaerobic conditions at 37°C for 24–48 hours. Select milky-white colonies for preliminary screening. Strains that are Gram-positive, H2O2 enzyme-negative, and catalase-negative can be preliminarily identified as lactic acid bacteria. Activate the 20 strains obtained from the initial screening and inoculate them at a 1% (v / v) inoculum in liquid MRS medium containing 0.1% bile salts at pH=3. Incubate at 37°C for 18 hours, and then count the viable cells. The number of viable cells is used as the evaluation criterion for the acid and bile salt tolerance of the strains. Lactic acid bacteria strains with strong acid and bile salt tolerance are preliminarily screened and further used for screening bacteriocinogenic strains. The results of acid and bile salt tolerance are shown in Table 1 below.

[0033] Table 1. Screening results of lactic acid bacteria with acid and bile salt tolerance

[0034] Based on the results of acid and bile salt tolerance, the 10 strains with the highest viable counts were selected for antibacterial testing. The seed culture of these 10 strains was inoculated into MRS liquid medium at a rate of 1% (v / v), and incubated statically at 37°C for 24 hours. An appropriate amount of fermentation broth was then centrifuged at 6000 rpm for 10 minutes, and the supernatant was collected. Four indicator pathogens, *Escherichia coli* (chicken coli), were then... Escherichia coli CVCC25926), Listeria monocytogenes ( Listeria monocytogenes CVCC2423), Salmonella pullorum ( Salmonella pullorum CVCC526), ​​Staphylococcus aureus ( Staphylococcus aureus The viable cell count of the CVCC2234 bacterial suspension was adjusted to 10. 7 CFU / mL, 0.1 mL of bacterial suspension was evenly spread on the surface of LB solid medium, with three plates for each indicator bacterium. One Oxford cup was placed on each plate, and 200 μL of supernatant was added to the cup. The plates were incubated at 37℃, and the inhibition zone diameter was observed and measured every 1 hour, with the maximum value recorded. The results are shown in Table 2. Strain number 11 showed the largest inhibition zone diameters against Listeria monocytogenes and Salmonella pullorum, at 28.5±0.5 mm and 24.3±0.2 mm, respectively. Based on these results, strain number 11 was selected for colony morphology observation, physiological and biochemical characteristics, and 16S rDNA sequence identification. The result was Lactobacillus johnsonii (…). Lactobacillus johnsonii The primers used for molecular biological PCR amplification were 27F (SEQ ID NO.3: 5'-AGAGTTTGATCCTGGCTCAG-3') and 1492R (SEQ ID NO.4: 5'-GGTTACCTTGTTACGACTT-3'). This bacterium was grown in MRS medium and anaerobically cultured at 37°C for 48 h. The cells were rod-shaped, arranged singly, in pairs, or in chains, with a size of approximately 0.69 × 1.35–3.75 μm, and Gram-positive. Figure 1 As shown; on MRS medium, colonies are milky white, moist, droplet-shaped, with irregular protrusions, opaque, and with irregular edges, such as... Figure 2 As shown in Table 3, the physiological and biochemical characteristics of the amplified product are shown in SEQ ID NO.5.

[0035] Table 2. Inhibition zone diameters (mm) of fermentation supernatant against indicator pathogens.

[0036] Table 3 Physiological and biochemical characteristics of Lactobacillus johnsonii

[0037] Note: "+" indicates a positive result; "-" indicates a negative result; "±" indicates a weak positive result.

[0038] 16S rDNA (Seq ID No. 5): The strain has been deposited with the China General Microbiological Culture Collection Center (Address: No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, Postcode: 100101) on May 14, 2025, with accession number CGMCC No. 34541, accession name: JLJ071, and classification: Lactobacillus johnsonii. Lactobacillus johnsonii .

[0039] Example 2. Stability determination of bacteriocins in Lactobacillus johnsonii For target strains with strong antibacterial activity, organic acid exclusion test, hydrogen peroxide exclusion test and bacteriocin stability test are carried out in sequence to clarify whether the antibacterial activity is dominated by bacteriocins and to verify the stability characteristics of bacteriocins.

