Construction and antimicrobial application of recombinant engineered probiotics producing microbialin MccY and MccJ25

Abstract

This invention belongs to the field of biotechnology and discloses three types of engineered probiotics recombinant with microbesin. These three types of engineered probiotics are: an engineered probiotic recombinant with microbesin MccJ25, an engineered probiotic recombinant with microbesin MccY, and an engineered probiotic recombinant with both microbesin MccJ25 and MccY. These three engineered probiotics share the characteristics of lacking resistance marker genes, exhibiting good growth activity, genetic stability, and antibacterial activity. Furthermore, this invention also provides a method for preparing these engineered probiotics and their applications.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to the construction and antibacterial application of recombinant engineered probiotics producing microbes MccY and MccJ25, and more specifically, to an engineered probiotic recombinantly integrated with microbes MccJ25, an engineered probiotic recombinantly integrated with microbes MccY, an engineered probiotic recombinantly integrated with microbes MccY and MccJ25, and their preparation methods and uses. Background Technology

[0002] Since the U.S. Food and Drug Administration (FDA) first approved antibiotics as feed additives in 1950, countries around the world have successively approved antibiotics for use in livestock production. However, the long-term irrational use of antibiotics can have certain negative impacts on livestock and humans, leading to problems such as the spread of drug resistance, drug residues, and environmental pollution. In October 2021, my country's Ministry of Agriculture and Rural Affairs issued the "National Action Plan for Reducing the Use of Veterinary Antibiotics." Therefore, the banning, restriction, reduction, and substitution of antibiotics are urgent, and the development of safe, efficient, and low-residue veterinary antibiotic reduction and substitution inhibitors is particularly important.

[0003] Microsporins are low-molecular-weight, highly stable bacterial peptides produced by Gram-negative bacteria and synthesized by ribosomes. They can be used to prevent and treat bacterial infections in animals. Microsporins are divided into two classes: Class I microsporins are less than 5 kDa and are encoded by plasmids, while Class II microsporins are approximately 5–10 kDa in size and can be either plasmid-encoded or chromosome-encoded. Microsporin MccJ25 belongs to Class I micropeptide microsporins, composed of 21 amino acids. Its synthesis involves the precursor gene mcjA, the splicing modification gene mcjB, the post-transcriptional modification gene mcjC, and the secretion and autoimmune gene mcjD. MccJ25 primarily kills bacteria by inhibiting RNA polymerase activity in cells, thereby terminating respiratory chain transcription. It exhibits highly efficient bactericidal activity against Salmonella and Escherichia coli. Therefore, genetic engineering to obtain engineered strains that can efficiently produce MccJ25 has significant application value.

[0004] The following is a case study on inserting microbial MccJ25 into engineered bacteria for the expression of microbial MccJ25:

[0005] The invention patent application with publication number CN113774006A is entitled to a highly efficient engineered strain expressing MccJ25 and its fermentation process.

[0006] Publication number CN114574412A, the subject of which is an engineered Bacillus subtilis strain expressing MccJ25 and its construction method and application;

[0007] The above schemes all use production bacteria. Among them, the production bacteria used in CN113774006A is Escherichia coli AGL027, and the production bacteria used in CN114574412A is Bacillus subtilis.

[0008] The producing bacteria themselves possess a very strong ability to express the target gene, which is determined by the characteristics of the producing bacteria themselves. Furthermore, the producing bacteria generally do not require growth within a living organism. As stated in paragraph 66 of the specification of CN113774006A: "Lactobacillus plantarum is cultured for 16-18 hours, resuspended in an equal volume of distilled water, treated with lysozyme for 10 minutes to completely lyse the cells, centrifuged, and the supernatant is obtained. The J25 content is determined by high-performance liquid chromatography." The method for obtaining J25 protein is to collect it after lysing and breaking the cells.

[0009] Escherichia coli Nissle 1917 (EcN) is the only FDA-approved live bacteria preparation for the treatment of enteritis. It is a non-pathogenic E. coli strain isolated in 1917 by German physician Alfred Nissle from the feces of a soldier fighting diarrhea. It possesses probiotic properties and, due to its ability to secrete microinfectious agents McCH47 and McCM, has the potential to be used as an antibiotic-reducing or antibiotic-alternative agent. It contains 2.5–25 × 10⁻⁶ mg / L. 9 A freeze-dried probiotic drug of live Escherichiacoli Nissle 1917 (trade name Mutaflor) with colony-forming units has been approved for manufacture and sale as a treatment for many gastrointestinal diseases.

[0010] Microbesin MccH47 and McCM have relatively narrow antibacterial spectra. To further improve the antibacterial spectrum and antimicrobial activity of probiotic EcN, further modification of EcN is necessary. The following challenges exist in genetic engineering EcN: 1. How to broaden the antibacterial spectrum; 2. How to introduce targeted microbesin-related genes into EcN; 3. How to ensure that the microbesin-related genes exert their corresponding effects without resistance markers (the presence of resistance markers implies a risk of horizontal transmission of resistance genes, making it unsuitable for use as a probiotic). Summary of the Invention

[0011] One of the objectives of this invention is to provide two types of engineered probiotics that are recombinant and integrated with microbes. These two types of engineered probiotics are an engineered probiotic that is recombinant and integrated with microbes MccJ25 and an engineered probiotic that is recombinant and integrated with microbes MccJ25 and MccY. Both types of engineered probiotics of this invention have the characteristics of having no resistance marker gene, good growth activity, genetic stability, and antibacterial activity.

[0012] Meanwhile, the engineered probiotics that recombinantly integrate MccJ25 have a high level of MccJ25 protein expression, which can significantly improve the antibacterial ability of the probiotic EcN.

[0013] The engineered probiotics recombinantly integrated with Microbesin MccY+MccJ25 have a broader spectrum of activity, effectively inhibiting Salmonella Typhimurium ATCC14028, Salmonella Enteritidis CVCC3377, Salmonella Pullorum CVCC1800, Salmonella Infantile CMCC50041, Salmonella Kentuckyii BNCC239114, Salmonella Londonii CVCC2206, Salmonella Corvallis CMCC50922, Shigella flexneri SF1015, Shigella sonnei SS1014, and Escherichia coli ATCC25922.

[0014] In addition, the present invention also provides a method for preparing the above-mentioned engineered probiotics and their uses.

[0015] To achieve the above objectives, the present invention provides an engineered probiotic with recombinant integration of the microbial MccJ25 gene, wherein the engineered probiotic is an Escherichia coli with the integrated microbial MccJ25 gene; the nucleotide sequence of the microbial MccJ25 gene is shown in SEQ ID NO: 4; and the Escherichia coli is Escherichia coli Nissle 1917.

[0016] In addition, the present invention also discloses a method for preparing the engineered probiotics as described above, which uses genome editing technology and gene knockout vector to integrate the SD-T7RNP homologous arm repair fragment and the MccJ25E homologous arm repair fragment into the genome of Escherichia coli Nissle 1917, thereby obtaining the microbial MccJ25 recombinant integrated engineered probiotic EcN-MccJ25.

[0017] The nucleotide sequence of the SD-T7RNP homologous arm repair fragment is shown in SEQ ID NO: 5; the MccJ25E homologous arm repair fragment is shown in SEQ ID NO: 19.

[0018] In the above preparation method, the method specifically includes:

[0019] (1) pREDCas9 plasmid was transformed into EcN competent cells, positive transformants EcN-cas9 were screened, and the positive transformants EcN-cas9 were prepared into competent cells; the competent cells were EcN-cas9 competent cells.

[0020] (2) The SD-T7RNP homologous arm repair fragment and the araBsgRNA plasmid were transformed into EcN-cas9 competent cells, and the recombinant strain EcNΔaraB-T7RNP-araBsgRNA-cas9 was obtained by screening with spectinomycin and ampicillin; the araBsgRNA plasmid was a pUC19 vector with an inserted sgRNA-araB fragment; the nucleotide sequence of the sgRNA-araB fragment is shown in SEQ ID NO: 33;

[0021] (3) The recombinant strain EcNΔaraB-T7RNP-araBsgRNA-cas9 was passaged to screen for strains that were insensitive to spectinomycin and sensitive to ampicillin to eliminate the araBsgRNA plasmid in the strains. The MccJ25E homologous arm repair fragment and endAsgRNA plasmid were then introduced. The recombinant strain EcNΔaraB-T7RNP-Cas9-ΔendA-MccJ25-endAsgRNA was obtained by screening with spectinomycin and ampicillin. The endAsgRNA plasmid was a pUC19 vector with an inserted sgRNA-endA fragment. The nucleotide sequence of the sgRNA-endA fragment is shown in SEQ ID NO: 34.

