Construction and antibacterial application of recombinant engineering probiotics capable of producing microbacteriocin MccY

Abstract

The invention belongs to the technical field of biology, and discloses an engineering probiotic recombined and integrated by microrhin MccY, and the engineering probiotic has the characteristics of no resistance marker gene, 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] This invention relates to the field of biotechnology, specifically to the construction and antibacterial application of microbial-producing MccY recombinant engineered probiotics, and more specifically, to a microbial-producing MccY recombinant engineered probiotic, its preparation method, and its uses. Background Technology

[0002] 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.

[0003] Microbesin MccH47 and MccM have relatively narrow antibacterial spectra. To further improve the antibacterial spectrum and antimicrobial activity of probiotic EcN, further modification of probiotic EcN is necessary. The following challenges exist in genetic engineering modification of probiotic EcN: 1. How to broaden the antibacterial spectrum; 2. How to introduce targeted microbesin-related genes into probiotic EcN; 3. How to ensure that the microbesin-related genes exert their corresponding effects without resistance markers (the presence of resistance markers means there is a risk of horizontal transmission of resistance genes, making it unsuitable for use as a probiotic). Summary of the Invention

[0004] One of the objectives of this invention is to provide an engineered probiotic with recombinant microbial agents. The engineered probiotics of this invention are characterized by the absence of resistance marker genes, good growth activity, genetic stability, and antibacterial activity.

[0005] Meanwhile, the engineered probiotics recombinantly integrated with microbesmectin MccY have a broad spectrum of activity. Furthermore, the engineered probiotics recombinantly integrated with microbesmectin MccY are far more sensitive to Salmonella typhimurium than the engineered probiotics recombinantly integrated with microbesmectin MccJ25 and the engineered probiotics recombinantly integrated with microbesmectin MccY+MccJ25.

[0006] In addition, the present invention also provides a method for preparing the above-mentioned engineered probiotics and their uses.

[0007] To achieve the above objectives, the present invention provides an engineered probiotic that is a recombinant and integrated microbesin MccY, wherein the engineered probiotic is an Escherichia coli that integrates the microbesin MccY gene; the nucleotide sequence of the microbesin MccY gene is shown in SEQ ID NO.5; and the Escherichia coli is Escherichia coli Nissle 1917.

[0008] The preparation method of the microbial MccY recombinant integrated engineered probiotic is as follows: using genome editing technology and gene knockout vector, the SD-T7RNP homologous arm repair fragment and the MccY homologous arm repair fragment are integrated into the genome of Escherichia coli Nissle 1917, thus obtaining the microbial MccY recombinant integrated engineered probiotic EcN-MccY.

[0009] The nucleotide sequence of the SD-T7RNP homologous arm repair fragment is shown in SEQ ID NO.4; the MccJ25E homologous arm repair fragment is shown in SEQ ID NO.5.

[0010] In the above preparation method, the method specifically includes:

[0011] (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;

[0012] (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. 6;

[0013] (3) The recombinant strain EcN-T7RNP-araBsgRNA-cas9 was passaged and screened 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 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.7.

[0014] (4) Eliminate the endAsgRNA plasmid in the recombinant strain EcN-T7RNP-pREDCas9-MccY-endAsgRNA to obtain the recombinant strain EcN-T7RNP-MccY-cas9;

[0015] (5) Eliminate the pREDCas9 plasmid in the recombinant strain EcN-T7RNP-MccY-cas9 to obtain the microbial MccY recombinant integrated probiotic EcN-MccJ25.

[0016] Furthermore, the present invention also discloses the use of engineered probiotics as described above in the preparation of antibacterial agents.

[0017] In the above-mentioned uses, the dosage form of the antibacterial preparation is one of powder, solution and granules.

[0018] Finally, the present invention also discloses an antibacterial preparation containing one or more of the engineered probiotics described above.

[0019] In the above-mentioned antibacterial agents, the dosage form of the antibacterial agent is one of powder, solution and granules.

