CN111961634A - Bacillus amyloliquefaciens engineering bacterium for efficiently inhibiting staphylococcus aureus - Google Patents
Bacillus amyloliquefaciens engineering bacterium for efficiently inhibiting staphylococcus aureus Download PDFInfo
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Abstract
The invention provides a bacillus amyloliquefaciens engineering bacterium for efficiently inhibiting staphylococcus aureus, which knocks out a Surfactin synthesis related gene on the inner surface of a genome of bacillus amyloliquefaciens HZ-12 by a genetic engineering methodsrfACGene, successfully obtained deletionsrfACGenetically modified Bacillus amyloliquefaciens HZ deltasrfACAnd the antibacterial activity of the bacillus amyloliquefaciens on staphylococcus aureus is obviously improved. Compared with Bacillus amyloliquefaciens HZ-12, the engineering strain prepared by the invention has an improved inhibiting effect on staphylococcus aureus by 64%.
Description
Technical Field
The invention relates to the field of genetic engineering, in particular to a bacillus amyloliquefaciens engineering bacterium for efficiently inhibiting staphylococcus aureus.
Background
Staphylococcus aureus is one of the most major food-borne pathogenic bacteria, often causes food poisoning and seriously harms human health, and the drug resistance of staphylococcus aureus is increased year by year due to the massive use of antibiotics. The adoption of probiotics capable of inhibiting staphylococcus aureus is an effective means for solving the drug resistance of the staphylococcus aureus. The bacillus has great development potential in the application of inhibiting staphylococcus aureus as a probiotic with broad-spectrum antibacterial activity.
The Bacillus amyloliquefaciens HZ-12 has an inhibiting effect on staphylococcus aureus. Bacillus amyloliquefaciens secretes a variety of substances that inhibit bacteria and fungi, of which Surfactin (Surfactin) is one of the most powerful biosurfactant, and has been shown to have good inhibitory effects on fungi, and to destroy the phospholipid membrane stability of fungal pathogens, resulting in cell lysis, but not to be sensitive to bacteria. The biosynthetic gene cluster of Surfactin consists of 4 synthetic genes, wherein srfAA, srfAB and srfAC code for non-ribosomal peptide synthetases, and particularly the srfAC gene is important in the synthesis of Surfactin. On the one hand, Surfactin as a surfactant can cause liquid fermentation to produce bubbles, and can influence the metabolism of thalli. On the other hand, the synthetic pathway of Surfactin is complex, the synthetic process consumes a large amount of energy, and the production of other metabolites can be reduced. Furthermore, Surfactin can also affect the motility of bacillus and the formation of its biofilm. It was concluded that blocking Surfactin would reduce its effect on the production of bacterial inhibitory substances by bacillus amyloliquefaciens, and further investigation is needed. The invention discovers for the first time that: by knocking out srfAC gene of Bacillus amyloliquefaciens HZ-12, synthesis of Surfactin is blocked, the inhibiting effect of the srfAC gene on staphylococcus aureus is obviously improved, and a strain of engineering bacteria capable of efficiently inhibiting staphylococcus aureus is obtained.
Disclosure of Invention
The invention aims to provide a bacillus amyloliquefaciens engineering bacterium for efficiently inhibiting staphylococcus aureus, which is used for knocking out a Surfactin synthesis related gene srfAC by a gene knocking-out technology so as to obviously improve the inhibiting effect on the staphylococcus aureus.
In order to achieve the purpose, the invention adopts the following technical measures:
a Bacillus amyloliquefaciens engineering bacterium for efficiently inhibiting staphylococcus aureus is obtained by knocking out a Bacillus amyloliquefaciens (Bacillus amyloliquefaciens) HZ-12 surfactin synthesis gene srfAC, and the specific construction method comprises the following steps:
1) taking the genome DNA of the bacillus amyloliquefaciens HZ-12 as a template, and carrying out PCR amplification on an upstream homology arm of an srfAC gene and a downstream homology arm of the srfAC gene;
2) connecting an upstream homology arm of the srfAC gene and a downstream homology arm of the srfAC gene together by overlap extension PCR to form a homology arm fusion fragment;
3) carrying out double enzyme digestion on the homologous arm fusion fragment and the plasmid T2(2) -ori by using XbaI and BamHI restriction endonucleases to obtain an enzyme digestion gene fragment and a linear plasmid fragment, and connecting the enzyme digestion gene fragment and the linear plasmid fragment by using DNA ligase to obtain a knockout plasmid T2(2) -delta srfAC;
4) transferring the knockout plasmid T2(2) -delta srfAC into Bacillus amyloliquefaciens HZ-12, and screening by taking kanamycin as a screening marker to obtain a positive transformant;
5) transferring the positive transformant to a culture medium for several times at the temperature of 45 ℃, and then carrying out colony PCR detection to obtain a positive single-exchange conjugant strain which generates complete single exchange with the upstream homologous arm of the srfAC gene or the downstream homologous arm of the srfAC gene and the genomic DNA of the bacillus amyloliquefaciens HZ-12;
6) selecting a positive single crossover binder strain, inoculating the positive single crossover binder strain in a culture medium which does not contain kanamycin and transferring the culture medium for a plurality of times, and screening by a PCR method to obtain the bacillus amyloliquefaciens HZ delta srfAC with the srfAC gene knocked out.