[0022] (4) The recombinant strain EcNΔaraB-T7RNP-cas9-ΔendA-MccJ25-endAsgRNA was passaged and cultured to screen out strains that were insensitive to spectinomycin and sensitive to ampicillin in order to eliminate the endAsgRNA plasmid in the strains, and the recombinant strain EcNΔaraB-T7RNP-ΔendA-MccJ25-cas9 was obtained.

[0023] (5) The recombinant strain EcNΔaraB-T7RNP-ΔendA-MccJ25-cas9 was passaged at 42℃ to screen out strains that were sensitive to both spectinomycin and ampicillin to eliminate the pREDCas9 plasmid in the strains, thus obtaining the microbestocin MccJ25 recombinant integrated engineered probiotic EcN-MccJ25.

[0024] The strains constructed in this invention do not carry any resistance markers, eliminating the risk of lateral transmission of resistance genes. Because they are expressed in a non-plasmid form, there is no need for antibiotics to lock onto the plasmid. The other two engineered probiotics of this invention also possess this characteristic.

[0025] Another type of engineered probiotic disclosed in this invention is an engineered probiotic that integrates microbesin MccY and MccJ25. The engineered probiotic is an *Escherichia coli* that integrates the microbesin MccY gene and the microbesin MccJ25 gene. The nucleotide sequence of the microbesin MccY gene is shown in SEQ ID NO: 3. The nucleotide sequence of the microbesin MccJ25 gene is shown in SEQ ID NO: 4. The *Escherichia coli* is *Escherichia coli* Nissle 1917.

[0026] The preparation method of the recombinant and integrated probiotics of microbes MccY and MccJ25 is as follows: using genome editing technology and gene knockout vector, the SD-T7RNP homologous arm repair fragment, the MccJ25X homologous arm repair fragment and the MccY homologous arm repair fragment are integrated into the genome of Escherichia coli Nissle 1917, thus obtaining the recombinant and integrated probiotics of microbes MccY and MccJ25 EcN-MccY-MccJ25;

[0027] The nucleotide sequence of the SD-T7RNP homologous arm repair fragment is shown in SEQ ID NO: 5; the MccJ25X homologous arm repair fragment is shown in SEQ ID NO: 26; and the MccY homologous arm repair fragment is shown in SEQ ID NO: 12.

[0028] The method is specifically as follows:

[0029] (1) pREDCas9 plasmid was transformed into EcN competent cells, positive transformants EcN-cas9 were screened, and the positive transformants EcN-cas9 were prepared into competent cells; the competent cells were EcN-cas9 competent cells.

[0030] (2) The SD-T7RNP homologous arm repair fragment and the araBsgRNA plasmid were transformed into EcN-cas9 competent cells, and the recombinant strain EcN-T7RNP-araBsgRNA-cas9 was obtained by screening with spectinomycin and ampicillin; the araBsgRNA plasmid was a pUC19 vector with an inserted sgRNA-araB fragment; the nucleotide sequence of the sgRNA-araB fragment is shown in SEQ ID NO: 33;

[0031] (3) The recombinant strain EcN-T7RNP-araBsgRNA-cas9 was passaged to screen for strains that were insensitive to spectinomycin and sensitive to ampicillin to eliminate the araBsgRNA plasmid in the strains. The MccY homologous arm repair fragment and endAsgRNA plasmid were then introduced. The recombinant strain EcN-T7RNP-pREDCas9-MccY-endAsgRNA was obtained by screening with spectinomycin and ampicillin. The recombinant strain EcN-T7RNP-pREDCas9-MccY-endAsgRNA was then used to prepare competent cells, which were EcN-T7RNP-MccY-cas9 competent cells. The endAsgRNA plasmid was a pUC19 vector with an inserted sgRNA-endA fragment. The nucleotide sequence of the sgRNA-endA fragment is shown in SEQ ID NO: 34.

[0032] (4) The MccJ25X homologous arm repair fragment and the xylAsgRNA plasmid were transformed into EcN-T7RNP-MccY-cas9 competent cells and screened with spectinomycin and ampicillin double resistance plates to obtain the recombinant strain EcN-T7RNP-pREDCas9-MccY-MccJ25-xylAsgRNA, in which MccY and MccJ25 were simultaneously integrated into the EcN genome; the xylAsgRNA plasmid was a pUC19 vector with an inserted sgRNA-xylA fragment, the nucleotide sequence of which is shown in SEQ ID NO: 35;

[0033] (5) Eliminate the pREDCas9 plasmid and xylAsgRNA plasmid from the recombinant strain EcN-T7RNP-pREDCas9-MccY-MccJ25-xylAsgRNA to obtain strain EcN-T7RNP-MccY-MccJ25. This strain EcN-T7RNP-MccY-MccJ25 is the recombinant and integrated probiotic strain EcN-MccY-MccJ25 containing microbicins MccY and MccJ25.

[0034] Furthermore, this invention also discloses the use of the three engineered probiotics described above in the preparation of antibacterial agents.

[0035] In the above-mentioned uses, the dosage form of the antibacterial preparation is one of powder, solution and granules.

[0036] Finally, the present invention also discloses an antibacterial preparation containing one or more of the three engineered probiotics described above.

[0037] In the above-mentioned antibacterial agents, the dosage form of the antibacterial agent is one of powder, solution and granules.

[0038] Beneficial effects

[0039] Compared with the prior art, the present invention has at least the following advantages:

[0040] 1. The common advantage of the three engineered probiotics of this invention is that they can be used as probiotics, meeting the following requirements for probiotics: 1. They are live bacteria capable of acting on the intestines of humans or animals; 2. The three engineered strains constructed by the method of this invention do not carry any resistance markers, eliminating the risk of horizontal transmission of resistance genes and ensuring their safety as probiotics; 3. The three engineered strains constructed by the method of this invention express microinfectious agents in a non-plasmid expression form, eliminating the need for antibiotics to lock the plasmids, allowing them to stably act on the intestines and be passaged, meeting the stability requirements for probiotic use.

[0041] Through the above optimizations, all three engineered probiotics of the present invention can improve the intestinal health of humans or animals and enhance the antibacterial ability of EcN against harmful bacteria.

[0042] 2. The unique advantage of the engineered probiotics with recombinant integration of microbesin MccJ25 of the present invention is that the engineered probiotics with recombinant integration of microbesin MccJ25 can achieve a high expression level of 25.31 mg / L for microbesin MccJ25, and can effectively inhibit Salmonella enteritidis CVCC3377, Shigella flexneri SF1015, and Escherichia coli ATCC25922. The engineered probiotics with recombinant integration of microbesin MccJ25 are far more sensitive to Salmonella enteritidis than engineered probiotics with recombinant integration of microbesin MccY and engineered probiotics with recombinant integration of microbesin MccY+MccJ25.

[0043] 3. The unique advantage of the engineered probiotics with recombinant integration of microbesin MccY of the present invention is that the engineered probiotics with recombinant integration of microbesin MccY can achieve a high expression level of 9.23 mg / L for microbesin MccY, and can effectively inhibit Salmonella Typhimurium ATCC14028, Salmonella Enteritidis CVCC3377, Salmonella Pullorum CVCC1800, Salmonella Infantile CMCC50041, Salmonella Kentuckyis BNCC239114, Salmonella Londonella CVCC2206, Salmonella Corvallis CMCC50922, and Shigella Sonnei SS1014. Its sensitivity against Salmonella Typhimurium is far superior to that of engineered probiotics with recombinant integration of microbesin MccJ25 and engineered probiotics with recombinant integration of microbesin MccY+MccJ25.

