Construction and antibacterial application of recombinant engineering probiotics for producing microbacteriocin MccY and MccJ25

Abstract

The invention belongs to the technical field of biology, and discloses engineering probiotics recombined and integrated by three kinds of microbacteriocin, and the engineering probiotics recombined and integrated by the three kinds of microbacteriocin are respectively engineering probiotics recombined and integrated by microbacteriocin MccJ25, engineering probiotics recombined and integrated by microbacteriocin MccY and engineering probiotics recombined and integrated by the microbacteriocin MccJ25 and the microbacteriocin MccY. The three engineering probiotics have no resistance marker genes together, and have the characteristics of good growth activity, genetic stability and antibacterial activity. Meanwhile, the invention also provides a preparation method and application of the engineering probiotics.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to the construction and antibacterial application of recombinant engineering probiotics producing microcins MccY and MccJ25, and more specifically to an engineering probiotic with recombinant integration of microcin MccJ25, an engineering probiotic with recombinant integration of microcin MccY, an engineering probiotic with recombinant integration of microcin MccY and MccJ25, and a preparation method and use thereof. Background Art

[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 the use of antibiotics in livestock production. However, the long-term irrational use of antibiotics will have certain negative effects on livestock and humans, causing 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 promulgated the "National Action Plan for Reducing the Use of Veterinary Antimicrobial Drugs". Therefore, banning, limiting, reducing and replacing antibiotics are urgent, and it is particularly important to develop safe, efficient, and low-residue veterinary antibiotic-reducing and antibiotic-replacing inhibitors.

[0003] Microcin is a low molecular weight, highly stable bacterial peptide produced by Gram-negative bacteria and synthesized by ribosomes. It can be used to prevent and treat bacterial infections in animals. Microcins are divided into two categories: Class I microcins are less than 5kDa and are encoded by plasmids, and Class II microcins are about 5-10kDa in size and can be divided into plasmid-encoded and chromosome-encoded. Microcin MccJ25 belongs to Class I micropeptide microcins and is 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 autoimmunity gene mcjD. MccJ25 kills bacteria mainly by inhibiting the activity of RNA polymerase in cells and thus terminating the transcription of the respiratory chain; it exhibits efficient bactericidal activity against Salmonella and Escherichia coli. Therefore, it is of great application value to obtain an engineered strain that can efficiently produce MccJ25 through genetic engineering.

[0004] The following scheme is used to describe the case of inserting microcin MccJ25 into engineered bacteria for expressing microcin MccJ25:

[0005] Publication number CN113774006A, the subject is an invention patent application for an engineered strain that efficiently expresses MccJ25 and its fermentation process;

[0006] Publication No. CN114574412A, the subject is a Bacillus subtilis engineered bacterium expressing MccJ25 and its construction method and application;

[0007] The above schemes are all production bacteria, among which the production bacteria used in CN113774006A is Escherichia coli AGL027, and the production bacteria used in CN114574412A is Bacillus subtilis;

[0008] The production bacteria itself has a very strong target gene expression ability, which is determined by the characteristics of the production bacteria itself. At the same time, the production bacteria generally do not need to grow in vivo, as described in paragraph 66 of the specification of CN113774006A: "Lactobacillus plantarum is cultured for 16-18 hours, resuspended in an equal volume of distilled water, lysozyme is added for 10 minutes, the cells are completely broken, centrifuged, the supernatant is obtained, and the J25 content is determined by high performance liquid chromatography"; the method for obtaining J25 protein is to collect the cells after lysing and breaking them.

[0009] Escherichia coli Nissle 1917 (EcN) is the only live bacterial preparation approved by the FDA for the treatment of enteritis. It is a non-pathogenic Escherichia coli isolated by German doctor Alfred Nissle from the feces of a soldier fighting diarrhea in 1917. It has probiotic properties and has the potential to be used as an antibiotic-reducing and antibiotic-replacing preparation because it can secrete microcins MccH47 and MccM. It contains 2.5-25×10 9 A probiotic drug containing 100 colony-forming units of live Escherichiacoli Nissle 1917 (trade name Mutaflor) is approved for production and sale as a treatment for many gastrointestinal diseases.

[0010] The antibacterial spectrum of microcins MccH47 and MccM is relatively narrow. In order to further improve the antibacterial spectrum and antibacterial ability of probiotic EcN, probiotic EcN needs to be further modified. There are the following difficulties in genetic engineering modification of probiotic EcN: 1. How to expand the antibacterial spectrum; 2. How to introduce the relevant genes of targeted microcins into probiotic EcN; 3. How to ensure that the relevant genes of microcins have no resistance markers while exerting the corresponding effects (the presence of resistance markers means that it has the risk of horizontal transmission of resistance genes and is not suitable for use as a probiotic). Summary of the invention

[0011] One of the purposes of the present invention is to provide three kinds of microcin recombinantly integrated engineered probiotics, the three kinds of recombinantly integrated engineered probiotics are respectively microcin MccJ25 recombinantly integrated engineered probiotics, microcin MccY recombinantly integrated engineered probiotics and microcin MccJ25, MccY recombinantly integrated engineered probiotics, the three kinds of engineered probiotics of the present invention have the characteristics of no resistance marker gene, good growth activity, genetic stability and antibacterial activity;

[0012] At the same time, the expression level of MccJ25 protein in the engineered probiotics with recombinant integration of microcin MccJ25 is high, which can significantly improve the antibacterial ability of the probiotic EcN.

[0013] The engineered probiotics with recombinant integration of microcin MccY have the characteristics of high broad-spectrum activity. At the same time, the sensitivity of the engineered probiotics with recombinant integration of microcin MccY to Salmonella typhimurium is far superior to that of the engineered probiotics with recombinant integration of microcin MccJ25 and the engineered probiotics with recombinant integration of microcin MccY+MccJ25.

[0014] The engineered probiotics with recombinant integration of microcin MccY+MccJ25 have a higher broad-spectrum and can effectively inhibit Salmonella typhimurium ATCC14028, Salmonella Enteritidis CVCC3377, Salmonella Pullorum CVCC1800, Salmonella Infantis CMCC50041, Salmonella Kentucky BNCC239114, Salmonella London CVCC2206, Salmonella Corvallis CMCC50922, Shigella flexneri SF1015, Shigella sonnei SS1014, and Escherichia coli ATCC25922.

[0015] At the same time, the present invention also provides a preparation method and use of the above-mentioned engineered probiotics.

[0016] To achieve the above object, the present invention provides an engineered probiotic with recombinant integration of microcin MccJ25, wherein the engineered probiotic is Escherichia coli with integrated microcin MccJ25 gene; the nucleotide sequence of the microcin MccJ25 gene is shown in SEQ ID NO: 19; and the Escherichia coli is Escherichia coli Nissle 1917.

[0017] In addition, the present invention also discloses a method for preparing the engineered probiotics as described above, using genome editing technology and gene knockout vectors to integrate the SD-T7RNP homology arm repair fragment and the MccJ25E homology arm repair fragment into the Escherichia coli Nissle 1917 genome, thereby obtaining the microcin MccJ25 recombinant integrated engineered probiotic EcN-MccJ25;

[0018] The nucleotide sequence of the SD-T7RNP homology arm repair fragment is shown in SEQ ID NO: 2; the MccJ25E homology arm repair fragment is shown in SEQ ID NO: 3.

[0019] In the above-mentioned preparation method, the method is specifically:

[0020] (1) transferring the pREDCas9 plasmid into EcN competent cells, screening to obtain positive transformants EcN-cas9, and preparing the positive transformants EcN-cas9 into competent cells; the competent cells are EcN-cas9 competent cells;

[0021] (2) The SD-T7RNP homology arm repair fragment and araBsgRNA plasmid were transferred into EcN-cas9 competent cells, and the recombinant strain EcNΔaraB-T7RNP-araBsgRNA-cas9 was screened using spectinomycin and ampicillin; the araBsgRNA plasmid was a pUC19 vector inserted with the sgRNA-araB fragment; the nucleotide sequence of the sgRNA-araB fragment was shown in SEQ ID NO: 33;

[0022] (3) The recombinant strain EcNΔaraB-T7RNP-araBsgRNA-cas9 was subcultured to screen out a strain that was insensitive to spectinomycin and sensitive to ampicillin to eliminate the araBsgRNA plasmid in the strain, and the MccJ25E homology arm repair fragment and the endAsgRNA plasmid were transferred into the strain. The recombinant strain EcNΔaraB-T7RNP-Cas9-ΔendA-MccJ25-endAsgRNA was screened using spectinomycin and ampicillin; the endAsgRNA plasmid was a pUC19 vector inserted with an sgRNA-endA fragment; the nucleotide sequence of the sgRNA-endA fragment was shown in SEQ ID NO: 34;

[0023] (4) subculturing the recombinant strain EcNΔaraB-T7RNP-cas9-ΔendA-MccJ25-endAsgRNA, screening out strains that are insensitive to spectinomycin and sensitive to ampicillin to eliminate the endAsgRNA plasmid in the strain, and obtaining the recombinant strain EcNΔaraB-T7RNP-ΔendA-MccJ25-cas9;

[0024] (5) The recombinant strain EcNΔaraB-T7RNP-ΔendA-MccJ25-cas9 was subcultured at 42°C, and strains sensitive to both spectinomycin and ampicillin were screened to eliminate the pREDCas9 plasmid in the strain, thereby obtaining the micromycin MccJ25 recombinant integrated engineered probiotic EcN-MccJ25.