[0020] Beneficial effects

[0021] Compared with the prior art, the present invention has at least the following advantages:

[0022] 1. The common advantage of the engineered probiotics of the present invention is that they can be used as probiotics, and they meet the following requirements of probiotics: 1. They are live bacteria and can act on the intestines of humans or animals; 2. The engineered bacteria 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 engineered bacteria constructed by the method of the present invention express microinfectious agents in a non-plasmid expression form, and there is no need for antibiotics to lock the plasmids. They can stably act on the intestines and be passaged, meeting the stability requirements for the use of probiotics.

[0023] Through the above optimizations, the 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.

[0024] 2. 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. Attached Figure Description

[0025] The present invention will be further described below with reference to the accompanying drawings and embodiments;

[0026] Figure 1A The plasmid map of the recombinant plasmid pUC19-T7RNP;

[0027] Figure 1B The plasmid map of the recombinant plasmid pUC19-MccY;

[0028] Figure 2A The plasmid map of the araBsgRNA plasmid;

[0029] Figure 2B The plasmid map of the endAsgRNA plasmid;

[0030] Figure 2C This is a plasmid map of the xylAsgRNA plasmid;

[0031] Figure 3A This is a diagram showing the PCR identification results of the EcN∆araB-T7RNP strain;

[0032] Figure 3B This is a diagram showing the PCR identification results of strain EcN∆araB-T7RN-∆endA-MccY;

[0033] Figure 4 This is a diagram of the inhibition zone of MccY recombinant and integrated probiotics in 0.5% LB soft agar containing Salmonella typhimurium;

[0034] Figure 5A This is a liquid chromatography result of the culture supernatant of EcN-MccY engineered probiotics;

[0035] Figure 5B This is a liquid chromatography-mass spectrometry result of the culture supernatant of EcN-MccY engineered probiotics;

[0036] Figure 6A This is a diagram of the inhibition zones of MccY recombinant and integrated probiotics expressed at different times;

[0037] Figure 6B This is a diagram of the inhibition zones of MccJ25 recombinant and integrated engineered probiotics at different expression times;

[0038] 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.

[0039] 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.

[0040] 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

[0041] 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.

[0042] 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).

[0043] The following is a detailed description.

[0044] Example 1: Construction of MccY Recombinant Integrative Engineered Probiotics

[0045] In this embodiment, Escherichia coli Nissle 1917 (EcN) was used as the substrate bacteria. Using pREDCas9 E. coli gene editing technology, a 20 bp SD sequence and a 2652 bp complete T7RNP sequence were integrated into the araB position of its genome. Subsequently, the MccY repair fragment was integrated into the endA position of its genome to obtain MccY recombination-integrated probiotics.

[0046] The SD nucleotide sequence is: 5'-CTAACTGGAAGAGGCACTAA-3' (SEQ ID NO.1).

[0047] The nucleotide sequence of the SD-T7RNP sequence is shown in SEQ ID NO.2;

[0048] The nucleotide sequence of the McCY repair fragment is shown in SEQ ID NO.3;

[0049] The specific construction method is as follows:

[0050] (1) Construction of pUC19-T7RNP vector and pUC19-MccY vector:

[0051] 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).

[0052] 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.

[0053] A 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.

[0054] (2) Construction of SD-T7RNP homologous arm repair fragment and optimized McCY homologous arm repair fragment:

[0055] 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 20 bp 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 20 bp SD sequence as templates, the SD-T7RNP homologous arm repair fragment was obtained by overlap PCR amplification using araBup-F and araBdown-R primers.

[0056] The obtained SD-T7RNP homologous arm repair fragment is shown in SEQ ID NO.4, and the primer sequences are shown in Table 1.

[0057] Table 1 Primer sequence information for SD-T7RNP integration

[0058] Primer name Sequence (5'-3') araBup-F 5'-tgacgaccgtagtgatgaat-3' araBup-R 5'-ttagtgcctcttccagttagtgacggaactggttattcgg-3' araBdown-F 5'-atgacacctgccacacaaat-3' araBdown-R 5'-tcagcgcatggctgaagacggtatg-3' T7RNP-F 5'-ctaactggaagaggcactaaatgaacacgattaacatcgc-3' T7RNP-R 5'-atttgtgtggcaggtgtcatcgttacgcgaacgcgaagtccg-3'

[0059] The PCR amplification systems are shown in Table 2. The overlapping PCR amplification systems are shown in Table 3.