The application of the bacillus amyloliquefaciens engineering bacterium HZ delta srfA prepared by the method in the efficient inhibition of staphylococcus aureus is as follows: adding the HZ delta srfAC fermentation liquor into the holes of a staphylococcus aureus plate, culturing at 37 ℃ for 16-18 h, and measuring the size of a bacteriostatic circle. The result shows that the inhibition capacity of the bacillus amyloliquefaciens engineering bacteria HZ delta srfAC on staphylococcus aureus is improved by 64 percent compared with that of the original bacteria.
The inventor tries to construct a knockout carrier for knocking Surfactin synthesis related gene srfAC for the first time, successfully obtains a bacillus amyloliquefaciens gene engineering strain lacking srfAC gene, obviously improves the inhibiting effect on staphylococcus aureus, and provides a new strategy for inhibiting staphylococcus aureus.
Compared with the prior art, the invention has the following advantages and effects:
1. the strain is subjected to genetic engineering modification by a traceless gene knockout method, no exogenous gene is introduced, no other accessory substances are generated, and the strain is harmless to human bodies and other organisms.
2. According to the invention, the knockout of srfAC gene is found for the first time, the inhibition effect of bacillus amyloliquefaciens on staphylococcus aureus can be obviously improved, and compared with the original bacillus amyloliquefaciens HZ-12, the inhibition effect of the bacillus amyloliquefaciens HZ delta srfAC on staphylococcus aureus is improved by 64%.
Drawings
FIG. 1 shows a verification band of Bacillus amyloliquefaciens HZ Δ srfAC with the srfAC gene knocked out in example 2, wherein a lane M is a DNA marker, lanes 1 and 2 are respectively Δ srfAC-KYF and Δ srfAC-KYR as primers, and the Bacillus amyloliquefaciens HZ Δ srfAC and HZ-12 are PCR products as templates.
Fig. 2 is a graph of the effect of blocking surfactin synthesis on the inhibition of b.
Detailed Description
In the following examples, the molecular biological test methods not specified in the specific conditions were performed according to the conventional conditions, as described in "molecular cloning protocols" (New York: Cold Spring Harbor).
Description of the biological Material:
bacillus amyloliquefaciens HZ-12 belongs to a disclosed biomaterial and has been reported in an article (Ruan dying, Li Lu, Zou Dian, Jiang Cong, Wen Zhiyou, Chen Shouwen, Deng Yu, Wei Xuetutan, (2019) metabolism engineering of Bacillus amyloliquefaciens for engineering of S-adenosylmethionine approach and the tricarboxyacrylic acid. biotechnol. biofuels 12,211), and is found in the agricultural bioengineering laboratory of Huazhong, university applicant has guaranteed to release the biomaterial to the public for twenty years from the application date for verification of the experiment.
Example 1: construction of temperature-sensitive knockout vectors
Designing upstream homology arm primers (srfAC-F1, srfAC-R1) and downstream homology arm primers (srfAC-F2, srfAC-R2) of srfAC gene according to the gene sequence of srfAC gene in the DNA sequence of Bacillus amyloliquefaciens HZ-12 genome; and taking the genome DNA of the bacillus amyloliquefaciens HZ-12 as a template, and respectively carrying out PCR amplification by using an upstream homologous arm primer and a downstream homologous arm primer of the srfAC gene to obtain an upstream homologous arm fragment of the srfAC gene and a downstream homologous arm fragment of the srfAC gene (the upstream homologous arm fragment of the srfAC gene is 666bp, and the downstream homologous arm fragment of the srfAC gene is 698 bp). The primer sequences are as follows:
srfAC-F1: CGCGGATCCCAGGCGGTTTGGAGTGTATT (containing BamHI cleavage site)
srfAC-R1：TTTGCTTCGTCAGGTCGTGCTTGAACCAATCCGTCAGAGGC
srfAC-F2：GCCTCTGACGGATTGGTTCAAGCACGACCTGACGAAGCAAA
srfAC-R2: GCTCTAGATCAGCCCGTAACCGAGAACC (containing XbaI cleavage site)
And (3) PCR system: ddH2O 25.0μL、5×TransStartTMFastPfu Buffer 5.0. mu. L, dNTPs 2.5.5. mu.L, FastPfu DNA Polymerase 1.0. mu.L, primer 11.0. mu.L, primer 21.0. mu.L, template DNA 0.5. mu.L.