[0025] The strain constructed by the present invention does not carry any resistance markers, and there is no risk of horizontal transmission of resistance genes. Because it is a non-plasmid expression form, there is no need for antibiotics to lock the plasmid. The other two engineered probiotics of the present invention also have this feature;

[0026] The second engineered probiotic disclosed in the present invention is an engineered probiotic with recombinant integration of microcin MccY. The engineered probiotic is Escherichia coli with integrated microcin MccY gene; the nucleotide sequence of the microcin MccY gene is shown in SEQ ID NO: 12; and the Escherichia coli is Escherichia coli Nissle 1917.

[0027] The preparation method of the engineered probiotics with recombinant integration of microcin MccY is as follows: using genome editing technology and gene knockout vector to integrate the SD-T7RNP homology arm repair fragment and the MccY homology arm repair fragment into the genome of Escherichia coli Nissle 1917, so as to obtain the engineered probiotics EcN-MccY with recombinant integration of microcin MccY;

[0028] The nucleotide sequence of the SD-T7RNP homology arm repair fragment is shown in SEQ ID NO: 5; the nucleotide sequence of the MccJ25E homology arm repair fragment is shown in SEQ ID NO: 12.

[0029] In the above-mentioned preparation method, the method is specifically:

[0030] (1) transferring the pREDCas9 plasmid into EcN competent cells, screening to obtain positive transformants EcN-cas9, and preparing the positive transformants EcN-cas9 into competent cells; the competent cells are EcN-cas9 competent cells;

[0031] (2) The SD-T7RNP homology arm repair fragment and araBsgRNA plasmid were transferred into EcN-cas9 competent cells, and the recombinant strain EcN-T7RNP-araBsgRNA-cas9 was screened using spectinomycin and ampicillin; the araBsgRNA plasmid was a pUC19 vector inserted with the sgRNA-araB fragment; the nucleotide sequence of the sgRNA-araB fragment was shown in SEQ ID NO: 33;

[0032] (3) The recombinant strain EcN-T7RNP-araBsgRNA-cas9 was subcultured to screen out a strain that was insensitive to spectinomycin and sensitive to ampicillin to eliminate the araBsgRNA plasmid in the strain, and the MccY homology arm repair fragment and the endAsgRNA plasmid were transferred into the strain. The recombinant strain EcN-T7RNP-pREDCas9-MccY-endAsgRNA was screened using spectinomycin and ampicillin; the endAsgRNA plasmid was a pUC19 vector inserted with an sgRNA-endA fragment; the nucleotide sequence of the sgRNA-endA fragment was shown in SEQ ID NO: 34;

[0033] (4) eliminating the endAsgRNA plasmid in the recombinant strain EcN-T7RNP-pREDCas9-MccY-endAsgRNA to obtain the recombinant strain EcN-T7RNP-MccY-cas9;

[0034] (5) Eliminate the pREDCas9 plasmid in the recombinant strain EcN-T7RNP-MccY-cas9 to obtain the microcin MccY recombinant integrated engineered probiotic EcN-MccJ25.

[0035] The third engineered probiotic disclosed in the present invention is an engineered probiotic with recombinant integration of microcin MccY and MccJ25. The engineered probiotic is Escherichia coli with integrated microcin MccY gene and microcin MccJ25 gene; the nucleotide sequence of the microcin MccY gene is shown in SEQ ID NO: 12; the nucleotide sequence of the microcin MccJ25 gene is shown in SEQ ID NO: 26; and the Escherichia coli is Escherichia coli Nissle 1917.

[0036] The preparation method of the engineered probiotics with recombinant integration of microcins MccY and MccJ25 is as follows: using genome editing technology and gene knockout vectors to integrate the SD-T7RNP homology arm repair fragment, the MccJ25X homology arm repair fragment and the MccY homology arm repair fragment into the genome of Escherichia coli Nissle 1917, so as to obtain the engineered probiotics EcN-MccY-MccJ25 with recombinant integration of microcins MccY and MccJ25;

[0037] The nucleotide sequence of the SD-T7RNP homology arm repair fragment is shown in SEQ ID NO: 5; the MccJ25X homology arm repair fragment is shown in SEQ ID NO: 26; and the MccY homology arm repair fragment is shown in SEQ ID NO: 12.

[0038] 9. The preparation method according to claim 8, characterized in that the method specifically comprises:

[0039] (1) transferring the pREDCas9 plasmid into EcN competent cells, screening to obtain positive transformants EcN-cas9, and preparing the positive transformants EcN-cas9 into competent cells; the competent cells are EcN-cas9 competent cells;

[0040] (2) The SD-T7RNP homology arm repair fragment and araBsgRNA plasmid were transferred into EcN-cas9 competent cells, and the recombinant strain EcN-T7RNP-araBsgRNA-cas9 was screened using spectinomycin and ampicillin; the araBsgRNA plasmid was a pUC19 vector inserted with the sgRNA-araB fragment; the nucleotide sequence of the sgRNA-araB fragment was shown in SEQ ID NO: 33;

[0041] (3) The recombinant strain EcN-T7RNP-araBsgRNA-cas9 was subcultured to screen out strains that were insensitive to spectinomycin and sensitive to ampicillin to eliminate the araBsgRNA plasmid in the strain, and the MccY homology arm repair fragment and the endAsgRNA plasmid were transferred into the strain, and the recombinant strain EcN-T7RNP-pREDCas9-MccY-endAsgRNA was screened using spectinomycin and ampicillin, and the recombinant strain EcN-T7RNP-pREDCas9-MccY-endAsgRNA was prepared into competent cells, which were EcN-T7RNP-MccY-cas9 competent cells; the endAsgRNA plasmid was a pUC19 vector inserted with an sgRNA-endA fragment; the nucleotide sequence of the sgRNA-endA fragment was shown in SEQ ID NO: 34;

[0042] (4) The MccJ25X homology arm repair fragment and the xylAsgRNA plasmid were transferred into the 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 inserted with the sgRNA-xylA fragment, and the nucleotide sequence of the sgRNA-xylA fragment was shown in SEQ ID NO: 35;

[0043] (5) Eliminating the pREDCas9 plasmid and the xylAsgRNA plasmid in the recombinant strain EcN-T7RNP-pREDCas9-MccY-MccJ25-xylAsgRNA to obtain the strain EcN-T7RNP-MccY-MccJ25, wherein the strain EcN-T7RNP-MccY-MccJ25 is the recombinant integrated engineering probiotic EcN-MccY-MccJ25 of microcin MccY and MccJ25

[0044] In addition, the present invention also discloses the use of the three engineered probiotics mentioned above in preparing antibacterial preparations.

[0045] In the above-mentioned use, the dosage form of the antibacterial preparation is one of powder, solution and granule.

[0046] Finally, the present invention also discloses an antibacterial preparation, which contains any one or more of the three engineered probiotics mentioned above.

[0047] In the above-mentioned antibacterial preparation, the dosage form of the antibacterial preparation is one of powder, solution and granule.

[0048] Beneficial Effects

[0049] Compared with the prior art, the present invention has at least the following advantages:

[0050] 1. The three engineered probiotics of the present invention have the common advantage that they can be used as probiotics, which meet the following requirements of probiotics: 1. They are live bacteria and can act on the intestines of humans or animals; 2. The three engineers constructed by the method of the present invention do not carry any resistance markers, and there is no risk of horizontal transmission of resistance genes, ensuring their safety as probiotics; 3. The three engineers constructed by the method of the present invention express microbactin in the form of non-plasmid expression, and do not require antibiotics to lock the plasmids. They can stably act on the intestines and be propagated, meeting the stability requirements for the use of probiotics;

[0051] Through the above optimization, the 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.