[0044] 4. The unique advantages of the engineered probiotics of the present invention, which recombinantly integrates microbes MccY and MccJ25, are as follows: the expression level of microbes MccY can reach a high level of 8.02 mg / L, and the expression level of microbes MccJ25 can reach a high level of 9.11 mg / L. This allows them to inhibit Salmonella Typhimurium ATCC14028, Salmonella Enteritidis CVCC3377, Salmonella Pullorum CVCC1800, and Salmonella Infantitidis CMCC50041. The probiotics recombinantly integrated with Microbes Y+MccJ25 effectively inhibited the growth of *Salmonella Kentuckyii* BNCC239114, *Salmonella Londonii* CVCC2206, *Salmonella Corvallisii* CMCC50922, *Shigella flexneri* SF1015, *Shigella sonnei* SS1014, and *Escherichia coli* ATCC25922. The broad-spectrum antibacterial activity of the probiotics recombinantly integrated with Microbes Y+MccJ25 was superior to that of the probiotics recombinantly integrated with Microbes Y+MccJ25. Attached Figure Description

[0045] The present invention will be further described below with reference to the accompanying drawings and embodiments;

[0046] Figure 1A The plasmid map of the recombinant plasmid pUC19-T7RNP;

[0047] Figure 1B The plasmid map of the recombinant plasmid pUC19-MccY;

[0048] Figure 1C The plasmid map of recombinant plasmid pUC19-MccJ25;

[0049] Figure 2A The plasmid map of the araBsgRNA plasmid;

[0050] Figure 2B The plasmid map of the endAsgRNA plasmid;

[0051] Figure 2C This is a plasmid map of the xylAsgRNA plasmid;

[0052] Figure 3A This is a diagram showing the PCR identification results of the EcNΔaraB-T7RNP strain;

[0053] Figure 3B This is a diagram showing the PCR identification results of strain EcNΔaraB-T7RN-ΔendA-MccY;

[0054] Figure 3C This is a diagram showing the PCR identification results of strain EcNΔaraB-T7RN-ΔendA-MccJ25;

[0055] Figure 3D This is a diagram showing the PCR identification results of strain EcNΔaraB-T7RN-ΔendA-MccY-ΔxylA-MccJ25.

[0056] Figure 4A This is a diagram of the inhibition zone of MccY recombinant and integrated probiotics in 0.5% LB soft agar containing Salmonella typhimurium;

[0057] Figure 4B This is a diagram of the inhibition zone of MccJ25 recombinant and integrated probiotic in 0.5% LB soft agar containing Salmonella enteritidis;

[0058] Figure 4C This is an inhibition zone diagram of MccY+MccJ25 recombinant and integrated probiotics in 0.5% LB soft agar containing Salmonella typhimurium and 0.5% LB soft agar containing Salmonella enteritidis.

[0059] Figure 5A This is a liquid chromatography result of the culture supernatant of EcN-MccY engineered probiotics;

[0060] Figure 5B This is a liquid chromatography-mass spectrometry result of the culture supernatant of EcN-MccY engineered probiotics;

[0061] Figure 5C This is a liquid chromatography result of the culture supernatant of EcN-MccJ25 engineered probiotics;

[0062] Figure 5D This is a liquid chromatography-mass spectrometry result of the culture supernatant of EcN-MccJ25 engineered probiotics;

[0063] Figure 5E This is a liquid chromatography result of the culture supernatant of the MccY+MccJ25 recombinant and integrated probiotic strain;

[0064] Figure 5F This is a liquid chromatography-mass spectrometry (LC-MS) result of the culture supernatant of the MccY+MccJ25 recombinant and integrated probiotic strain.

[0065] Figure 5G This is a liquid chromatography result of MccJ25 in the culture supernatant of MccY+MccJ25 recombinant and integrated probiotics;

[0066] Figure 5H This is a liquid chromatography-mass spectrometry (LC-MS) result of MccJ25 in the culture supernatant of MccY+MccJ25 recombinant and integrated probiotics.

[0067] Figure 6A This is a diagram of the inhibition zones of MccY recombinant and integrated probiotics expressed at different times;

[0068] Figure 6B This is a diagram of the inhibition zones of MccJ25 recombinant and integrated engineered probiotics at different expression times;

[0069] Figure 6C This is a diagram showing the inhibition zones of MccY+MccJ25 recombinant and integrated engineered probiotics at different expression times against Salmonella typhimurium.

[0070] Figure 6D This is a diagram showing the inhibition zones of MccY+MccJ25 recombinant and integrated engineered probiotics at different expression times against Salmonella enteritidis.

[0071] Figure 7 Comparative graph showing the inhibitory effects of MccY secreted by MccY recombinant engineered bacteria, MccJ25 recombinant engineered bacteria, and MccY+MccJ25 recombinant engineered bacteria on Gram-negative bacteria such as Salmonella Typhimurium, Salmonella Pullorum, Salmonella Enteritidis, Salmonella Infantile, Salmonella Kentuckyis, Salmonella London, Salmonella Corvallis, Escherichia coli, Shigella Sonnei, and Shigella fulminatus; and on Gram-positive bacteria such as Bacillus aureus and Bacillus subtilis. Detailed Implementation

[0072] The present invention will be further described below with reference to embodiments, but this does not constitute any limitation on the present invention. Any limited modifications made within the scope of the claims of the present invention are still within the scope of the claims of the present invention.

[0073] In the following examples, whole-genome synthesis, primer synthesis, and sequencing were all performed by Suzhou Genewiz Biotechnology Co., Ltd. Molecular biology experiments, such as competent cell preparation and transformation, were conducted in accordance with *Molecular Cloning: A Laboratory Manual* (3rd edition).

[0074] The following is a detailed description.

[0075] Example 1: Construction of MccY and MccJ25 recombinant-integrated engineered probiotics, and a dual recombinant-integrated engineered probiotic strain using both MccY and MccJ25.

[0076] In this embodiment, *Escherichia coli* Nissle 1917 (EcN) was used as the substrate bacterium. Using pREDCas9 *E. coli* gene editing technology, a 20bp SD sequence and a 2652bp complete T7RNP sequence were integrated into the araB position of its genome. Subsequently, the MccY repair fragment and the MccJ25 repair fragment were integrated into the endA position of its genome, respectively, to obtain MccY recombinant-integrated engineered probiotics and MccJ25 recombinant-integrated engineered probiotics. Based on the obtained MccY recombinant-integrated engineered probiotics, the MccJ25 repair fragment was integrated into the xylA position of the bacterium's genome, resulting in a MccY and MccJ25 dual recombinant-integrated engineered probiotic.

[0077] The SD nucleotide sequence is: 5'-CTAACTGGAAGAGGCACTAA-3' (SEQ ID NO: 1).

[0078] The nucleotide sequence of the SD-T7RNP sequence is shown in SEQ ID NO: 2;

[0079] The nucleotide sequence of the McCY repair fragment is shown in SEQ ID NO: 3;

[0080] The nucleotide sequence of the McJ25 repair fragment is shown in SEQ ID NO: 4;

[0081] The specific construction method is as follows:

[0082] (1) Construction of pUC19-T7RNP, pUC19-MccY, and pUC19-MccJ25 vectors:

[0083] Referring to NCBI number CP081489, the nucleotide sequence of the modified gene T7RNP was synthesized. The SD sequence (SEQ ID NO: 1) was linked with the T7RNP nucleotide sequence to obtain the target gene SD-T7RNP sequence (the sequence is shown in SEQ ID NO: 2 above).

[0084] The sequence shown in SEQ ID NO: 2 was introduced into the pUC19 cloning plasmid to obtain the recombinant plasmid pUC19-SD-T7RNP containing the sequence shown in SEQ ID NO: 2. The plasmid map of the obtained recombinant plasmid pUC19-SD-T7RNP is shown below. Figure 1A Show.

[0085] The recombinant plasmid pUC19-MccY containing the sequence shown in SEQ ID NO: 3 was synthesized. The plasmid map of the obtained recombinant plasmid pUC19-MccY is shown below. Figure 1B As shown.

[0086] The recombinant plasmid pUC19-MccJ25 containing the sequence shown in SEQ ID NO: 4 was synthesized. The plasmid map of the obtained recombinant plasmid pUC19-MccJ25 is shown below. Figure 1C As shown.

[0087] (2) Constructing the SD-T7RNP homologous arm repair fragment, the optimized MccY homologous arm repair fragment, the optimized MccJ25E homologous arm repair fragment, and the optimized MccJ25X homologous arm repair fragment:

[0088] Using the EcN genome (NCBI ID NZ_CP007799.1) as a template, the upstream homologous arm araBup was obtained by PCR amplification using araBup-F and araBup-R primers; the downstream homologous arm araBdown was obtained by PCR amplification using the EcN genome as a template, using araBdown-F and araBdown-R primers. Using the recombinant plasmid pUC19-SD-T7RNP as a template, a complete T7RNP fragment with a 20bp SD sequence was obtained by PCR amplification using T7RNP-F and T7RNP-R primers. Using the upstream homologous arm araBup, the downstream homologous arm araBdown, and the complete T7RNP fragment with a 20bp SD sequence as templates, the SD-T7RNP homologous arm repair fragment was obtained by overlap PCR amplification using araBup-F and araBdown-R primers.

[0089] The obtained SD-T7RNP homologous arm repair fragment is shown in SEQ ID NO:5, and the primer sequences are shown in Table 1.