[0060] Table 2 PCR amplification system

[0061] 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

[0062] Table 3 Overlap PCR Amplification System

[0063] 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

[0064] 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.

[0065] 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.

[0066] The obtained MccY homologous arm repair fragment is shown in SEQ ID NO.5, and the primer sequences are shown in Table 4.

[0067] Table 4 Primer sequence information for MccY integration

[0068] Primer name Sequence (5'-3') endAyup-F 5'-cgctggaacatattgaacgt-3' endAyup-R 5'-gggatcgagatctcgatcctttgataccttcggccaatgc-3' endAydown-F 5'-atgggacaagatgtatccag-3' endAydown-R 5'-ttcatctgccgataaaccgc-3' MccY-F 5'-aggatcgagatctcgatccc-3' MccY-R 5'-ctggatacatcttgtcccatttattctgtagctgatgccag-3'

[0069] The PCR amplification system and procedure, and the overlapping PCR amplification system and procedure are the same as those in the above embodiments.

[0070] (3) Construct araBsgRNA plasmid, endAsgRNA plasmid, and xylAsgRNA plasmid:

[0071] The sgRNA-araB fragment was designed, and its sequence is shown in SEQ ID NO.6. The sequence shown in SEQ ID NO.6 was cloned into the pUC19 vector by General Biotechnology (Anhui) Co., Ltd., thus obtaining the araBsgRNA plasmid (plasmid map shown). Figure 2A (As shown).

[0072] As described above, an sgRNA-endA fragment was designed, with the sequence shown in SEQ ID NO.7. Weitong Bio (Anhui) Co., Ltd. cloned the sequence shown in SEQ ID NO.7 into the pUC19 vector, thereby obtaining the endAsgRNA plasmid (plasmid map shown in...). Figure 2B (As shown).

[0073] The sgRNA-xylA fragment was designed, and its sequence is shown in SEQ ID NO.8. Weitong Bio (Anhui) Co., Ltd. cloned the sequence shown in SEQ ID NO.8 into the pUC19 vector, thereby obtaining the xylAsgRNA plasmid (plasmid map shown in...). Figure 2C (As shown).

[0074] (4) Construction of EcN-cas9 strain containing pREDCas9 plasmid:

[0075] 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.

[0076] The sequences of pREDCas9-test-F and pREDCas9-test-R are shown in Table 5.

[0077] Table 5 Primer information used for identification of EcN-cas9 strains

[0078] Primer name Sequence (5'-3') pREDCas9-test-F 5'-ttcctaatcagcccggcatttc-3' pREDCas9-test-R 5'-acgacataatgcaggccttc-3'

[0079] (5) Construction of EcN∆araB-pREDCas9-T7RNP-araBsgRNA

[0080] The obtained EcN-cas9 strain was induced to OD600 = 0.6-0.8 with 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) 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. The successfully transformed strain was identified as the recombinant strain EcN-T7RNP-araBsgRNA-cas9.

[0081] The sequences of araB-test-F and araB-test-R are shown in Table 6.

[0082] Table 6 Primer information for identification of recombinant strain EcN∆araB-T7RNP-araBsgRNA-cas9

[0083] Primer name Sequence (5'-3') araB-test-F 5'- attcacaacctgccctaaac-3' araB-test-R 5'-caccttcatgatgcgaagca-3'

[0084] (6) Elimination of araBsgRNA plasmid:

[0085] The recombinant strain EcN∆araB-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., strains 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 EcN∆araB-T7RNP-cas9 with araBsgRNA plasmid eliminated.

[0086] The sequences of sgRNA-test-F and sgRNA-test-R are shown in Table 7.

[0087] Table 7 Primer information for identification of recombinant strain EcN-T7RNP-cas9 used for araBsgRNA plasmid elimination.