[0052] 2. The unique advantage of the engineered probiotics with recombinant integration of microcin MccJ25 of the present invention is that the expression amount of microcin MccJ25 by the engineered probiotics with recombinant integration of microcin MccJ25 can reach a high expression level of 25.31 mg / L, and can effectively inhibit Salmonella enteritidis CVCC3377, Shigella flexneri SF1015, and Escherichia coli ATCC25922. The sensitivity of the engineered probiotics with recombinant integration of microcin MccJ25 to Salmonella enteritidis is far superior to that of the engineered probiotics with recombinant integration of microcin MccY and the engineered probiotics with recombinant integration of microcin MccY+MccJ25.

[0053] 3. The unique advantage of the engineered probiotics with recombinant integration of microcin MccY of the present invention is that the expression amount of microcin MccY by the engineered probiotics with recombinant integration of microcin MccY can reach a high expression level of 9.23 mg / L, and can effectively inhibit Salmonella typhimurium ATCC14028, Salmonella enteritidis CVCC3377, Salmonella pullorum CVCC1800, Salmonella infantis CMCC50041, Salmonella Kentucky BNCC239114, Salmonella London CVCC2206, Salmonella Corvallis CMCC50922, and Shigella sonnei SS1014, and its sensitivity to Salmonella typhimurium is far superior to that of the engineered probiotics with recombinant integration of microcin MccJ25 and the engineered probiotics with recombinant integration of microcin MccY+MccJ25;

[0054] 4. The unique advantages of the engineered probiotics with recombinant integration of microcin MccY and MccJ25 of the present invention are: the expression level of microcin MccY can reach a high expression level of 8.02 mg / L, and the expression level of microcin MccJ25 can reach a high expression level of 9.11 mg / L, which can be used to treat Salmonella typhimurium ATCC14028, Salmonella enteritidis CVCC3377, Salmonella pullorum CVCC1800, and Salmonella infantum CMCC50041. , Kentucky Salmonella BNCC239114, London Salmonella CVCC2206, Corvallis Salmonella CMCC50922, Shigella flexneri SF1015, Shigella sonnei SS1014, and Escherichia coli ATCC25922. The broad-spectrum antibacterial activity of the engineered probiotics recombinantly integrated with microcin MccY+MccJ25 is better than that of the engineered probiotics recombinantly integrated with microcin MccY and the engineered probiotics recombinantly integrated with microcin MccJ25. BRIEF DESCRIPTION OF THE DRAWINGS

[0055] The present invention is further described below in conjunction with the accompanying drawings and embodiments;

[0056] Figure 1A is the plasmid map of the recombinant plasmid pUC19-T7RNP;

[0057] Figure 1B is the plasmid map of the recombinant plasmid pUC19-MccY;

[0058] Figure 1C is the plasmid map of the recombinant plasmid pUC19-MccJ25;

[0059] Figure 2A This is the plasmid map of araBsgRNA plasmid;

[0060] Figure 2Bis the plasmid map of the endAsgRNA plasmid;

[0061] Figure 2C This is the plasmid map of the xylAsgRNA plasmid;

[0062] Figure 3A This is a graph of the PCR identification results of the EcNΔaraB-T7RNP strain;

[0063] Figure 3B This is a graph showing the PCR identification results of the EcNΔaraB-T7RN-ΔendA-MccY strain;

[0064] Figure 3C This is a graph showing the PCR identification results of the EcNΔaraB-T7RN-ΔendA-MccJ25 strain;

[0065] Figure 3D This is a graph of the PCR identification results of the EcNΔaraB-T7RNP strain;

[0066] Figure 3E This is a graph showing the PCR identification results of the EcNΔaraB-T7RN-ΔendA-MccY-ΔxylA-MccJ25 strain;

[0067] Figure 4A This is the inhibition zone diagram of MccY recombinant integrated engineering probiotics in 0.5% LB soft agar containing Salmonella typhimurium;

[0068] Figure 4B This is the inhibition zone diagram of MccJ25 recombinant integrated engineering probiotics in 0.5% LB soft agar containing Salmonella enteritidis;

[0069] Figure 4C It is the inhibition zone diagram of MccY+MccJ25 recombinant integrated engineering probiotics in 0.5% LB soft agar containing Salmonella typhimurium and 0.5% LB soft agar containing Salmonella enteritidis;

[0070] Figure 5A This is the liquid chromatography result of the culture supernatant of EcN-MccY engineered probiotics;

[0071] Figure 5B This is the liquid chromatography mass spectrometry result of the culture supernatant of EcN-MccY engineered probiotics;

[0072] Figure 5C This is the liquid chromatography result of the culture supernatant of EcN-MccJ25 engineered probiotics;

[0073] Figure 5D This is the liquid chromatography mass spectrometry result of the culture supernatant of EcN-MccJ25 engineered probiotics;

[0074] Figure 5E This is the liquid chromatography result of the supernatant of MccY+MccJ25 recombinant integrated engineering probiotic culture;

[0075] Fig. 5F This is the liquid chromatography mass spectrometry result of the supernatant of MccY+MccJ25 recombinant integrated engineering probiotic culture;

[0076] Figure 5G This is the liquid chromatography result of MccJ25 in the supernatant of MccY+MccJ25 recombinant integrated engineering probiotic culture;

[0077] Figure 5H This is the liquid chromatography mass spectrometry result of MccJ25 in the supernatant of MccY+MccJ25 recombinant integrated engineering probiotic culture;

[0078] Fig. 6A It is the inhibition zone diagram of MccY recombinant integrated engineering probiotics with different expression time;

[0079] Figure 6B It is the inhibition zone diagram of MccJ25 recombinant integrated engineering probiotics with different expression time;

[0080] Figure 6C This is the inhibition zone diagram of the MccY+MccJ25 recombinant integrated engineering probiotics with different expression times against Salmonella typhimurium;

[0081] Fig.6D This is the inhibition zone diagram of MccY+MccJ25 recombinant integrated engineering probiotics with different expression time against Salmonella enteritidis;

[0082] Figure 7 This is a comparison of the inhibitory effects of MccY secreted by MccY recombinant integrated engineering bacteria, MccJ25 recombinant integrated engineering bacteria and MccY+MccJ25 recombinant integrated engineering bacteria on Gram-negative bacteria Salmonella typhimurium, Salmonella pullorum, Salmonella Enteritidis, Salmonella infantis, Salmonella Kentucky, Salmonella London, Salmonella Corvallis, Escherichia coli, Shigella sonnei, Shigella fonni; and Gram-positive bacteria Bacillus aureus and Bacillus subtilis. DETAILED DESCRIPTION

[0083] The present invention will be further described below in conjunction with the embodiments, but it does not constitute any limitation to the present invention. Any limited number of modifications made within the scope of the claims of the present invention are still within the scope of the claims of the present invention.

[0084] In the following examples, the whole gene synthesis, primer synthesis and sequencing were all completed by Suzhou Jinweizhi Biotechnology Co., Ltd. The molecular biology experiment related content, such as competent cell preparation and transformation, was carried out with reference to the Molecular Cloning Experiment Guide (3rd Edition).

[0085] A detailed description is given below.

[0086] Example 1 Construction of MccY recombinant integrated engineering probiotics, MccJ25 recombinant integrated engineering probiotics, and MccY and MccJ25 dual recombinant integrated engineering probiotics

[0087] In this embodiment, Escherichia coli Nissle 1917 (EcN) was used as the base bacteria, and the pREDCas9 Escherichia coli gene editing technology was used to integrate the SD sequence 20bp and the complete T7RNP sequence 2652bp into the araB position of its genome. Subsequently, the MccY repair fragment and the MccJ25 repair fragment were respectively integrated into the endA position of its genome to obtain MccY recombinant integrated engineering probiotics and MccJ25 recombinant integrated engineering probiotics; based on the obtained MccY recombinant integrated engineering probiotics, the MccJ25 repair fragment was integrated into the xylA position of the bacterial genome to obtain MccY and MccJ25 dual recombinant integrated engineering probiotics.

[0088] The SD nucleotide sequence is: 5'-CTAACTGGAAGAGGCACTAA-3' (SEQ ID NO: 1).