[0090] Table 1 Primer sequence information for SD-T7RNP integration.

[0091] Primer name Sequence (5'-3') araBup-F 5'-tgacgaccgtagtgatgaat-3'(SEQ ID NO:6) araBup-R 5'-ttagtgcctcttccagttagtgacggaactggttattcgg-3'(SEQ ID NO:7) araBdown-F 5'-atgacacctgccacacaaat-3'(SEQ ID NO:8) araBdown-R 5'-tcagcgcatggctgaagacggtatg-3'(SEQ ID NO:9) T7RNP-F 5'-ctaactggaagaggcactaaatgaacacgattaacatcgc-3'(SEQ ID NO:10) T7RNP-R 5'-atttgtgtggcaggtgtcatcgttacgcgaacgcgaagtccg-3' (SEQ ID NO: 11)

[0092] The PCR amplification systems are shown in Table 2. The overlapping PCR amplification systems are shown in Table 3.

[0093] Table 2 PCR amplification system

[0094] Components Dosage template 1μL Upstream primer (10 μmol / L) 1μL Downstream primer (10 μmol / L) 1μL 2×Phanta Max Master Mix 10μL ddH2O Add to 20μL

[0095] Table 3 Overlap PCR Amplification System

[0096] Components Dosage Upstream homology arm 1μL Downstream homology arm 1μL T7RNP fragment 1μL Upstream primer (10 μmol / L) 2μL Downstream primer (10 μmol / L) 2μL 2×Phanta Max Master Mix 25μL ddH2O Add to 50μL

[0097] The PCR amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 56℃ annealing for 15 s, 72℃ extension for 30 s, 35 cycles; and a final extension at 72℃ for 5 min. The overlap PCR amplification program was as follows: 95℃ pre-denaturation for 5 min; 95℃ denaturation for 15 s, 56℃ annealing for 15 s, 72℃ extension for 1 min, 40 cycles; and a final extension at 72℃ for 5 min.

[0098] Following the above procedures, using the EcN genome as a template, and employing primers endAyup-F and endAyup-R, the upstream homologous arm endAyup was obtained by PCR amplification; using the EcN genome as a template, and employing primers endAydown-F and endAydown-R, the downstream homologous arm endAydown was obtained by PCR amplification. Using the recombinant plasmid pUC19-MccY as a template, and employing primers MccY-F and MccY-R, the target gene MccY sequence fragment was obtained by PCR amplification. Using the upstream homologous arm endAyup, the downstream homologous arm endAydown, and the modified gene MccY sequence fragment as templates, and employing primers endAyup-F and endAydown-R, overlapping PCR amplification was used to obtain the MccY sequence homologous arm repair fragment.

[0099] The obtained MccY homologous arm repair fragment is shown in SEQ ID NO:12, and the primer sequences are shown in Table 4.

[0100] Table 4 Primer sequence information for McCY integration

[0101] Primer name Sequence (5'-3') endAyup-F 5'-cgctggaacatattgaacgt-3'(SEQ ID NO:13) endAyup-R 5'-gggatcgagatctcgatcctttgataccttcggccaatgc-3' (SEQ ID NO: 14) endAydown-F 5'-atgggacaagatgtatccag-3'(SEQ ID NO:15) endAydown-R 5'-ttcatctgccgataaaccgc-3'(SEQ ID NO:16) MccY-F 5'-aggatcgagatctcgatccc-3'(SEQ ID NO:17) MccY-R 5'-ctggatacatcttgtcccatttattctgtagctgatgccag-3'(SEQ ID NO:18)

[0102] The PCR amplification system and procedure, and the overlapping PCR amplification system and procedure are the same as those in the above embodiments.

[0103] Following the above procedures, using the EcN genome as a template, and employing primers endAjup-F and endAjup-R, the upstream homologous arm endAjup was obtained by PCR amplification; using the EcN genome as a template, the downstream homologous arm endAjdown was obtained by PCR amplification using primers endAjdown-F and endAjdown-R. Using the recombinant plasmid pUC19-MccJ25 as a template, and employing primers MccJ25E-F and MccJ25E-R, the target gene MccJ25 sequence fragment was obtained by PCR amplification. Using the upstream homologous arm endAjup, the downstream homologous arm endAjdown, and the modified gene MccJ25 sequence fragment as templates, and employing primers endAjup-F and endAjdown-R, the MccJ25E sequence homologous arm repair fragment was obtained by overlapping PCR amplification.

[0104] The obtained MccJ25E homologous arm repair fragment is shown in SEQ ID NO:19, and the primer sequences are shown in Table 5.

[0105] Table 5 Primer sequence information for McCJ25 integration

[0106] Primer name Sequence (5'-3') endAjup-F 5'-cgctggaacatattgaacgt-3'(SEQ ID NO:20) endAjup-R 5'-gggatcgagatctcgatcctttgataccttcggccaatgc-3' (SEQ ID NO: 21) endAjdown-F 5'-atgggacaagatgtatccag-3'(SEQ ID NO:22) endAjdown-R 5'-ttcatctgccgataaaccgc-3'(SEQ ID NO:23) McJ25E-F 5'-aggatcgagatctcgatccc-3'(SEQ ID NO:24) McJ25E-R 5'-ctggatacatcttgtcccatttattcagtaacagaagccag-3'(SEQ ID NO:25)

[0107] The PCR amplification system and procedure, and the overlapping PCR amplification system and procedure are the same as those in the above embodiments.

[0108] Following the above procedures, using the EcN genome as a template, and using xylAjup-F and xylAjup-R as primers, the upstream homologous arm xylAjup was obtained by PCR amplification; using the EcN genome as a template, and using xylAjdown-F and xylAjdown-R as primers, the downstream homologous arm xylAjdown was obtained by PCR amplification. Using the recombinant plasmid pUC19-MccJ25 as a template, and using MccJ25X-F and MccJ25X-R as primers, the target gene MccJ25 sequence fragment was obtained by PCR amplification. Using the upstream homologous arm xylAjup, the downstream homologous arm xylAjdown, and the modified gene MccJ25 sequence fragment as templates, and using xylAjup-F and xylAjdown-R as primers, the MccJ25X sequence homologous arm repair fragment was obtained by overlapping PCR amplification.

[0109] The obtained MccJ25X homologous arm repair fragment is shown in SEQ ID NO:26, and the primer sequences are shown in Table 6.

[0110] Table 6 Primer sequence information for McCJ25 integration

[0111] Primer name Sequence (5'-3') xylAjup-F 5'-ggaacaatatcgaccagggc-3'(SEQ ID NO:27) xylAjup-R 5'-gggatcgagatctcgatccttgagccttcataacgaacgc-3' (SEQ ID NO: 28) xylAjdown-F 5'-ctcaggaacataatttgtctccg-3'(SEQ ID NO:29) xylAjdown-R 5'-tcgctgccttcgtataatgc-3'(SEQ ID NO:30) McJ25X-F 5'-aggatcgagatctcgatccc-3'(SEQ ID NO:31) McJ25X-R 5'-cggagacaaattatgttcctgagttattctgtagctgatgccag-3' (SEQ ID NO: 32)

[0112] The PCR amplification system and procedure, and the overlapping PCR amplification system and procedure are the same as those in the above embodiments.

[0113] (3) Constructing araBsgRNA plasmid, endAsgRNA plasmid, and xylAsgRNA plasmid:

[0114] The sgRNA-araB fragment was designed, with the sequence shown in SEQ ID NO: 33. The sequence shown in SEQ ID NO: 33 was cloned into the pUC19 vector by General Biotechnology (Anhui) Co., Ltd., thus obtaining the araBsgRNA plasmid (plasmid map shown). Figure 2A (As shown).

[0115] As described above, an sgRNA-endA fragment was designed, with the sequence shown in SEQ ID NO: 34. Weitong Biotechnology (Anhui) Co., Ltd. cloned the sequence shown in SEQ ID NO: 34 into the pUC19 vector, thereby obtaining the endAsgRNA plasmid (plasmid map shown in...). Figure 2B (As shown).

[0116] The sgRNA-xylA fragment was designed, with the sequence shown in SEQ ID NO: 35. Weitong Bio (Anhui) Co., Ltd. cloned the sequence shown in SEQ ID NO: 35 into the pUC19 vector, thereby obtaining the xylAsgRNA plasmid (plasmid map shown). Figure 2C (As shown).