[0040] After activation, *Lactobacillus johnsonii* strain JLJ071 was inoculated into MRS liquid medium at a 1% inoculum and cultured at 37°C for 24 h. The fermentation broth was centrifuged at 6000 rpm for 15 min, and the supernatant was filtered through a 0.22 μm filter membrane and stored at 4°C for later use. *Listeria monocytogenes* and *Salmonella pullorum* were used as indicator bacteria for the antibacterial test. After activation of the seed culture, they were inoculated into LB broth at a 1% inoculum and cultured at 37°C for 18 h, adjusting the viable count to 10⁻⁶. 7 CFU / mL available for use.

[0041] (1) Organic acid and hydrogen peroxide elimination test The pH of MRS liquid medium was adjusted to be the same as that of the supernatant using 1 mol / L NaOH and HCl solution. The supernatant was used as a control. Oxford cup inhibition tests were performed on two indicator bacteria, Listeria monocytogenes and Salmonella, and the diameter of the inhibition zone was measured to evaluate the effect of organic acids on the antibacterial effect of bacteriocins. The pH of the supernatant was adjusted to the optimal pH of catalase 7.0 using 1 mol / L NaOH and HCl solution. Catalase was added to make the final concentration 1 mg / mL and the mixture was incubated at 37°C for 2 h. The supernatant without catalase treatment was used as a blank control for the antibacterial test.

[0042] (2) Protease stability test Take four 1 mL aliquots of supernatant and adjust the pH of each solution to the optimal pH values ​​for trypsin, papain, pepsin, and proteinase K (trypsin: 7.4; papain: 6.5; pepsin: 4.5; proteinase K: 7.5). Then add the four proteins to each solution to a final concentration of 1 mg / mL. After incubating the solutions in a 37°C water bath for 2 hours, adjust the pH back to the original value. Use the untreated supernatant as a blank control and perform antibacterial tests using the Oxford cup method.

[0043] (3) Temperature stability determination The supernatant was incubated in water at 40℃, 60℃, and 80℃ for 4 hours, and at 100℃ for 30 minutes. The supernatant at room temperature was used as a blank control, and the Oxford cup method was used for antibacterial testing.

[0044] (4) pH stability determination Take four 1 mL aliquots of supernatant and adjust their pH to 3.0, 4.0, 5.0, 6.0, 7.0, and 8.0 respectively using 1 mol / L NaOH and HCl solutions. After standing at room temperature for 1 h, adjust the supernatant back to its original pH. Using the untreated supernatant as a blank control, determine the antibacterial activity of bacteriocins under different pH treatments using the Oxford cup method.

[0045] Stability testing of bacteriocin production by the strain is shown in [link to relevant documentation]. Figures 3-6 .Depend on Figure 3 As can be seen, in the organic acid exclusion test, compared with the control group, the inhibition zone diameter of the supernatant against Listeria was 26.5 mm, while the inhibition zone diameter against Salmonella was 18.0 mm; in the hydrogen peroxide exclusion test, the inhibition zone diameter against both indicator bacteria remained above 24.1 mm. The results indicate that the antibacterial substances in the fermentation supernatant of the strains were not acidic metabolites or hydrogen peroxide. Figure 4 It is evident that after treatment with four proteases—trypsin, pepsin, papain, and proteinase K—except for pepsin, which had a significant impact on the diameter of the inhibition zone, the diameter of the inhibition zone in the supernatant decreased to varying degrees after treatment with the other three enzymes, but still remained above 19.1 mm. This indicates that the antibacterial substance in the fermentation supernatant of the strain is protein, which is sensitive to proteases but still exhibits a certain degree of tolerance. Figure 5 As can be seen, after temperature treatment (40℃, 60℃, 80℃, and 100℃), the inhibition zone diameters of the supernatant against both indicator bacteria were all above 20.5 mm, indicating that the active substance has very strong thermal stability. Figure 6 As can be seen, after acid and alkali treatment (pH 3, 4, 5, 6, 7, 8), the diameter of the inhibiting bacteria in the supernatant remained above 23.2 mm, and its antibacterial activity did not decrease significantly, indicating that the active substance has a certain degree of tolerance to acids and alkalis. The above research results indicate that the antibacterial substance in the fermentation supernatant of this strain is bacteriocin, and it has strong acid and alkali resistance and high temperature resistance.