[0089] The nucleotide sequence of SD-T7RNP sequence is shown in SEQ ID NO: 2;

[0090] The nucleotide sequence of the MccY repair fragment is shown in SEQ ID NO: 3;

[0091] The nucleotide sequence of the MccJ25 repair fragment is shown in SEQ ID NO: 4;

[0092] The specific construction method is:

[0093] (1) Construction of pUC19-T7RNP vector, pUC19-MccY, and pUC19-MccJ25 vector:

[0094] With reference to NCBI No. CP081489, the modified gene T7RNP nucleotide sequence was synthesized, and the SD sequence (SEQ ID NO: 1) was connected to the T7RNP nucleotide sequence to obtain the target gene SD-T7RNP sequence (the sequence is shown in the above SEQ ID NO: 2).

[0095] The sequence shown in SEQ ID NO: 2 was introduced into the pUC19 cloning plasmid to obtain a 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 in Figure 1A Show.

[0096] 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 in Figure 1B shown.

[0097] 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 in Figure 1C shown.

[0098] (2) Construction of SD-T7RNP homology arm repair fragment, optimized MccY homology arm repair fragment, optimized MccJ25E homology arm repair fragment and optimized MccJ25X homology arm repair fragment:

[0099] Using the EcN genome (NCBI number NZ_CP007799.1) as a template, araBup-F and araBup-R as primers, the upstream homology arm araBup was obtained by PCR amplification; using the EcN genome as a template, araBdown-F and araBdown-R as primers, the downstream homology arm araBdown was obtained by PCR amplification. Using the recombinant plasmid pUC19-SD-T7RNP as a template, T7RNP-F and T7RNP-R as primers, a complete T7RNP fragment with a 20bp SD sequence was obtained by PCR amplification. Using the upstream homology arm araBup, the downstream homology arm araBdown and the complete T7RNP fragment with a 20bp SD sequence as templates, araBup-F and araBdown-R as primers, the SD-T7RNP homology arm repair fragment was obtained by overlapping PCR amplification.

[0100] Among them, the obtained SD-T7RNP homology arm repair fragment is shown in SEQ ID NO:5, and the sequences of each primer are shown in Table 1.

[0101] Table 1 SD-T7 RNP integration primer sequence information

[0102]

[0103]

[0104] The PCR amplification system is shown in Table 2. The overlapping PCR amplification system is shown in Table 3.

[0105] Table 2 PCR amplification system

[0106] 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 20 μL

[0107] Table 3 Overlapping PCR amplification system

[0108] 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 50 μL

[0109] The PCR amplification program was as follows: 95°C pre-denaturation for 5 min; 95°C denaturation for 15 s, 56°C annealing for 15 s, 72°C extension for 30 s, 35 cycles; 72°C post-extension for 5 min. The overlapping PCR amplification system program was as follows: 95°C pre-denaturation for 5 min; 95°C denaturation for 15 s, 56°C annealing for 15 s, 72°C extension for 1 min, 40 cycles; 72°C post-extension for 5 min.

[0110] According to the above operation, the upstream homology arm endAyup was obtained by PCR amplification using the EcN genome as a template, endAyup-F and endAyup-R as primers; the downstream homology arm endAydown was obtained by PCR amplification using the EcN genome as a template, endAydown-F and endAydown-R as primers. The target gene MccY sequence fragment was obtained by PCR amplification using the recombinant plasmid pUC19-MccY as a template, MccY-F and MccY-R as primers. The upstream homology arm endAyup, downstream homology arm endAydown and modified gene MccY sequence fragment were used as templates, endAyup-F and endAydown-R were used as primers, and overlapping PCR amplification was used to obtain the MccY sequence homology arm repair fragment.

[0111] Among them, the obtained MccY homology arm repair fragment is shown in SEQ ID NO:12, and the sequences of each primer are shown in Table 4.

[0112] Table 4 MccY integration primer sequence information

[0113] 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)

[0114] The PCR amplification system and procedure, and the overlapping PCR amplification system and procedure are the same as those in the above embodiment.

[0115] According to the above operation, the upstream homology arm endAjup was obtained by PCR amplification using the EcN genome as a template, endAjup-F and endAjup-R as primers; the downstream homology arm endAjdown was obtained by PCR amplification using the EcN genome as a template, endAjdown-F and endAjdown-R as primers. The target gene MccJ25 sequence fragment was obtained by PCR amplification using the recombinant plasmid pUC19-MccJ25 as a template, MccJ25E-F and MccJ25E-R as primers. The upstream homology arm endAjup, downstream homology arm endAjdown and modified gene MccJ25 sequence fragment were used as templates, endAjup-F and endAjdown-R were used as primers, and overlapping PCR amplification was used to obtain the MccJ25E sequence homology arm repair fragment.

[0116] Among them, the obtained MccJ25E homology arm repair fragment is shown in SEQ ID NO:19, and the sequences of each primer are shown in Table 5.

[0117] Table 5 MccJ25 integration primer sequence information

[0118] 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) MccJ25E-F 5'-aggatcgagatctcgatccc-3'(SEQ ID NO:24) MccJ25E-R 5'-ctggatacatcttgtcccatttattcagtaacagaagccag-3'(SEQ ID NO:25)

[0119] The PCR amplification system and procedure, and the overlapping PCR amplification system and procedure are the same as those in the above embodiment.

[0120] According to the above operation, the upstream homology arm xylAjup was obtained by PCR amplification using the EcN genome as a template, xylAjup-F and xylAjup-R as primers; the downstream homology arm xylAjdown was obtained by PCR amplification using the EcN genome as a template, xylAjdown-F and xylAjdown-R as primers. The target gene MccJ25 sequence fragment was obtained by PCR amplification using the recombinant plasmid pUC19-MccJ25 as a template, MccJ25X-F and MccJ25X-R as primers. The upstream homology arm xylAjup, downstream homology arm xylAjdown and modified gene MccJ25 sequence fragment were used as templates, and xylAjup-F and xylAjdown-R were used as primers to obtain the MccJ25X sequence homology arm repair fragment by overlapping PCR amplification.

[0121] Among them, the obtained MccJ25X homology arm repair fragment is shown in SEQ ID NO:26, and the sequences of each primer are shown in Table 6.

[0122] Table 6 MccJ25 integration primer sequence information

[0123] 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) MccJ25X-F 5'-aggatcgagatctcgatccc-3'(SEQ ID NO:31) MccJ25X-R 5'-cggagacaaattatgttcctgagttattctgtagctgatgccag-3' (SEQ ID NO: 32)

[0124] The PCR amplification system and procedure, and the overlapping PCR amplification system and procedure are the same as those in the above embodiment.

[0125] (3) Construction of araBsgRNA plasmid, endAsgRNA plasmid, and xylAsgRNA plasmid:

[0126] The sgRNA-araB fragment was designed, and the sequence was shown in SEQ ID NO: 33. General Biotech (Anhui) Co., Ltd. was commissioned to clone the sequence shown in SEQ ID NO: 33 into the pUC19 vector to obtain the araBsgRNA plasmid (the plasmid map is shown in Figure 2A shown).

[0127] According to the above, the sgRNA-endA fragment was designed, and the sequence is shown in SEQ ID NO: 34. General Biotechnology (Anhui) Co., Ltd. cloned the sequence shown in SEQ ID NO: 34 into the pUC19 vector to obtain the endAsgRNA plasmid (the plasmid map is shown in Figure 2B shown).

[0128] The sgRNA-xylA fragment was designed, and the sequence was shown in SEQ ID NO: 35. General Biotechnology (Anhui) Co., Ltd. cloned the sequence shown in SEQ ID NO: 35 into the pUC19 vector to obtain the xylAsgRNA plasmid (the plasmid map is shown in Figure 2C shown).

[0129] (4) Construction of EcN-cas9 strain containing pREDCas9 plasmid:

[0130] EcN competent cells were prepared according to the Molecular Cloning Experiment Guide (3rd edition), and pREDCas9 plasmid (purchased from addgene) was transferred into EcN competent cells by electroporation, and transformed by screening with spectinomycin resistance plates to obtain transformants. PCR amplification was performed with pREDCas9-test-F and pREDCas9-test-R primers to identify whether the transfer was successful. The strain with successful transfer was the EcN-cas9 strain containing the pREDCas9 plasmid.

[0131] Among them, the sequences of pREDCas9-test-F and pREDCas9-test-R are shown in Table 7.

[0132] Table 7 Primer information used for EcN-cas9 strain identification

[0133] 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)

[0134] (5) Construction of EcNΔaraB-pREDCas9-T7RNP-araBsgRNA

[0135] Take the EcN-cas9 strain obtained above, induce the strain to OD600 = 0.6-0.8 with 0.5mM isopropyl-β-D-thiogalactoside (IPTG), prepare EcN-cas9 competent cells, and transfer the SD sequence and the complete T7RNP homology arm repair fragment and araBsgRNA plasmid into the EcN-cas9 competent cells by electroporation, and screen with spectinomycin (50μg / mL) and ampicillin (100μg / mL) double resistance plates to obtain transformants. PCR amplification was performed with araB-test-F and araB-test-R primers to identify whether the transfer was successful. The strain with successful transfer was the recombinant strain EcN-T7RNP-araBsgRNA-cas9.