[0117] (4) Construction of EcN-cas9 strain containing pREDCas9 plasmid:

[0118] EcN competent cells were prepared according to *Molecular Cloning: A Laboratory Manual* (3rd edition). The pREDCas9 plasmid (purchased from Addgene) was transformed into the EcN competent cells via electroporation. Transformants were obtained by screening with spectinomycin-resistant plates. PCR amplification was performed using pREDCas9-test-F and pREDCas9-test-R primers to confirm successful transformation. Successfully transformed strains were identified as EcN-cas9 strains containing the pREDCas9 plasmid.

[0119] The sequences of pREDCas9-test-F and pREDCas9-test-R are shown in Table 7.

[0120] Table 7 Primer information for EcN-cas9 strain identification

[0121] Primer name Sequence (5'-3') pREDCas9-test-F 5'-ttcctaatcagcccggcatttc-3'(SEQ ID NO:36) pREDCas9-test-R 5'-acgacataatgcaggccttc-3'(SEQ ID NO:37)

[0122] (5) Construction of EcNΔaraB-pREDCas9-T7RNP-araBsgRNA

[0123] The obtained EcN-cas9 strain was induced with 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) to an OD600 of 0.6-0.8 to prepare EcN-cas9 competent cells. The SD sequence, the complete T7RNP homologous arm repair fragment, and the araBsgRNA plasmid were then transformed into the EcN-cas9 competent cells via electroporation. Transformants were obtained by screening with spectinomycin (50 μg / mL) and ampicillin (100 μg / mL) double antibiotic plates. PCR amplification was performed using araB-test-F and araB-test-R primers to confirm successful transformation. Successfully transformed strains were identified as the recombinant strain EcN-T7RNP-araBsgRNA-cas9.

[0124] The sequences of araB-test-F and araB-test-R are shown in Table 8.

[0125] Table 8 Primer information for identification of recombinant strain EcNΔaraB-T7RNP-araBsgRNA-cas9

[0126] Primer name Sequence (5'-3') araB-test-F 5'-attcacaacctgccctaaac-3' (SEQ ID NO:38) araB-test-R 5'-caccttcatgatgcgaagca-3'(SEQ ID NO:39)

[0127] (6) Elimination of araBsgRNA plasmid:

[0128] The recombinant strain EcNΔaraB-T7RNP-araBsgRNA-cas9 was continuously passaged on spectinomycin-resistant plates and cultured at 30°C. Single colonies were picked and stab-tested on spectinomycin-resistant plates and spectinomycin and ampicillin-resistant plates to screen for strains that were insensitive to spectinomycin but sensitive to ampicillin (i.e., those that survived on spectinomycin-resistant plates but could not survive on spectinomycin and ampicillin-resistant plates). PCR amplification was performed using sgRNA-test-F and sgRNA-test-R primers to identify whether the transformation was successful. The successfully transformed strain was the recombinant strain EcNΔaraB-T7RNP-cas9 with the araBsgRNA plasmid eliminated.

[0129] The sequences of sgRNA-test-F and sgRNA-test-R are shown in Table 9.

[0130] Table 9 Primer information for identification of recombinant strain EcN-T7RNP-cas9 used for araBsgRNA plasmid elimination.

[0131]

[0132]

[0133] (7) Construction of MccY recombinant integration engineered probiotic strain EcNΔaraB-T7RNP-cas9-ΔendA-MccY-endAsgRNA:

[0134] Following the above procedures, the recombinant strain EcNΔaraB-T7RNP-cas9, with the araBsgRNA plasmid eliminated, was induced with 0.5 mM IPTG to an OD600 of 0.6-0.8. EcNΔaraB-T7RNP-cas9 competent cells were prepared. The optimized MccY sequence repair fragment and the endAsgRNA plasmid were then transformed into EcNΔaraB-T7RNP-cas9 competent cells via electroporation. Screening was performed using spectinomycin and ampicillin-containing plates; surviving bacteria were identified as transformants. PCR amplification was performed using endA-F and endA-R primers to confirm successful transformation. Successfully transformed strains were identified as the recombinant strain EcNΔaraB-T7RNP-cas9-ΔendA-MccY-endAsgRNA, which integrates the optimized MccY sequence into the EcN genome.

[0135] The sequences of endA-test-F and endA-test-R are shown in Table 10.

[0136] Table 10 Primer information for identification of EcNΔaraB-T7RNP-cas9-ΔendA-MccY / MccJ25-endAsgRNA

[0137] Primer name Sequence (5'-3') endA-test-F 5'-tgagtgccggtccgctgatt-3'(SEQ ID NO:42) endA-test-R 5'-tgtgcttccagcaacatagc-3'(SEQ ID NO:43)

[0138] (8) MccJ25 Recombinant Integrative Probiotic Strains

[0139] Construction of EcNΔaraB-T7RNP-cas9-ΔendA-MccJ25-endAsgRNA:

[0140] Following the above procedure, the recombinant strain EcNΔaraB-T7RNP-cas9 with the araBsgRNA plasmid eliminated was induced with 0.5 mM IPTG to an OD600 of 0.6-0.8 to prepare EcNΔaraB-T7RNP-cas9 competent cells. The MccJ25E homologous arm repair fragment and the endAsgRNA plasmid were then transformed into the EcNΔaraB-T7RNP-cas9 competent cells by electroporation. The cells were then screened using spectinomycin and ampicillin double antibiotic plates, and the surviving bacteria were the transformants.

[0141] PCR amplification was performed using endA-test-F and endA-test-R primers (as shown in Table 10) to identify whether the transformation was successful. The successfully transformed strain was the recombinant strain EcNΔaraB-T7RNP-cas9-ΔendA-MccJ25-endAsgRNA, which integrated the optimized MccJ25 sequence into the EcN genome.

[0142] (9) Elimination of endAsgRNA plasmid:

[0143] The recombinant strains EcNΔaraB-T7RNP-ΔendA-MccY-endAsgRNA-cas9 and EcNΔaraB-T7RNP-ΔendA-MccJ25-endAsgRNA-cas9 were continuously passaged on spectinomycin-resistant plates and cultured at 30°C. Single colonies were picked and stab-tested on spectinomycin-resistant plates and spectinomycin and ampicillin-resistant plates to screen for strains that were insensitive to spectinomycin but sensitive to ampicillin (i.e., strains that survived on spectinomycin-resistant plates but not on spectinomycin and ampicillin-resistant plates). PCR amplification was performed using sgRNA-test-F and sgRNA-test-R primers (as shown in Table 9) to identify successful transformation. The successfully transformed strains were the recombinant strains with the endAsgRNA plasmid eliminated.

[0144] EcNΔaraB-T7RNP-ΔendA-MccY-cas9 and EcNΔaraB-T7RNP-ΔendA-MccJ25-cas9.

[0145] (10) Elimination of pREDCas9 plasmid:

[0146] The recombinant strains obtained above, EcNΔaraB-T7RNP-ΔendA-MccY-cas9 and EcNΔaraB-T7RNP-ΔendA-MccJ25-cas9, were continuously passaged on antibiotic-free plates and cultured at 42°C. Single colonies were picked and stab-dropped onto antibiotic-free and spectinomycin-resistant plates, respectively, to screen for spectinomycin-sensitive strains. PCR amplification was performed using sgRNA-test-F and sgRNA-test-R primers to confirm successful transformation. The successfully transformed strains were the recombinant strains EcNΔaraB-T7RNP-ΔendA-MccY and EcNΔaraB-T7RNP-ΔendA-MccJ25 (i.e., Microbicin MccY recombinant integrated engineered probiotic and MccJ25 recombinant integrated engineered probiotic) with the pREDCas9 and endAsgRNA plasmids eliminated.

[0147] (11) Construction of MccY+MccJ25 recombinant and integrated engineered probiotic strain EcNΔaraB-T7RNP-cas9-ΔendA-MccY-ΔxylA-MccJ25-xylAsgRNA:

[0148] Following the above procedure, the recombinant strain EcNΔaraB-T7RNP-ΔendA-MccY-cas9, which had its endAsgRNA plasmid eliminated, was induced with 0.5 mM IPTG to an OD600 of 0.6-0.8 to prepare EcNΔaraB-T7RNP-ΔendA-MccY-cas9 competent cells. The optimized McJ25X homologous arm repair fragment and xylAsgRNA plasmid were then transformed into EcNΔaraB-T7RNP-ΔendA-MccY-cas9 competent cells via electroporation. Transformants were obtained by screening with spectinomycin and ampicillin dual-resistance plates. PCR amplification was performed using xylA-test-F and xylA-test-R primers to identify whether the transformation was successful. The successfully transformed strain was the recombinant strain EcNΔaraB-T7RNP-pREDCas9-ΔendA-MccY-ΔxylA-MccJ25-xylAsgRNA, which simultaneously integrated the optimized MccY and MccJ25 sequences into the EcN genome.