[0088] Primer name Sequence (5'-3') sgRNA-test-F 5'-cgccagggttttcccagtcacgac-3' sgRNA-test-R 5'-agcggataacaatttcacacagga-3'

[0089] (7) Construction of MccY recombinant integrated engineered probiotic strain EcN∆araB-T7RNP-cas9-∆endA-MccY-endAsgRNA:

[0090] Following the above procedures, the recombinant strain EcN∆araB-T7RNP-cas9, with the araBsgRNA plasmid eliminated, was induced with 0.5 mM IPTG to an OD600 of 0.6-0.8. EcN∆araB-T7RNP-cas9 competent cells were prepared. The optimized MccY sequence repair fragment and the endAsgRNA plasmid were then transformed into the EcN∆araB-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 EcN∆araB-T7RNP-cas9-∆endA-MccY-endAsgRNA, which integrates the optimized MccY sequence into the EcN genome.

[0091] The sequences of endA-test-F and endA-test-R are shown in Table 8.

[0092] Table 8 Primer information for identification of EcN∆araB-T7RNP-cas9-∆endA-MccY / MccJ25-endAsgRNA

[0093] Primer name Sequence (5'-3') endA-test-F 5'-tgagtgccggtccgctgatt-3' endA-test-R 5'-tgtgcttccagcaacatagc-3'

[0094] (8) Elimination of endAsgRNA plasmid:

[0095] The recombinant strain EcN∆araB-T7RNP-∆endA-MccY-endAsgRNA-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., strains 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 (as shown in Table 7) to identify successful transformation. The successfully transformed strains were the recombinant strains with endAsgRNA plasmid eliminated.

[0096] EcNΔaraB-T7RNP-ΔendA-MccY-cas9.

[0097] (9) Elimination of pREDCas9 plasmid:

[0098] The recombinant strain EcN∆araB-T7RNP-∆endA-MccY-cas9 obtained above was continuously passaged on antibiotic-free plates and cultured at 42℃. Single colonies were picked and stab-dropped onto antibiotic-free plates and spectinomycin-resistant plates respectively to screen for strains sensitive to spectinomycin. PCR amplification was performed using sgRNA-test-F and sgRNA-test-R primers to confirm successful transformation. The successfully transformed strain was the recombinant strain EcN∆araB-T7RNP-∆endA-MccY (i.e., Microsin MccY recombinant integrated engineered probiotic) with pREDCas9 and endAsgRNA plasmids eliminated.

[0099] Figures 3A to 3B They are respectively:

[0100] Figure 3A This is a diagram showing the PCR identification results of the EcN∆araB-T7RNP strain;

[0101] Figure 3B This is a diagram showing the PCR identification results of strain EcN∆araB-T7RN-∆endA-MccY.

[0102] Example 2: Sensitivity determination of MccY recombinant and integrated engineered probiotics

[0103] The constructed MccY recombinant integrated engineered probiotic was inoculated into 20 mL of LB medium, and 1 M m IPTG was added to induce expression. The culture was then incubated at 37°C for 24 hours at 200 rpm / min. After centrifugation, bacterial cells were removed, yielding the expression supernatants of MccY recombinant integrated engineered probiotics, MccJ25 recombinant integrated engineered probiotics, 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°C for later use.

[0104] 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 culture was then poured into petri dishes and allowed to stand for 30 min until solidification. 10 μL of the supernatant of the MccY recombinant engineered probiotic obtained by serial dilution in the above steps was then spotted onto 0.5% LB soft agar containing Salmonella Typhimurium. Similarly, 10 μL of the supernatant of the MccJ25 recombinant engineered probiotic obtained by serial dilution in the above steps was spotted onto 0.5% LB soft agar containing Salmonella Enteritidis. The supernatant of the MccY+MccJ25 recombinant and integrated engineered probiotics obtained by serial dilution in the above steps was spotted onto 0.5% LB soft agar containing Salmonella enteritidis and 0.5% LB soft agar containing Salmonella typhimurium, respectively. After drying, the samples were placed in an incubator at 37°C for 16 hours and the inhibition zone was observed.

[0105] The results are as follows Figure 4 As shown;

[0106] Figure 4 This is a diagram of the inhibition zone of MccY recombinant and integrated probiotics in 0.5% LB soft agar containing Salmonella typhimurium;

[0107] It was found that the 1×, 2×, 10×, and 20× supernatants of the McCY recombinant and integrated probiotic strain had significant antibacterial effects against Salmonella typhimurium, while the 100× supernatant had a weak antibacterial effect against Salmonella typhimurium.