[0136] Among them, the sequences of araB-test-F and araB-test-R are shown in Table 8.

[0137] Table 8 Primer information used for identification of recombinant strain EcNΔaraB-T7RNP-araBsgRNA-cas9

[0138] 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)

[0139] (6) Elimination of araBsgRNA plasmid:

[0140] The above-mentioned recombinant strain EcNΔaraB-T7RNP-araBsgRNA-cas9 was serially passaged on spectinomycin-resistant plates and cultured at 30°C. Single colonies were picked and punctured on spectinomycin-resistant plates and spectinomycin and ampicillin double-resistant plates to screen out strains that were insensitive to spectinomycin and sensitive to ampicillin (i.e., strains that survived on spectinomycin-resistant plates but could not survive on spectinomycin and ampicillin double-resistant plates). PCR amplification was performed using sgRNA-test-F and sgRNA-test-R primers to identify whether the transfer was successful. The strain with successful transfer was the recombinant strain EcNΔaraB-T7RNP-cas9 with araBsgRNA plasmid eliminated.

[0141] Among them, the sequences of sgRNA-test-F and sgRNA-test-R are shown in Table 9.

[0142] Table 9 Primer information for identification of recombinant strain EcN-T7RNP-cas9 for araBsgRNA plasmid elimination

[0143]

[0144]

[0145] (7) Construction of MccY recombinant integration engineering probiotic strain EcNΔaraB-T7RNP-cas9-ΔendA-MccY-endAsgRNA:

[0146] Referring to the above operation, the recombinant strain EcNΔaraB-T7RNP-cas9 obtained above with the araBsgRNA plasmid eliminated was taken, and the strain was induced to OD600 = 0.6-0.8 with 0.5mM IPTG to prepare EcNΔaraB-T7RNP-cas9 competent cells, and the optimized MccY sequence repair fragment and the endAsgRNA plasmid were transferred into the EcNΔaraB-T7RNP-cas9 competent cells by electroporation, and screened with spectinomycin and ampicillin double resistance plates, and the surviving bacteria were transformants. PCR amplification was performed with endA-F and endA-R primers to identify whether the transfer was successful. The successfully transferred strain was the recombinant strain EcNΔaraB-T7RNP-cas9-ΔendA-MccY-endAsgRNA in which the optimized MccY sequence was integrated into the EcN genome.

[0147] Among them, the sequences of endA-test-F and endA-test-R are shown in Table 10.

[0148] Table 10 Primer information for EcNΔaraB-T7RNP-cas9-ΔendA-MccY / MccJ25-endAsgRNA identification

[0149] 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)

[0150] (8)MccJ25 recombinant integrated probiotic strain

[0151] Construction of EcNΔaraB-T7RNP-cas9-ΔendA-MccJ25-endAsgRNA:

[0152] Referring to the above operation, the recombinant strain EcNΔaraB-T7RNP-cas9 with the araBsgRNA plasmid eliminated was taken, and the strain was induced to OD600 = 0.6-0.8 with 0.5mM IPTG to prepare EcNΔaraB-T7RNP-cas9 competent cells. The MccJ25E homology arm repair fragment and the endAsgRNA plasmid were transferred into the EcNΔaraB-T7RNP-cas9 competent cells by electroporation, and screened with spectinomycin and ampicillin double resistance plates. The surviving bacteria were the transformants.

[0153] PCR amplification was performed using endA-test-F and endA-test-R primers (as shown in Table 10) to identify whether the transfer was successful. The successfully transferred strain was the recombinant strain EcNΔaraB-T7RNP-cas9-ΔendA-MccJ25-endAsgRNA in which the optimized MccJ25 sequence was integrated into the EcN genome.

[0154] (9) Elimination of endAsgRNA plasmid:

[0155] The above-mentioned recombinant strains EcNΔaraB-T7RNP-ΔendA-MccY-endAsgRNA-cas9 and EcNΔaraB-T7RNP-ΔendA-MccJ25-endAsgRNA-cas9 were serially passaged on spectinomycin-resistant plates and cultured at 30°C. Single colonies were picked and punctured on spectinomycin-resistant plates and spectinomycin and ampicillin double-resistant plates to screen out strains that were insensitive to spectinomycin and sensitive to ampicillin (i.e., strains that survived on spectinomycin-resistant plates but could not survive on spectinomycin and ampicillin double-resistant plates). PCR amplification was performed using sgRNA-test-F and sgRNA-test-R primers (as shown in Table 9) to identify whether the transfer was successful. The strains that were successfully transferred were recombinant strains with the endAsgRNA plasmid eliminated:

[0156] EcNΔaraB-T7RNP-ΔendA-MccY-cas9 and EcNΔaraB-T7RNP-ΔendA-MccJ25-cas9.

[0157] (10) Elimination of pREDCas9 plasmid:

[0158] The above-obtained recombinant strains: EcNΔaraB-T7RNP-ΔendA-MccY-cas9 and EcNΔaraB-T7RNP-ΔendA-MccJ25-cas9 were continuously passaged on non-resistant plates, cultured at 42°C, and single colonies were picked and punctured on non-resistant plates and spectinomycin-resistant plates, respectively, to screen out spectinomycin-sensitive strains. PCR amplification was performed using sgRNA-test-F and sgRNA-test-R primers to identify whether the transfer was successful. The successfully transferred strains were the recombinant strains EcNΔaraB-T7RNP-ΔendA-MccY and EcNΔaraB-T7RNP-ΔendA-MccJ25 (i.e., microcin MccY recombinant integrated engineering probiotics and MccJ25 recombinant integrated engineering probiotics) that eliminated pREDCas9 and endAsgRNA plasmids.

[0159] (11) Construction of MccY+MccJ25 recombinant integration engineering probiotic strain EcNΔaraB-T7RNP-cas9-ΔendA-MccY-ΔxylA-MccJ25-xylAsgRNA:

[0160] Referring to the above operation, the recombinant strain EcNΔaraB-T7RNP-ΔendA-MccY-cas9 with the endAsgRNA plasmid eliminated was taken, and the strain was induced to OD600 = 0.6-0.8 with 0.5mM IPTG to prepare EcNΔaraB-T7RNP-ΔendA-MccY-cas9 competent cells, and the optimized MccJ25X homology arm repair fragment and xylAsgRNA plasmid were transferred into EcNΔaraB-T7RNP-ΔendA-MccY-cas9 competent cells by electroporation, and screened with spectinomycin and ampicillin double resistance plates to obtain transformants. 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, in which the optimized MccY and MccJ25 sequences were simultaneously integrated into the EcN genome.

[0161] Among them, the sequences of xylA-test-F and xylA-test-R are shown in Table 11.

[0162] Table 11 Primer information for EcNΔaraB-T7RNP-cas9-ΔendA-MccY-ΔxylA-MccJ25-xylAsgRNA identification

[0163] 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)

[0164] (12) Elimination of pREDCas9 and BsgRNA double plasmids:

[0165] The above-obtained recombinant strain EcNΔaraB-T7RNP-ΔendA-MccY-ΔxylA-MccJ25-xylAsgRNA-cas9 was continuously passaged on a non-resistant plate, cultured at 42°C, and single colonies were picked and punctured on a non-resistant plate, a spectinomycin-resistant plate, and a kanamycin-resistant plate, respectively, to screen out strains sensitive to spectinomycin and ampicillin. PCR amplification was performed using sgRNA-test-F and sgRNA-test-R primers to identify whether the transfer was successful. The successfully transferred strain was EcNΔaraB-T7RNP-ΔendA-MccY-ΔxylA-MccJ25 (i.e., microcin MccY-MccJ25 recombinant integrated engineering probiotics) that eliminated the pREDCas9 and xylAsgRNA plasmids.

[0166] FIG. 3A to FIG. 3E They are:

[0167] Figure 3A This is a graph of the PCR identification results of the EcNΔaraB-T7RNP strain;

[0168] Figure 3B This is a graph showing the PCR identification results of the EcNΔaraB-T7RN-ΔendA-MccY strain;

[0169] Figure 3C This is a graph showing the PCR identification results of the EcNΔaraB-T7RN-ΔendA-MccJ25 strain;

[0170] Figure 3D This is a graph showing the PCR identification results of the EcNΔaraB-T7RN-ΔendA-MccY-ΔxylA-MccJ25 strain;

[0171] Figure 3E This is the PCR identification result of EcNΔaraB-T7RN-ΔendA-MccY-ΔxylA-MccJ25 strain.