[0149] The sequences of xylA-test-F and xylA-test-R are shown in Table 11.

[0150] Table 11 Primer information for identification of EcNΔaraB-T7RNP-cas9-ΔendA-MccY-ΔxylA-MccJ25-xylAsgRNA

[0151] Primer name Sequence (5'-3') xylA-test-F 5'-gaggcaactacagcatcaat-3'(SEQ ID NO:44) xylA-test-R 5'-cgagctgcagtgattaaagc-3'(SEQ ID NO:45)

[0152] (12) Elimination of pREDCas9 and BsgRNA dual plasmids:

[0153] The recombinant strain EcNΔaraB-T7RNP-ΔendA-MccY-ΔxylA-MccJ25-xylAsgRNA-cas9 obtained above was continuously passaged on antibiotic-free plates and cultured at 42℃. Single colonies were picked and stab-tested onto antibiotic-free plates, spectinomycin-resistant plates, and kanamycin-resistant plates to screen for strains sensitive to spectinomycin and ampicillin. PCR amplification was performed using sgRNA-test-F and sgRNA-test-R primers to confirm successful transformation. The successfully transformed strain was EcNΔaraB-T7RNP-ΔendA-MccY-ΔxylA-MccJ25 (i.e., Microsin MccY-MccJ25 recombinant integrated probiotic) with pREDCas9 and xylAsgRNA plasmids eliminated.

[0154] Figures 3A to 3D They are respectively:

[0155] Figure 3A This is a diagram showing the PCR identification results of the EcNΔaraB-T7RNP strain;

[0156] Figure 3B This is a diagram showing the PCR identification results of strain EcNΔaraB-T7RN-ΔendA-MccY;

[0157] Figure 3C This is a graph showing the PCR identification results of strain EcNΔaraB-T7RN-ΔendA-MccJ25;

[0158] Figure 3D This is a diagram showing the PCR identification results of strain EcNΔaraB-T7RN-ΔendA-MccY-ΔxylA-MccJ25.

[0159] Example 2: Sensitivity determination of MccY recombinant and integrated engineered probiotics, MccJ25 recombinant and integrated engineered probiotics, and MccY+MccJ25 recombinant and integrated engineered probiotics.

[0160] The constructed MccY recombinant integrated engineered probiotics, MccJ25 recombinant integrated engineered probiotics, and MccY+MccJ25 recombinant integrated engineered probiotics were inoculated into 20 mL LB medium, and 1 Mm IPTG inducer was added for expression induction. The mixture was then incubated at 37℃ for 24 hours at 200 rpm / min, centrifuged, and the bacterial cells were removed to obtain the expression supernatants of the MccY, MccJ25, and MccY+MccJ25 recombinant integrated engineered probiotics. These supernatants were serially diluted with PBS at 2-fold (2×), 10-fold (10×), 20-fold (20×), 100-fold (100×), and 200-fold (200×), respectively, and sterilized by filtration through a 0.22 μm filter. The supernatants were stored at -20℃ for later use.

[0161] Salmonella Typhimurium (strain number ATCC 14028) and Salmonella Cyclotropii Enteritidis (strain number CVCC3377) were revived on LB agar plates. Single colonies were picked and cultured until OD600 = 0.8. The bacterial suspensions were then inoculated into 0.5% LB soft agar at a ratio of 1:1000. The culture was then poured into petri dishes and allowed to stand for 30 minutes until the medium solidified. 10 μL of the supernatant from the serially diluted MccY recombinant engineered probiotics obtained in the above steps was then spotted onto 0.5% LB soft agar containing Salmonella Typhimurium. 10 μL of the supernatant from the MccY recombinant engineered probiotics obtained in the above steps was then spotted onto 0.5% LB soft agar containing Salmonella Typhimurium. Take 10 μL of the supernatant of the MccJ25 recombinant and integrated engineered probiotics obtained by serial dilution in the above steps, and spot it onto 0.5% LB soft agar containing Salmonella enteritidis. Take 10 μL of the supernatant of the MccY+MccJ25 recombinant and integrated engineered probiotics obtained by serial dilution in the above steps, and spot it onto 0.5% LB soft agar containing Salmonella enteritidis and 0.5% LB soft agar containing Salmonella typhimurium, respectively. After drying, place it in an incubator at 37°C for 16 hours and observe the inhibition zone.

[0162] The results are as follows Figures 4A to 4C As shown;

[0163] Figure 4A This is a diagram of the inhibition zone of MccY recombinant and integrated probiotics in 0.5% LB soft agar containing Salmonella typhimurium;

[0164] Figure 4B This is a diagram of the inhibition zone of MccJ25 recombinant and integrated probiotic in 0.5% LB soft agar containing Salmonella enteritidis;

[0165] Figure 4C This is an inhibition zone diagram of the MccY+MccJ25 recombinant and integrated probiotic in 0.5% LB soft agar containing Salmonella typhimurium and 0.5% LB soft agar containing Salmonella enteritidis.

[0166] It was found that the 1×, 2×, 10×, and 20× supernatants of the MccY recombinant and integrated engineered probiotic strain had significant antibacterial effects against Salmonella typhimurium, while the 100× supernatant had a weak antibacterial effect against Salmonella typhimurium. The 1×, 2×, 10×, and 20× supernatants of the MccJ25 recombinant and integrated engineered probiotic strain had significant antibacterial effects against Salmonella enteritidis, while the 100× supernatant had a weak antibacterial effect against Salmonella enteritidis. The 1×, 2×, and 10× supernatants of the MccY+MccJ25 recombinant and integrated engineered probiotic strain had significant antibacterial effects against both Salmonella enteritidis and Salmonella typhimurium, while the 20× supernatant had a weak antibacterial effect against Salmonella enteritidis.

[0167] Example 3: Quantitative analysis of MccY recombinant integrative engineered probiotics, MccJ25 recombinant integrative engineered probiotics, and MccY+MccJ25 recombinant integrative engineered probiotics using high-performance liquid chromatography (HPLC).

[0168] One mL of supernatant from MccY recombinant integrated engineered probiotics, MccJ25 recombinant integrated engineered probiotics, and MccY+MccJ25 recombinant integrated engineered probiotics expressed for 24 hours was collected, and the concentrations of MccY and MccJ25 in the supernatant were detected using ultra-high performance liquid chromatography-quadrupole tandem time-of-flight mass spectrometry (Agilent Technologies, USA, model: UPLC1290-6540BQ-TOF). Mobile phase A: 100% acetonitrile, initial concentration 5% (v / v); mobile phase B: 100% water plus 0.2% (v / v) formic acid, initial concentration 95% (v / v); column temperature 25℃; detection wavelength 214 nm; flow rate 0.5 mL / min; gradient elution was performed according to Table 12.

[0169] Table 12

[0170] Time (min) A(％) B(％) 20 50 50 30 90 10 35 10 90

[0171] The results are as follows Figures 5A to 5D As shown, mass spectrometry analysis of the MccY recombinant and integrated engineered probiotics revealed a characteristic ion peak at 1113.73, consistent with the target substance MccY, indicating successful expression and secretion of MccY in the engineered bacteria, with a protein concentration of 24.88 mg / L. Mass spectrometry analysis of the MccJ25 recombinant and integrated engineered probiotics revealed a characteristic ion peak at 1054.685, consistent with the target substance MccJ25, indicating successful expression and secretion of MccJ25 in the engineered bacteria, with a protein concentration of 34.12 mg / L. Mass spectrometry analysis of the MccY+MccJ25 recombinant and integrated engineered probiotics revealed a characteristic ion peak at 1113.73, consistent with the target substance MccY, indicating successful expression and secretion of MccY in the engineered bacteria, with a protein concentration of 9.80 mg / L. Mass spectrometry analysis of the MccY+MccJ25 recombinant and integrated engineered probiotics revealed a characteristic ion peak at 1054.685, consistent with the target substance MccJ25, indicating successful expression and secretion of MccJ25 in the engineered bacteria, with a protein concentration of 9.11 mg / L.