[0046] Example 3. Construction of engineered bacteria for heterologous bacteriocin expression The complete genome sequence of *Lactobacillus johnsonii* was obtained and its gene sequence was aligned using NCBI BLAST. The amino acid sequence encoding the bacteriocin gene was determined as shown in SEQ ID No. 1, and the nucleotide sequence as shown in SEQ ID No. 2. Primers were designed based on this sequence, and a heterologous bacteriocin-producing strain was constructed through PCR cloning, purification, and insertion into an expression vector. Online BlastP alignment analysis (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) showed that the bacteriocin shared 81.14% amino acid sequence homology with identified Class III bacteriocins, indicating that it is a novel bacteriocin, and this is the first time the inventors have researched, identified, or reported it.

[0047] SEQ ID No.1: MIGKETQIRLVNKLENIHHVVVQASSIVGSNVFALQLLHRQSDVVVYQTPNDSETVTFDEDHHILYLKGHGSAGTATTHTQTWIWSTENHKWTVGTTPKHHGNTTWTTWIARVTWPGTQTQVFAFNTDLPFLSYWNRVGWGYDDGGTYYPGDDLVTVEWTPSP AGHYILIASTLINTTGYWALGDLNTVTNWLNEANEKADDINNENTTCFGAVKVPHFNVQKIVSISGYGSDDNRVIYIMSQPSPFTTFLGFQRQGKPRSIIKIPWGMVDPMKWSVVNLDLSMKLDALDFCTEMEGIQVTSDCLYLMVAYHQRNSDLTTLMNRIYQVEKF SEQ ID No.2: (1) PCR amplification of bacteriocinogen gene Lactobacillus johnsonii JLJ071 was inoculated into MRS liquid medium and cultured statically at 37°C for 18 h. The bacterial sludge was harvested by centrifugation, and the genome was extracted using a bacterial genomic DNA extraction kit, following the kit instructions. Using the extracted genome as a template, primer F (Seq ID No. 6) (5'-CACCGTTAATTAACCCGG) was used to extract the genome. GGATCC ATGATTGGAAAAGAAACACAAATA-3', underscore is Bam H Ⅰ restriction site) and R (Seq ID No. 7): 5'-GGAGAAAAAACCCC GGATCC CTAAAATTTTTCAACTTGGTAAATA-3', underlined Bam Amplification of the bacteriocinogen gene using HⅠ restriction enzyme sites. The PCR reaction system was as follows: DNA template, 0.5 μL; primer F, 2 μL; primer R, 2 μL; 2×Mix, 25 μL; dNTP, 1 μL; Taq enzyme, 0.5 μL; ddH2O, 19 μL. PCR amplification conditions were: 95℃ pre-denaturation for 3 min; 95℃ denaturation for 15 s; 56℃ annealing for 15 s; 72℃ extension for 30 s; 30 cycles; 72℃ extension for 5 min. After the reaction, 5 μL of product was taken and analyzed by electrophoresis on a 1% agarose gel. The expected size of the PCR product was recovered by gel excision.

[0048] (2) Connection Use restriction endonucleases Bam H Ⅰ was used to digest the pAM1 plasmid at 37℃ for 2 h. The reaction system was: pAM1 plasmid, 10 μL; Bam H Ⅰ 2μL; 10×CutSmart buffer, 2μL; ddH2O, 6μL. After enzyme digestion and recovery, the purified linear plasmid fragment and gene fragment were ligated at a 1:3 molar ratio with T4 DNA ligase at 50℃ for 45 min, and then introduced into *E. coli* DH5α competent cells using the heat shock transformation method. Single colonies were picked and inoculated into 500μL of LB liquid medium (containing 25ug / mL ampicillin antibiotic), and cultured at 37℃, 220rpm for 1 h on a shaker. Colony PCR was then performed to identify positive clones. Single colonies that were positive by colony PCR were sent to Beijing Qingke Biotechnology Co., Ltd. for sequencing verification.