[0172] Example 2 Sensitivity determination of MccY recombinant integrated engineering probiotics, MccJ25 recombinant integrated engineering probiotics, and MccY+MccJ25 recombinant integrated engineering probiotics

[0173] The constructed MccY recombinant integrated engineering probiotics, MccJ25 recombinant integrated engineering probiotics, and MccY+MccJ25 recombinant integrated engineering probiotics were inoculated in 20 mL LB medium, 1 Mm IPTG inducer was added for induction expression, and the cells were shaken at 200 rpm / min at 37°C for 24 hours, centrifuged, and the cells were removed to obtain the expression supernatant of MccY recombinant integrated engineering probiotics, MccJ25 recombinant integrated engineering probiotics, and MccY+MccJ25 recombinant integrated engineering probiotics. The supernatants were gradiently diluted with PBS by 2 times (2×), 10 times (10×), 20 times (20×), 100 times (100×), and 200 times (200×), and filtered with a 0.22 μm filter for sterilization and stored at -20°C for later use.

[0174] Salmonella typhimurium (strain number ATCC 14028) and Salmonella enterica serovar Enteritidis (strain number CVCC3377) were revived in LB plates, and single colonies were selected and cultured to OD600 = 0.8. The bacterial liquid was inoculated into 0.5% LB soft agar medium at a ratio of 1:1000, and then poured into a culture dish and allowed to stand for 30 minutes. After the culture medium solidified, 10 μL of the supernatant of the MccY recombinant integrated engineering probiotics obtained by the multiple dilution in the above steps was taken and spotted in 0.5% LB soft agar containing Salmonella typhimurium; 10 Take 10 μL of the supernatant of the MccJ25 recombinant integrated probiotics obtained by the multiple dilution in the above step, and spot it on 0.5% LB soft agar containing Salmonella Enteritidis; take 10 μL of the supernatant of the MccY+MccJ25 recombinant integrated probiotics obtained by the multiple dilution in the above step, and spot it on 0.5% LB soft agar containing Salmonella Enteritidis and 0.5% LB soft agar containing Salmonella Typhimurium respectively; after drying, place it in a 37°C incubator for 16 hours, and observe the inhibition zone.

[0175] The results are as follows FIG. 4A to FIG. 4C As shown;

[0176] Figure 4A This is the inhibition zone diagram of MccY recombinant integrated engineering probiotics in 0.5% LB soft agar containing Salmonella typhimurium;

[0177] Figure 4B This is the inhibition zone diagram of MccJ25 recombinant integrated engineering probiotics in 0.5% LB soft agar containing Salmonella enteritidis;

[0178] Figure 4C It is the inhibition zone diagram of MccY+MccJ25 recombinant integrated engineering probiotics in 0.5% LB soft agar containing Salmonella typhimurium and 0.5% LB soft agar containing Salmonella enteritidis.

[0179] It can be found that the 1×, 2×, 10×, and 20× supernatants of the MccY recombinant integrated engineering probiotic strain have significant antibacterial effects on Salmonella typhimurium, and the 100× supernatant has a weak antibacterial effect on Salmonella typhimurium; the 1×, 2×, 10×, and 20× supernatants of the MccJ25 recombinant integrated engineering probiotic strain have significant antibacterial effects on Salmonella enteritidis, and the 100× supernatant has a weak antibacterial effect on Salmonella enteritidis; the 1×, 2×, and 10× supernatants of the MccY+MccJ25 recombinant integrated engineering probiotic strain have significant antibacterial effects on Salmonella enteritidis and Salmonella typhimurium, and the 20× supernatant has a weak antibacterial effect on Salmonella enteritidis.

[0180] Example 3 Quantitative analysis of MccY recombinant integrated probiotics, MccJ25 recombinant integrated probiotics, and MccY+MccJ25 recombinant integrated probiotics using high pressure liquid chromatography

[0181] Take 1 mL of the supernatant of the MccY recombinant integrated probiotics, MccJ25 recombinant integrated probiotics, and MccY+MccJ25 recombinant integrated probiotics expressed for 24 hours, and use ultra-high pressure liquid chromatography-quadrupole tandem time-of-flight mass spectrometry (Agilent, USA, model: UPLC1290-6540BQ-TOF) to detect the concentration of MccY and MccJ25 in the supernatant. 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°C; detection wavelength 214nm; flow rate 0.5mL / min; gradient elution according to Table 12.

[0182] Table 12

[0183] Time (min) A(％) B(％) 20 50 50 30 90 10 35 10 90

[0184] The results are as follows FIG. 5A to FIG. 5DAs shown, the mass spectrometry analysis of the MccY recombinant integrated engineering probiotics showed a characteristic ion peak 1113.73 consistent with the target substance MccY, MccY was successfully expressed and secreted in the engineering bacteria, and the protein concentration was 24.88 mg / L; the mass spectrometry analysis of the MccJ25 recombinant integrated engineering probiotics showed a characteristic ion peak 1054.685 consistent with the target substance MccJ25, MccJ25 was successfully expressed and secreted in the engineering bacteria, and the protein concentration was 34.12 mg / L; the mass spectrometry analysis of the MccY+MccJ25 recombinant integrated engineering probiotics showed a characteristic ion peak 1113.73 consistent with the target substance MccY, MccY was successfully expressed and secreted in the engineering bacteria, and the protein concentration was 9.80 mg / L; the characteristic ion peak 1054.685 consistent with the target substance MccJ25 was shown, MccJ25 was successfully expressed and secreted in the engineering bacteria, and the protein concentration was 9.11 mg / L.

[0185] in, Figure 5A This is the liquid chromatography result of the culture supernatant of EcN-MccY engineered probiotics;

[0186] Figure 5B This is the liquid chromatography mass spectrometry result of the culture supernatant of EcN-MccY engineered probiotics;

[0187] Figure 5C This is the liquid chromatography result of the culture supernatant of EcN-MccJ25 engineered probiotics;

[0188] Figure 5D This is the liquid chromatography mass spectrometry result of the culture supernatant of EcN-MccJ25 engineered probiotics;

[0189] Figure 5E This is the liquid chromatography result of MccY in the supernatant of MccY+MccJ25 recombinant integrated engineering probiotic culture;

[0190] Fig. 5F This is the liquid chromatography mass spectrometry result of MccY in the supernatant of MccY+MccJ25 recombinant integrated engineering probiotic culture;

[0191] Figure 5G This is the liquid chromatography result of MccJ25 in the supernatant of MccY+MccJ25 recombinant integrated engineering probiotic culture;

[0192] Figure 5H This is the liquid chromatography-mass spectrometry result of MccJ25 in the culture supernatant of MccY+MccJ25 recombinant integrated engineering probiotics.

[0193] Example 4 Detection of the antibacterial activity of MccY recombinant integrated engineering probiotics, MccJ25 recombinant integrated engineering probiotics, and MccY+MccJ25 recombinant integrated engineering probiotics at different expression times

[0194] 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, respectively, and 1 mM IPTG inducer was added to induce expression. The culture was shaken at 200 rpm / min at 37°C for expression. 1 mL of bacterial solution was taken at 4, 8, 12, 16, 20, and 24 hours, respectively. The bacterial solution at different time periods was centrifuged to remove the bacteria, the supernatant was taken, and filtered and sterilized with a 0.22 μm filter, and stored at -20°C for later use.

[0195] Salmonella typhimurium (strain number ATCC 14028) and a ring of Salmonella enteritidis (strain number CVCC3377) were revived in LB plates, and single colonies were picked and cultured to OD600 = 0.8, and the bacterial liquid was inoculated into 0.5% LB soft agar medium at a ratio of 1:1000, and then poured into a culture dish and allowed to stand for 30 minutes. After the culture medium solidified, the plate was divided into 6 equal areas, and 10 μL of the supernatant expressed by MccY recombinant integrated engineering probiotics and MccY+MccJ25 recombinant integrated engineering probiotics at 4, 8, 12, 16, 20, and 24 h was spotted on each equal surface of the soft agar containing Salmonella typhimurium in a clockwise direction, and 10 μL of the supernatant expressed by MccY recombinant integrated engineering probiotics and MccY+MccJ25 recombinant integrated engineering probiotics were spotted on each equal surface of the soft agar containing Salmonella enteritidis. The supernatants of MccJ25 recombinant integrated engineering probiotics and MccY+MccJ25 recombinant integrated engineering probiotics at 4, 8, 12, 16, 20, and 24 hours were dried for 10 minutes, then inverted and placed in a 37°C constant temperature incubator for overnight culture to observe the antibacterial effect.