[0172] in, Figure 5A This is a liquid chromatography result of the culture supernatant of EcN-MccY engineered probiotics;

[0173] Figure 5B This is a liquid chromatography-mass spectrometry result of the culture supernatant of EcN-MccY engineered probiotics;

[0174] Figure 5C This is a liquid chromatography result of the culture supernatant of EcN-MccJ25 engineered probiotics;

[0175] Figure 5D This is a liquid chromatography-mass spectrometry result of the culture supernatant of EcN-MccJ25 engineered probiotics;

[0176] Figure 5E This is a liquid chromatography result of MccY in the culture supernatant of MccY+MccJ25 recombinant and integrated probiotics;

[0177] Figure 5F This is a liquid chromatography-mass spectrometry (LC-MS) result of MccY in the culture supernatant of the MccY+MccJ25 recombinant and integrated probiotic strain.

[0178] Figure 5G This is a liquid chromatography result of MccJ25 in the culture supernatant of MccY+MccJ25 recombinant and integrated probiotics;

[0179] Figure 5H This is a liquid chromatography-mass spectrometry (LC-MS) result of MccJ25 in the culture supernatant of the MccY+MccJ25 recombinant and integrated probiotic.

[0180] Example 4: Detection of antibacterial activity of MccY recombinant integrative engineered probiotics, MccJ25 recombinant integrative engineered probiotics, and MccY+MccJ25 recombinant integrative engineered probiotics at different expression times.

[0181] The constructed MccY recombinant integrated engineered probiotics, MccJ25 recombinant integrated engineered probiotics, and MccY+MccJ25 recombinant integrated engineered probiotics were inoculated into 20 mL LB medium, and 1 mM IPTG inducer was added for expression. The culture was then placed in a shaker at 37 °C at 200 rpm / min for expression. 1 mL of bacterial culture was taken at 4, 8, 12, 16, 20, and 24 hours, and the bacterial culture at different time points was centrifuged, the bacterial cells were removed, the supernatant was collected, and the culture was filtered through a 0.22 μm filter for sterilization. The culture was then stored at -20 °C for later use.

[0182] Salmonella typhimurium (strain number ATCC 14028) and Salmonella enteritidis (strain number CVCC3377) were revived on LB agar plates. Single colonies were picked and cultured until OD600 = 0.8. The bacterial suspensions were then inoculated into 0.5% LB soft agar at a ratio of 1:1000. The agar plates were then poured into Petri dishes and allowed to stand for 30 min until the medium solidified. The Petri dishes were then divided into 6 equal parts. 10 μL of the supernatant expressed by MccY recombinant engineered probiotics and MccY+MccJ25 recombinant engineered probiotics at 4, 8, 12, 16, 20, and 24 h was spotted onto the surface of each agar plate containing Salmonella typhimurium, and 10 μL of the supernatant expressed by MccY+MccJ25 recombinant engineered probiotics was spotted onto the surface of each agar plate containing Salmonella enteritidis. The supernatants expressed by MccJ25 recombinant integrated engineered probiotics and MccY+MccJ25 recombinant integrated engineered probiotics at 4, 8, 12, 16, 20, and 24 hours were air-dried for 10 minutes and then inverted and incubated overnight at 37°C to observe the antibacterial effect.

[0183] As shown in Figure 6, the supernatant of MccY recombinant integrated engineered probiotics, MccJ25 recombinant integrated engineered probiotics, and MccY+MccJ25 recombinant integrated engineered probiotics showed inhibition zones from 4 to 24 hours after expression, and the antibacterial effect became more obvious with increasing time.

[0184] Figure 6A This is a diagram of the inhibition zones of MccY recombinant and integrated probiotics expressed at different times;

[0185] Figure 6B This is a diagram of the inhibition zones of MccJ25 recombinant and integrated engineered probiotics at different expression times;

[0186] Figure 6C This is a diagram showing the inhibition zones of MccY+MccJ25 recombinant and integrated engineered probiotics at different expression times against Salmonella typhimurium.

[0187] Figure 6D This is a diagram showing the inhibition zones of MccY+MccJ25 recombinant and integrated engineered probiotics at different expression times against Salmonella enteritidis.

[0188] Example 5: Antibacterial activity test of MccY recombinant engineered probiotics, MccJ25 recombinant engineered probiotics, and MccY+MccJ25 recombinant engineered probiotics.

[0189] The MccY, MccJ25, and MccY+MccJ25 recombinant-integrated probiotic strains were revived in LB agar plates. One loopful of each strain was scraped into 1 mL of sterile water and mixed thoroughly. 10 μL of the bacterial suspension was then punctured into an LB agar plate, air-dried, and incubated at 37°C for 24 h. After incubation, the plate was removed and irradiated with ultraviolet light for 1 h.

[0190] Salmonella typhimurium ATCC14028, Salmonella pullorum CVCC1800, Salmonella enteritidis CVCC3377, Salmonella infantis CMCC50041, Salmonella Kentuckyii BNCC239114, Salmonella Londonii CVCC2206, Salmonella Corvallis CMCC50922, Escherichia coli DH5α, Escherichia coli BL21, Escherichia coli ATCC25922, Shigella sonnei SS1014, Shigella flexneri SF1015, Staphylococcus aureus ATCC6538, and Bacillus subtilis ATCC6633 were resuscitated on LB agar plates. Single colonies of each test strain were picked and cultured until OD600 = 0.8. The bacterial solution was then inoculated into 0.5% LB soft agar medium at a ratio of 1:1000. The inoculated agar was then poured into LB plates (pre-irradiated with ultraviolet light) containing MccY recombinant integrated engineered probiotics, MccJ25 recombinant integrated engineered probiotics, and MccY+MccJ25 recombinant integrated engineered probiotics. After solidification for 30 minutes, the plates were incubated at 37°C for 16 hours, and the inhibition zone was observed.

[0191] The results are as follows Figure 7 As shown, after 24 hours of colonization and culture of the EcN-MccY recombinant and integrated probiotics, a large amount of MccY was secreted, which could inhibit the growth of Salmonella Typhimurium ATCC14028, Salmonella Enteritidis CVCC3377, Salmonella Pullorum CVCC1800, Salmonella Infantile CMCC50041, Salmonella Kentuckyis BNCC239114, Salmonella Londonii CVCC2206, Salmonella Corvallis CMCC50922, and Shigella Sonnei SS1014, forming inhibition zones of varying sizes; however, the inhibitory effect on Escherichia coli ATCC25922, Shigella flexneri SF1015, Bacillus subtilis BS1011, and Staphylococcus aureus ATCC6538 was not obvious.

[0192] After colonization and culture of the EcN-MccJ25 recombinant and integrated probiotics for 24 hours, a large amount of MccJ25 was secreted, which could inhibit the growth of Salmonella enteritidis CVCC3377, Shigella flexneri SF1015, and Escherichia coli ATCC25922, forming inhibition zones of varying sizes; however, the inhibitory effect on Salmonella typhimurium ATCC14028, Salmonella pullorum CVCC1800, Salmonella infantis CMCC50041, Salmonella Kentuckyis BNCC239114, Salmonella Londonii CVCC2206, Salmonella Corvallis CMCC50922, Shigella sonnei SS1014, Bacillus subtilis BS1011, and Staphylococcus aureus ATCC6538 was not obvious.

[0193] After colonization and culture of the EcN-MccY+MccJ25 recombinant and integrated probiotic strain for 24 hours, it secreted a large amount of MccY and MccJ25, which could inhibit the growth of Salmonella Typhimurium ATCC14028, Salmonella Enteritidis CVCC3377, Salmonella Pullorum CVCC1800, Salmonella Infantile CMCC50041, Salmonella Kentuckyii BNCC239114, Salmonella Londonii CVCC2206, Salmonella Corvallis CMCC50922, Shigella flexneri SF1015, Shigella sonnei SS1014, and Escherichia coli ATCC25922, forming inhibition zones of varying sizes; however, the inhibitory effect on Bacillus subtilis BS1011 and Staphylococcus aureus ATCC6538 was not obvious.

[0194] In summary, the advantages of this invention are:

[0195] 1. The common advantage of the three engineered probiotics of this invention is that they can be used as probiotics, meeting the following requirements for probiotics: 1. They are live bacteria capable of acting on the intestines of humans or animals; 2. The three engineered strains constructed by the method of this invention do not carry any resistance markers, eliminating the risk of horizontal transmission of resistance genes and ensuring their safety as probiotics; 3. The three engineered strains constructed by the method of this invention express microinfectious agents in a non-plasmid expression form, eliminating the need for antibiotics to lock the plasmids, allowing them to stably act on the intestines and be passaged, meeting the stability requirements for probiotic use.

[0196] Through the above optimizations, all three engineered probiotics of the present invention can improve the intestinal health of humans or animals and enhance the antibacterial ability of EcN against harmful bacteria.