[0049] (3) Transformation and screening Plasmids were extracted from strains with correct sequences and transformed into YPG30 yeast competent cells using chemical transformation. Positive transformants were screened by culturing on SD medium (without Ura3) plates at 30°C for 3 days to obtain the genetically engineered Lactobacillus johnsonii strain JLJ071.

[0050] Preparation of competent yeast cells: YPG30 Saccharomyces cerevisiae cells were inoculated into 10 mL of YPD medium and cultured overnight at 30°C and 220 rpm. The bacterial suspension was then inoculated into YPD medium at a 10% inoculation ratio and cultured for approximately 5 hours at 30°C and 220 rpm until the logarithmic growth phase was reached. The cells were centrifuged at 800×g for 2 min, the supernatant was discarded, and the cells were resuspended in 3 mL of ddH2O by pipetting and aspiration. The cells were then centrifuged at 800×g for 2 min, and the supernatant was discarded. The cells were resuspended in 500 μL of 0.1 M LiAc solution to obtain the competent yeast cell suspension.

[0051] Transformation method: Mix 2 μL of recombinant plasmid and 50 μL of heat-denatured 2 mg / mL salmon sperm DNA by pipetting and then add to 100 μL of competent cell solution and gently tap to mix. Add 240 μL of 50% PEG3350 solution and mix by inverting. Incubate at room temperature for 30 min, heat shock at 42℃ for 15 min, incubate on ice for 5 min, centrifuge at 800×g for 2 min, discard the supernatant, and resuspend in YPD medium.

[0052] (4) SDS-PAGE identification of target proteins in yeast From Ura3-SD - Single colonies were selected from solid culture media. The constructed *Lactobacillus johnsonii* engineered bacteria were transferred to 5 mL of LYPR (20 g / L raffinose, 10 g / L imported yeast extract, 20 g / L peptone) medium and incubated at 30°C and 220 rpm for 16 h. Then, 1 mL of YPR medium was transferred to 3 mL of YPG (20 g / L galactose, 10 g / L imported yeast extract, 20 g / L peptone) medium and incubated at 30°C and 220 rpm for 4 h. The YPR and YPG cultures were centrifuged at 4°C and 12000 rpm for 1 min, respectively. The supernatants were collected and filtered for sterilization. Proteins were extracted from the sterilized supernatants using a protein extraction kit and identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results showed that the bacteriocin molecular weight was 57 kDa (…). Figure 7 Protein bands appeared after galactose induction, but no protein bands appeared after raffinose induction.

[0053] (5) Comparison of stability and antibacterial activity with existing recombinant bacteriocins Based on the Class III bacteriocins most closely related to the sequence of this invention published in the NCBI database (…), Lactobacillus johnsoniiThe amino acid sequence of the bacteriocin was obtained (SEQ ID No. 8), converted into a DNA sequence, and synthesized. Then, according to the method in Example 3 above, a bacteriocin heterologous expression engineered bacterium (hereinafter referred to as J-1) was constructed and induced to express. The engineered bacterium *Lactobacillus johnsonii* JLJ071 and engineered bacterium J-1 were inoculated into YPG for culture. The fermentation broth was centrifuged at 4℃ and 6000×g for 30 min. The supernatant was collected for stability and antibacterial activity tests of *Lactobacillus johnsonii*.

[0054] SEQ ID No.8: MIGRETQIRLVNKLENIHHVVVQASAIDGSNVFALQLLHKQSDVVVYQTPNDSETVTFDEDHPILYLKGPNSAGTAGGHTQTWIQSGENNKWFVGTKPKRHGNTYWTTQIARVTVPGYQTQVFANNTDLPRLSYLNRAGAGYGDGGTVYPGKDLVRVEATVSPN GHYFLIASIDINHTGYFALYDLNEVNNKLDEAEEKAEDINIENLTCLGAFKVPHFNDQKIVSIQGYGIDDNRDIYISSQPSQPSPHTTFLGFPRQGKPREIIKIPWGMVDPDKWSVVNLDNSLKLDALDFCTEFEGIQVTSDCLYLTVAYHQRNSDLTTLMNRIYQVEKF The Oxford cup method was used to detect the antibacterial effects of bacteriocins produced by the two engineered bacteria against four pathogens. Table 4 shows that, compared with J- bacteria, the JLJ071 engineered bacteria had better antibacterial effects against the four indicator pathogens, with the best effects against Listeria monocytogenes and Salmonella pullorum, with inhibition diameters reaching 29.5 and 24.7 mm, respectively.