[0196] The results are shown in Figure 6. In the MccY recombinant integrated engineering probiotics, MccJ25 recombinant integrated engineering probiotics, and MccY+MccJ25 recombinant integrated engineering probiotics, the supernatants expressed from 4 to 24 hours all had inhibition zones, and the inhibition effect became more obvious as time went on;

[0197] Fig. 6A It is the inhibition zone diagram of MccY recombinant integrated engineering probiotics with different expression time;

[0198] Figure 6B It is the inhibition zone diagram of MccJ25 recombinant integrated engineering probiotics with different expression time;

[0199] Figure 6C This is the inhibition zone diagram of the MccY+MccJ25 recombinant integrated engineering probiotics with different expression times against Salmonella typhimurium;

[0200] Fig.6DThis is the inhibition zone diagram of MccY+MccJ25 recombinant integrated engineering probiotics with different expression time against Salmonella enteritidis;

[0201] Example 5 Antibacterial activity test of MccY recombinant integrated engineering probiotics, MccJ25 recombinant integrated engineering probiotics, and MccY+MccJ25 recombinant integrated engineering probiotics

[0202] Resuscitate MccY recombinant integrated engineering probiotic strain, MccJ25 recombinant integrated engineering probiotic strain, and MccY+MccJ25 recombinant integrated engineering probiotic strain in LB plates. Use an inoculation loop to scrape a loop of each strain into 1 mL of sterile water and pipette to mix, take 10 μL of bacterial solution and pierce the LB plate, dry it, and place it in a 37°C incubator for 24 hours. After the incubation, take it out and irradiate it with ultraviolet light for 1 hour.

[0203] Salmonella Typhimurium ATCC14028, Salmonella Pullorum CVCC1800, Salmonella Enteritidis CVCC3377, Salmonella Infantis CMCC50041, Salmonella Kentucky BNCC239114, Salmonella London 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 revived in LB plates respectively. A single colony of each test strain was picked and shake-cultured until OD600 = 0.8, and the bacterial solution was inoculated into 0.5% LB soft agar medium at a ratio of 1:1000, and then poured into the LB plates (pre-irradiated with ultraviolet light) punctured with MccY recombinant integrated engineered probiotics, MccJ25 recombinant integrated engineered probiotics, and MccY + MccJ25 recombinant integrated engineered probiotics in the above step, respectively. After standing for 30 minutes to solidify, it was placed in a 37°C incubator for 16 hours to observe the inhibition zone.

[0204] The results are as follows Figure 7 As shown, after 24 hours of colonization and culture, the EcN-MccY recombinant integrated engineered probiotics secreted a large amount of MccY, which could inhibit the growth of Salmonella Typhimurium ATCC14028, Salmonella Enteritidis CVCC3377, Salmonella Pullorum CVCC1800, Salmonella Infantis CMCC50041, Salmonella Kentucky BNCC239114, Salmonella London CVCC2206, Salmonella Corvallis CMCC50922, and Shigella sonnei SS1014, forming inhibition zones of varying sizes; but the inhibitory effect on Escherichia coli ATCC25922, Shigella flexneri SF1015, Bacillus subtilis BS1011, and Staphylococcus aureus ATCC6538 was not obvious.

[0205] After 24 hours of colonization and culture, the EcN-MccJ25 recombinant integrated engineered probiotics secreted a large amount of MccJ25, which could inhibit the growth of Enteritidis Salmonella CVCC3377, Shigella flexneri SF1015, and Escherichia coli ATCC25922, forming inhibition zones of varying sizes; but the inhibitory effect on Salmonella typhimurium ATCC14028, Salmonella pullorum CVCC1800, Salmonella infantis CMCC50041, Salmonella Kentucky BNCC239114, Salmonella London CVCC2206, Salmonella Corvallis CMCC50922, Shigella sonnei SS1014, Bacillus subtilis BS1011, and Staphylococcus aureus ATCC6538 was not obvious.

[0206] After 24 hours of colonization and cultivation, the EcN-MccY+MccJ25 recombinant integrated engineered probiotics secreted a large amount of MccY and MccJ25, which could inhibit the growth of Salmonella Typhimurium ATCC14028, Salmonella Enteritidis CVCC3377, Salmonella Pullorum CVCC1800, Salmonella Infantis CMCC50041, Salmonella Kentucky BNCC239114, Salmonella London CVCC2206, Salmonella Corvallis CMCC50922, Shigella flexneri SF1015, Shigella sonnei SS1014, and Escherichia coli ATCC25922, forming inhibition zones of varying sizes; but the inhibitory effect on Bacillus subtilis BS1011 and Staphylococcus aureus ATCC6538 was not obvious.

[0207] In summary, the advantages of the present invention are:

[0208] 1. The three engineered probiotics of the present invention have the common advantage that they can be used as probiotics, which meet the following requirements of probiotics: 1. They are live bacteria and can act on the intestines of humans or animals; 2. The three engineers constructed by the method of the present invention do not carry any resistance markers, and there is no risk of horizontal transmission of resistance genes, ensuring their safety as probiotics; 3. The three engineers constructed by the method of the present invention express microbactin in the form of non-plasmid expression, and do not require antibiotics to lock the plasmids. They can stably act on the intestines and be propagated, meeting the stability requirements for the use of probiotics;

[0209] Through the above optimization, the 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.

[0210] 2. The unique advantage of the engineered probiotics with recombinant integration of microcin MccJ25 of the present invention is that the expression amount of microcin MccJ25 by the engineered probiotics with recombinant integration of microcin MccJ25 can reach a high expression level of 25.31 mg / L, and can effectively inhibit Salmonella enteritidis CVCC3377, Shigella flexneri SF1015, and Escherichia coli ATCC25922. The sensitivity of the engineered probiotics with recombinant integration of microcin MccJ25 to Salmonella enteritidis is far superior to that of the engineered probiotics with recombinant integration of microcin MccY and the engineered probiotics with recombinant integration of microcin MccY+MccJ25.

[0211] 3. The unique advantage of the engineered probiotics with recombinant integration of microcin MccY of the present invention is that the expression amount of microcin MccY by the engineered probiotics with recombinant integration of microcin MccY can reach a high expression level of 9.23 mg / L, and can effectively inhibit Salmonella typhimurium ATCC14028, Salmonella enteritidis CVCC3377, Salmonella pullorum CVCC1800, Salmonella infantis CMCC50041, Salmonella Kentucky BNCC239114, Salmonella London CVCC2206, Salmonella Corvallis CMCC50922, and Shigella sonnei SS1014, and its sensitivity to Salmonella typhimurium is far superior to that of the engineered probiotics with recombinant integration of microcin MccJ25 and the engineered probiotics with recombinant integration of microcin MccY+MccJ25;

[0212] 4. The unique advantages of the engineered probiotics with recombinant integration of microcin MccY and MccJ25 of the present invention are: the expression level of microcin MccY can reach a high expression level of 8.02 mg / L, and the expression level of microcin MccJ25 can reach a high expression level of 9.11 mg / L, which can be used to treat Salmonella typhimurium ATCC14028, Salmonella enteritidis CVCC3377, Salmonella pullorum CVCC1800, and Salmonella infantum CMCC50041. , Kentucky Salmonella BNCC239114, London Salmonella CVCC2206, Corvallis Salmonella CMCC50922, Shigella flexneri SF1015, Shigella sonnei SS1014, and Escherichia coli ATCC25922. The broad-spectrum antibacterial activity of the engineered probiotics recombinantly integrated with microcin MccY+MccJ25 is better than that of the engineered probiotics recombinantly integrated with microcin MccY and the engineered probiotics recombinantly integrated with microcin MccJ25.

[0213] The embodiments presented herein are only embodiments selected according to the combination of all possible embodiments. The appended claims should not be limited by the embodiments describing the present invention. Some numerical ranges used in the claims include sub-ranges therein, and the changes in these ranges should also be covered by the appended claims.

Claims

1. An engineered probiotic with recombinant integration of microcin MccJ25, characterized in that: The engineered probiotics are Escherichia coli integrated with the microcin MccJ25 gene; the nucleotide sequence of the microcin MccJ25 gene is shown in SEQ ID NO: 19; and the Escherichia coli is Escherichia coli Nissle 1917.