[0108] Example 3: Quantitative analysis of McY recombinant and integrated probiotics using high-performance liquid chromatography (HPLC).

[0109] One mL of the supernatant from the MccY recombinant integrated probiotic strain expressed for 24 hours was collected, and the concentrations of MccY and MccJ25 in the supernatant were detected using an ultra-high performance liquid chromatography-quadrupole tandem time-of-flight mass spectrometer (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 9.

[0110] Table 9

[0111] Time (min) A（%） B（%） 20 50 50 30 90 10 35 10 90

[0112] The results are as follows Figures 5A to 5B As shown, mass spectrometry analysis of the MccY recombinant and integrated engineered probiotics revealed a characteristic ion peak of 1113.73 consistent with the target substance MccY, indicating that MccY was successfully expressed and secreted in the engineered bacteria, with a protein concentration of 24.88 mg / L.

[0113] in, Figure 5A This is a liquid chromatography result of the culture supernatant of EcN-MccY engineered probiotics;

[0114] Figure 5B This is a liquid chromatography-mass spectrometry (LC-MS) result of the culture supernatant of EcN-MccY engineered probiotics.

[0115] 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.

[0116] 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 ℃ 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 ℃ for later use.

[0117] 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 in a 37 ℃ constant temperature incubator to observe the antibacterial effect.

[0118] The results are as follows Figures 6A-6D As shown, in MccY recombinant integrated engineered probiotics, MccJ25 recombinant integrated engineered probiotics, and MccY+MccJ25 recombinant integrated engineered probiotics, the supernatant expressed for 4 hours to 24 hours all showed inhibition zones, and the antibacterial effect became more obvious with increasing time.

[0119] Figure 6A This is a diagram of the inhibition zones of MccY recombinant and integrated probiotics expressed at different times;

[0120] Figure 6B This is a diagram of the inhibition zones of MccJ25 recombinant and integrated engineered probiotics at different expression times;

[0121] 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.

[0122] 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.

[0123] Example 5: Antibacterial activity test of MccY recombinant integrated engineered probiotics, MccJ25 recombinant integrated engineered probiotics, and MccY+MccJ25 recombinant integrated engineered probiotics.

[0124] The MccY, MccJ25, and MccY+MccJ25 recombinant-integrated probiotic strains were revived in LB agar plates. One loopful of each strain was transferred to 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 irradiated with ultraviolet light for 1 h.

[0125] 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 inoculated into 0.5% LB soft agar medium at a ratio of 1:1000. Then, the inoculated bacterial solution was 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 by standing for 30 min, the plates were placed in an incubator at 37℃ for 16 h and the inhibition zone was observed.

[0126] 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 Londonae 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.

[0127] 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.

[0128] 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.

[0129] In summary, the advantages of this invention are:

[0130] 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.

[0131] 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.

[0132] 2. 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.

[0133] 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. A recombinant and integrated microbial probiotic containing MccY, characterized in that, The engineered probiotic is an Escherichia coli that integrates the microinfectious drug MccY gene; the nucleotide sequence of the microinfectious drug MccY gene is shown in SEQ ID NO.5; the Escherichia coli is Escherichia coli Nissle 1917.

2. A method for preparing engineered probiotics as described in claim 1, characterized in that, Using genome editing technology and gene knockout vectors, the SD-T7RNP homologous arm repair fragment and the MccY homologous arm repair fragment were integrated into the genome of Escherichia coli Nissle 1917, thus obtaining the microbesin MccY recombinant integrated probiotic EcN-MccY. The nucleotide sequence of the SD-T7RNP homologous arm repair fragment is shown in SEQ ID NO.4; the MccJ25E homologous arm repair fragment is shown in SEQ ID NO.

5.

3. The preparation method according to claim 2, characterized in that, The method is specifically 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 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.6; (3) The recombinant strain EcN-T7RNP-araBsgRNA-cas9 was passaged and screened 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 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.

7. (4) Eliminate 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 microbial MccY recombinant integrated probiotic EcN-MccJ25.

4. Use of the engineered probiotics as described in claim 1 to prepare antibacterial preparations.

5. An antibacterial agent, characterized in that, It contains the engineered probiotics as described in claim 1.

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