[0197] 2. The unique advantage of the engineered probiotics with recombinant integration of microbesin MccJ25 of the present invention is that the engineered probiotics with recombinant integration of microbesin MccJ25 can achieve a high expression level of 25.31 mg / L for microbesin MccJ25, and can effectively inhibit Salmonella enteritidis CVCC3377, Shigella flexneri SF1015, and Escherichia coli ATCC25922. The engineered probiotics with recombinant integration of microbesin MccJ25 are far more sensitive to Salmonella enteritidis than engineered probiotics with recombinant integration of microbesin MccY and engineered probiotics with recombinant integration of microbesin MccY+MccJ25.

[0198] 3. The unique advantage of the engineered probiotics with recombinant integration of microbesin MccY of the present invention is that the engineered probiotics with recombinant integration of microbesin MccY can achieve a high expression level of 9.23 mg / L for microbesin MccY, and can effectively inhibit Salmonella Typhimurium ATCC14028, Salmonella Enteritidis CVCC3377, Salmonella Pullorum CVCC1800, Salmonella Infantile CMCC50041, Salmonella Kentuckyis BNCC239114, Salmonella Londonella CVCC2206, Salmonella Corvallis CMCC50922, and Shigella Sonnei SS1014. Its sensitivity against Salmonella Typhimurium is far superior to that of engineered probiotics with recombinant integration of microbesin MccJ25 and engineered probiotics with recombinant integration of microbesin MccY+MccJ25.

[0199] 4. The unique advantages of the engineered probiotics of the present invention, which recombinantly integrates microbes MccY and MccJ25, are as follows: the expression level of microbes MccY can reach a high level of 8.02 mg / L, and the expression level of microbes MccJ25 can reach a high level of 9.11 mg / L. This allows them to inhibit Salmonella Typhimurium ATCC14028, Salmonella Enteritidis CVCC3377, Salmonella Pullorum CVCC1800, and Salmonella Infantitidis CMCC50041. The probiotics recombinantly integrated with Microbes Y+MccJ25 effectively inhibited the growth of *Salmonella Kentuckyii* BNCC239114, *Salmonella Londonii* CVCC2206, *Salmonella Corvallisii* CMCC50922, *Shigella flexneri* SF1015, *Shigella sonnei* SS1014, and *Escherichia coli* ATCC25922. The broad-spectrum antibacterial activity of the probiotics recombinantly integrated with Microbes Y+MccJ25 was superior to that of the probiotics recombinantly integrated with Microbes Y+MccJ25.

[0200] The embodiments presented herein are merely selected implementations based on combinations of all possible embodiments. The appended claims should not be limited to the embodiments described herein. Some numerical ranges used in the claims include sub-ranges within them, and variations within these ranges should also be covered by the appended claims.

Claims

1. An engineered probiotic containing recombinant and integrated microbialin MccJ25, characterized in that, The engineered probiotic is an Escherichia coli that integrates the microcrystal MccJ25 gene; the nucleotide sequence of the microcrystal MccJ25 gene is shown in SEQ ID NO: 4; the Escherichia coli is Escherichia coli Nissle 1917; The preparation method of the engineered probiotics is as follows: (1) pREDCas9 plasmid was transformed into EcN competent cells, positive transformants EcN-cas9 were screened, and the positive transformants EcN-cas9 were prepared into competent cells; the competent cells were EcN-cas9 competent cells; (2) The SD-T7RNP homologous arm repair fragment and araBsgRNA plasmid were transformed into EcN-cas9 competent cells, and recombinant strain EcN∆ was obtained by screening with spectinomycin and ampicillin. araB -T7RNP-araBsgRNA-cas9; the araBsgRNA plasmid is a pUC19 vector with an inserted sgRNA-araB fragment; the nucleotide sequence of the sgRNA-araB fragment is shown in SEQ ID NO: 33; (3) For the recombinant strain EcN∆ araB The -T7RNP-araBsgRNA-cas9 strain was passaged to screen for strains insensitive to spectinomycin and sensitive to ampicillin, thus eliminating the araBsgRNA plasmid in the strains. The MccJ25E homologous arm repair fragment and endAsgRNA plasmid were then introduced, and recombinant strain EcN∆ was obtained through screening with spectinomycin and ampicillin. araB -T7RNP-cas9-∆ endA -MccJ25-endAsgRNA; the endAsgRNA plasmid is a pUC19 vector with an inserted sgRNA-endA fragment; the nucleotide sequence of the sgRNA-endA fragment is shown in SEQ ID NO: 34; (4) For the recombinant strain EcN∆ araB -T7RNP-cas9-∆ endA -MccJ25-endAsgRNA was used for subculturing to screen for strains that were insensitive to spectinomycin and sensitive to ampicillin, thereby eliminating the endAsgRNA plasmid in the strains and obtaining the recombinant strain EcN∆. araB -T7RNP-∆ endA -MccJ25-cas9; (5) For the recombinant strain EcN∆ araB -T7RNP-∆ endA -MccJ25-cas9 was passaged at 42℃ to screen for strains sensitive to both spectinomycin and ampicillin to eliminate the pREDCas9 plasmid in the strains, thus obtaining the microbestos MccJ25 recombinant integrated engineered probiotic EcN-MccJ25. The nucleotide sequence of the SD-T7RNP homologous arm repair fragment is shown in SEQ ID NO: 5; the MccJ25E homologous arm repair fragment is shown in SEQ ID NO:

19.

2. An engineered probiotic composed of recombinant and integrated microbes MccY and MccJ25, characterized in that, The engineered probiotic is an *Escherichia coli* strain integrating the microbesin MccY gene and the microbesin MccJ25 gene; the nucleotide sequence of the microbesin MccY gene is shown in SEQ ID NO: 3; the nucleotide sequence of the microbesin MccJ25 gene is shown in SEQ ID NO: 4; the *Escherichia coli* strain is *Escherichia coli* Nissle 1917. The preparation method of the engineered probiotics is as follows: (1) pREDCas9 plasmid was transformed into EcN competent cells, positive transformants EcN-cas9 were screened, and the positive transformants EcN-cas9 were prepared into competent cells; the competent cells were EcN-cas9 competent cells; (2) The SD-T7RNP homologous arm repair fragment and araBsgRNA plasmid were transformed into EcN-cas9 competent cells, and the recombinant strain EcN-T7RNP-araBsgRNA-cas9 was obtained by screening with spectinomycin and ampicillin; the araBsgRNA plasmid was a pUC19 vector with an inserted sgRNA-araB fragment; the nucleotide sequence of the sgRNA-araB fragment is shown in SEQ ID NO: 33; (3) The recombinant strain EcN-T7RNP-araBsgRNA-cas9 was passaged and screened to remove the araBsgRNA plasmid from the strains. The MccY homologous arm repair fragment and endAsgRNA plasmid were then introduced. The recombinant strain EcN-T7RNP-pREDCas9-MccY-endAsgRNA was obtained by screening with spectinomycin and ampicillin. The recombinant strain EcN-T7RNP-pREDCas9-MccY-endAsgRNA was then used to prepare competent cells, which were EcN-T7RNP-MccY-cas9 competent cells. The endAsgRNA plasmid was a pUC19 vector with an inserted sgRNA-endA fragment. The nucleotide sequence of the sgRNA-endA fragment is shown in SEQ ID NO:

34. (4) The MccJ25X homologous arm repair fragment and xylAsgRNA plasmid were transformed into EcN-T7RNP-MccY-cas9 competent cells and screened with spectinomycin and ampicillin double resistance plates to obtain the recombinant strain EcN-T7RNP-pREDCas9-MccY-MccJ25-xylAsgRNA, in which MccY and MccJ25 were simultaneously integrated into the EcN genome; the xylAsgRNA plasmid was a pUC19 vector with an inserted sgRNA-xylA fragment, the nucleotide sequence of which is shown in SEQ ID NO: 35; (5) Eliminate the pREDCas9 plasmid and xylAsgRNA plasmid in the recombinant strain EcN-T7RNP-pREDCas9-MccY-MccJ25-xylAsgRNA to obtain strain EcN-T7RNP-MccY-MccJ25. The strain EcN-T7RNP-MccY-MccJ25 is the recombinant and integrated probiotic EcN-MccY-MccJ25 of microbesin MccY and MccJ25. The nucleotide sequence of the SD-T7RNP homologous arm repair fragment is shown in SEQ ID NO: 5; the MccJ25X homologous arm repair fragment is shown in SEQ ID NO: 26; and the MccY homologous arm repair fragment is shown in SEQ ID NO:

12.

3. An antibacterial agent, characterized in that, It contains engineered probiotics as described in claim 1 or 2.

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