[0055] Table 4 Comparison of the antibacterial activity of the two bacteriocins (mm)

[0056] Note: Different lowercase letters in the superscript of data from the same row indicate significant differences. P <0.05, indicating no significant difference ( ). P >0.05).

[0057] Listeria monocytogenes was selected, and the Oxford cup method was used to conduct stability tests on the bacteriocins produced by the two engineered strains. The bacteriocin produced by the JLJ071 engineered strain served as the control group, and the bacteriocin produced by the J-1 engineered strain served as the experimental group, with five replicates in each group. The diameter (mm) of the inhibition zone against Listeria monocytogenes was measured before and after different treatments, and the antibacterial stability of the two bacteriocins was calculated. As shown in Table 5, the diameter of the inhibition zone before and after treatment with the bacteriocin produced by the JLJ071 engineered strain of this invention is larger than that of the bacteriocin produced by the J-1 engineered strain. From the stability results, except for slightly poor stability after proteinase K treatment, the bacteriocins produced by both the JLJ071 and J-1 engineered strains showed good stability to different treatments, reaching over 50%. Specifically, the bacteriocin produced by the JLJ071 engineered strain of this invention showed significantly higher stability to pH 3.0, 80℃, and pepsin than the bacteriocin produced by the J-1 engineered strain. In summary, compared with the control engineered bacteria, the bacteriocins produced by the engineered bacteria constructed in this invention exhibit good antibacterial properties and stability.

[0058] Table 5. Comparison of the stability of the two bacteriocins

[0059] Note: Different lowercase letters in the superscript of data from the same row indicate significant differences. P <0.05), with no letter indicating no significant difference.

Claims

1. A novel type III bacteriocin-producing Lactobacillus johnsonii ( Lactobacillus johnsonii JLJ071, its accession number is CGMCC No. 34541.

2. A bacteriocin, characterized in that, The amino acid sequence of this bacteriocin is as shown in SEQ ID No.

1.

3. The gene encoding the bacteriocin of claim 2.

4. The gene according to claim 3, characterized in that, The nucleotide sequence of the gene is shown in SEQ ID No.

2.

5. An expression vector containing the gene of claim 3 or 4.

6. The engineered bacteria that transform the vector of claim 5.

7. A method for preparing a novel type III bacteriocinogen-producing engineered bacterium, characterized in that, Includes the following steps: (1) Construction of bacteriocin expression vector Using the genomic DNA of strain JLJ071 as a template, upstream and downstream primers for bacteriocin amplification were designed to amplify the bacteriocin gene fragment; the linear plasmid fragment pAM1 and the PCR product were ligated in one step, and the ligation product was transformed into Escherichia coli DH5α by heat shock transformation, and positive transformants were screened and verified. (2) Introduce yeast and verify its function. The recombinant expression vector plasmid was transformed and introduced into YPG30 yeast, transferred to SD medium plates without Ura3, and cultured at 30°C for 3 days. The target protein was extracted and identified by SDS-PAGE, thus obtaining the recombinant engineered bacteria expressing the bacteriocinogen regulatory gene.

8. A product containing the JLJ071 strain of claim 1, the bacteriocin of claim 2, or the engineered bacteria of claim 6.

9. The product as described in claim 8, characterized in that, The product is a feed additive or feed.

10. The use of the JLJ071 strain of claim 1, the bacteriocin of claim 2, or the engineered bacteria of claim 6 in the preparation of products for preventing and treating diseases caused by Gram-negative bacteria.

11. The application as described in claim 10, characterized in that, The Gram-negative bacteria include Listeria monocytogenes and Salmonella pullorum.

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