2. A method for preparing the engineered probiotics according to claim 1, characterized in that: The SD-T7RNP homology arm repair fragment and the MccJ25E homology arm repair fragment were integrated into the Escherichia coli Nissle 1917 genome using genome editing technology and gene knockout vectors, thus obtaining the microcin MccJ25 recombinant integration engineering probiotic EcN-MccJ25; The nucleotide sequence of the SD-T7RNP homology arm repair fragment is shown in SEQ ID NO: 5; the MccJ25E homology arm repair fragment is shown in SEQ ID NO:

19.

3. The preparation method according to claim 2, characterized in that: The method is specifically as follows: (1) transferring the pREDCas9 plasmid into EcN competent cells, screening to obtain positive transformants EcN-cas9, and preparing the positive transformants EcN-cas9 into competent cells; the competent cells are EcN-cas9 competent cells; (2) The SD-T7RNP homology arm repair fragment and araBsgRNA plasmid were transferred into EcN-cas9 competent cells, and the recombinant strain EcNΔaraB-T7RNP-araBsgRNA-cas9 was screened using spectinomycin and ampicillin; the araBsgRNA plasmid was a pUC19 vector inserted with the sgRNA-araB fragment; the nucleotide sequence of the sgRNA-araB fragment was shown in SEQ ID NO: 33; (3) The recombinant strain EcNΔaraB-T7RNP-araBsgRNA-cas9 was subcultured to screen out a strain that was insensitive to spectinomycin and sensitive to ampicillin to eliminate the araBsgRNA plasmid in the strain, and the MccJ25E homology arm repair fragment and the endAsgRNA plasmid were transferred into the strain. The recombinant strain EcNΔaraB-T7RNP-cas9-ΔendA-MccJ25-endAsgRNA was screened using spectinomycin and ampicillin; the endAsgRNA plasmid was a pUC19 vector inserted with an sgRNA-endA fragment; the nucleotide sequence of the sgRNA-endA fragment was shown in SEQ ID NO: 34; (4) subculturing the recombinant strain EcNΔaraB-T7RNP-cas9-ΔendA-MccJ25-endAsgRNA, screening out strains that are insensitive to spectinomycin and sensitive to ampicillin to eliminate the endAsgRNA plasmid in the strain, and obtaining the recombinant strain EcN-T7RNP-MccJ25-cas9; (5) The recombinant strain EcNΔaraB-T7RNP-cas9-ΔendA-MccJ25 was subcultured at 42°C, and strains sensitive to both spectinomycin and ampicillin were screened to eliminate the pREDCas9 plasmid in the strain, thereby obtaining the microbacterium MccJ25 recombinant integrated engineered probiotic EcN-MccJ25.

4. An engineered probiotic with recombinant integration of microcin MccY, characterized in that: The engineered probiotics are Escherichia coli integrated with the microcin MccY gene; the nucleotide sequence of the microcin MccY gene is shown in SEQ ID NO: 12; and the Escherichia coli is Escherichia coli Nissle 1917.

5. A method for preparing the engineered probiotics according to claim 4, characterized in that: The SD-T7RNP homology arm repair fragment and the MccY homology arm repair fragment were integrated into the genome of Escherichia coli Nissle 1917 using genome editing technology and gene knockout vectors, thus obtaining the microcin MccY recombinant integration engineered probiotic EcN-MccY; The nucleotide sequence of the SD-T7RNP homology arm repair fragment is shown in SEQ ID NO: 5; the nucleotide sequence of the MccJ25E homology arm repair fragment is shown in SEQ ID NO:

12.

6. The preparation method according to claim 5, characterized in that: The method is specifically as follows: (1) transferring the pREDCas9 plasmid into EcN competent cells, screening to obtain positive transformants EcN-cas9, and preparing the positive transformants EcN-cas9 into competent cells; the competent cells are EcN-cas9 competent cells; (2) The SD-T7RNP homology arm repair fragment and araBsgRNA plasmid were transferred into EcN-cas9 competent cells, and the recombinant strain EcN-T7RNP-araBsgRNA-cas9 was screened using spectinomycin and ampicillin; the araBsgRNA plasmid was a pUC19 vector inserted with the sgRNA-araB fragment; the nucleotide sequence of the sgRNA-araB fragment was shown in SEQ ID NO: 33; (3) The recombinant strain EcN-T7RNP-araBsgRNA-cas9 was subcultured to screen out a strain that was insensitive to spectinomycin and sensitive to ampicillin to eliminate the araBsgRNA plasmid in the strain, and the MccY homology arm repair fragment and the endAsgRNA plasmid were transferred into the strain. The recombinant strain EcN-T7RNP-pREDCas9-MccY-endAsgRNA was screened using spectinomycin and ampicillin; the endAsgRNA plasmid was a pUC19 vector inserted with an sgRNA-endA fragment; the nucleotide sequence of the sgRNA-endA fragment was shown in SEQ ID NO: 34; (4) eliminating the endAsgRNA plasmid in the recombinant strain EcN-T7RNP-pREDCas9-MccY-endAsgRNA to obtain the recombinant strain EcN-T7RNP-MccY-cas9; (5) Eliminate the pREDCas9 plasmid in the recombinant strain EcN-T7RNP-MccY-cas9 to obtain the microcin MccY recombinant integrated engineered probiotic EcN-MccJ25.

7. An engineered probiotic with recombinant integration of microcins MccY and MccJ25, characterized in that: The engineered probiotics are Escherichia coli integrated with the microcin MccY gene and the microcin MccJ25 gene; the nucleotide sequence of the microcin MccY gene is shown in SEQ ID NO: 12; the nucleotide sequence of the microcin MccJ25 gene is shown in SEQ ID NO: 26; and the Escherichia coli is Escherichia coli Nissle 1917.

8. A method for preparing the engineered probiotics according to claim 7, characterized in that: The SD-T7RNP homology arm repair fragment, the MccJ25X homology arm repair fragment and the MccY homology arm repair fragment were integrated into the Escherichia coli Nissle 1917 genome using genome editing technology and gene knockout vectors, thus obtaining the microcin MccY and MccJ25 recombinant integration engineering probiotic EcN-MccY-MccJ25; The nucleotide sequence of the SD-T7RNP homology arm repair fragment is shown in SEQ ID NO: 5; the MccJ25X homology arm repair fragment is shown in SEQ ID NO: 26; and the MccY homology arm repair fragment is shown in SEQ ID NO:

12.

9. The preparation method according to claim 8, characterized in that: The method is specifically as follows: (1) transferring the pREDCas9 plasmid into EcN competent cells, screening to obtain positive transformants EcN-cas9, and preparing the positive transformants EcN-cas9 into competent cells; the competent cells are EcN-cas9 competent cells; (2) The SD-T7RNP homology arm repair fragment and araBsgRNA plasmid were transferred into EcN-cas9 competent cells, and the recombinant strain EcN-T7RNP-araBsgRNA-cas9 was screened using spectinomycin and ampicillin; the araBsgRNA plasmid was a pUC19 vector inserted with the sgRNA-araB fragment; the nucleotide sequence of the sgRNA-araB fragment was shown in SEQ ID NO: 33; (3) The recombinant strain EcN-T7RNP-araBsgRNA-cas9 was subcultured to screen out strains that were insensitive to spectinomycin and sensitive to ampicillin to eliminate the araBsgRNA plasmid in the strain, and the MccY homology arm repair fragment and the endAsgRNA plasmid were transferred into the strain, and the recombinant strain EcN-T7RNP-pREDCas9-MccY-endAsgRNA was screened using spectinomycin and ampicillin, and the recombinant strain EcN-T7RNP-pREDCas9-MccY-endAsgRNA was prepared into competent cells, which were EcN-T7RNP-MccY-cas9 competent cells; the endAsgRNA plasmid was a pUC19 vector inserted with an sgRNA-endA fragment; the nucleotide sequence of the sgRNA-endA fragment was shown in SEQ ID NO: 34; (4) The MccJ25X homology arm repair fragment and the xylAsgRNA plasmid were transferred into the 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 inserted with the sgRNA-xylA fragment, and the nucleotide sequence of the sgRNA-xylA fragment was 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 the strain EcN-T7RNP-MccY-MccJ25, which is the microcin MccY and MccJ25 recombinant integrated engineering probiotic EcN-MccY-MccJ25.

10. Use of the engineered probiotics according to claim 1, 4 or 7 for preparing an antibacterial preparation.

11. An antibacterial preparation, characterized in that: Contains the engineered probiotics as described in claim 1, 4 or 7.

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