And (3) PCR reaction conditions: 5min at 95 ℃; 30s at 95 ℃, 30s at 50-60 ℃, 30-60 s at 72 ℃ and 30-35 circulation numbers; 5min at 72 ℃.
According to the PCR reaction system and conditions, a primer pair (srfAC-F1/srfAC-R1) is utilized to carry out PCR amplification to obtain an srfAC upstream gene fragment (666bp) with a sequence shown in SEQ ID NO.2, and a primer pair (srfAC-F2/srfAC-R2) is utilized to carry out PCR amplification to obtain an srfAC downstream gene fragment (698bp) with a sequence shown in SEQ ID NO. 3. The upstream and downstream homology arms were ligated together by overlap extension PCR to form a homology arm fusion fragment (1364 bp).
The homologous arm fusion fragment and the plasmid T2(2) -ori were digested simultaneously with XbaI and BamHI restriction enzymes to obtain a digested gene fragment and a linear plasmid fragment. Subjecting the enzyme-cut gene fragment and the linear plasmid fragment to T4DNA ligase is used for ligation to obtain a ligation product; the ligation product is transferred into Escherichia coli DH5 alpha by calcium chloride transformation method, screening is carried out on LB culture medium (peptone 10g/L, yeast powder 5g/L, NaCl 10g/L, pH 7.2) containing kanamycin (20 mu g/mL) resistance under the condition of 37 ℃, transformants are obtained by screening, colony PCR verification and plasmid PCR verification are carried out on the transformants (the used primers are T2-F and T2-R), and the gene knockout vector T2(2) -delta srfAC is obtained.
Wherein the sequences of T2-F and T2-R are:
T2-F：ATGTGATAACTCGGCGTA
T2-R：GCAAGCAGCAGATTACGC。
example 2: construction of gene knockout engineering bacterium HZ delta srfAC
Transferring the knockout vector T2(2) -delta srfAC into Bacillus amyloliquefaciens HZ-12, screening by a culture medium containing kanamycin resistance at 37 ℃, screening to obtain a transformant, and carrying out colony PCR verification on the transformant (the used primers are T2-F and T2-R) to obtain a positive transformant (namely the Bacillus amyloliquefaciens HZ-12 transferred with the knockout vector T2(2) -delta srfAC).
Wherein the sequences of T2-F and T2-R are:
T2-F：ATGTGATAACTCGGCGTA
T2-R：GCAAGCAGCAGATTACGC
and (3) carrying out transfer culture on the positive transformant on a kanamycin-resistant culture medium at the temperature of 45 ℃ for 3 times, wherein each culture time is 12h, carrying out colony PCR (polymerase chain reaction) detection on the single-crossover strain by taking T2-F and delta srfAC-KYR as primers (or taking T2-R and delta srfAC-KYF as primers), and amplifying a band with the length of 1657bp or 2704bp to obtain the single-crossover strain.
Wherein the sequences of the delta srfAC-KYF and the delta srfAC-KYR are as follows:
ΔsrfAC-KYF：ATACAATGCCCTGCGAGAA、
ΔsrfAC-KYR：CGGCGGGAACGCAAACAGT
the single-crossover strain was inoculated and cultured, and transformants were picked for colony PCR verification (primers. DELTA. srfAC-KYF and. DELTA. srfAC-KYR) after several transfer cultures in a medium containing no kanamycin at 37 ℃ as shown in FIG. 1. Then, DNA sequencing is carried out on the positive transformant for further verification, and the srfAC knockout strain (i.e. the bacillus amyloliquefaciens HZ delta srfAC) with successful double crossover is obtained.
Example 3: blocking the influence of surfactin synthesis on HZ-12 antibacterial effect
And (3) preparing a staphylococcus aureus plate by a bacteria mixing method, punching a hole on the solidified and cooled staphylococcus aureus plate by using a sterilized puncher, and picking out the culture medium in the agar hole by using a sterile needle after the hole is punched. The distance between the centers of the holes is more than 20mm, and the distance between the centers of the holes and the peripheral source of the culture medium is more than 15 mm. mu.L of the HZ.DELTA.srfAC broth was pipetted into agar wells using a pipette gun, the plates were covered, and 3 replicates were added. And (4) measuring the size of the inhibition zone after culturing for 16-18 h in an incubator at 37 ℃. The results are shown in fig. 2, and the inhibition capacity of the bacillus amyloliquefaciens engineering bacteria HZ delta srfAC on staphylococcus aureus is improved by 64 percent compared with that of the original bacteria.
Sequence listing
<110> university of agriculture in Huazhong
<120> bacillus amyloliquefaciens engineering bacterium for efficiently inhibiting staphylococcus aureus
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1047
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgagccagt tcaaaaaaga tcaagttcag gacatgtatt atttgtcgcc gatgcaggag 60
ggaatgctgt ttcatactct cctgaatccc ggccaaagct tttacatcga acaaatgaca 120
atgagagtaa aaggcagctt gaatatcaaa tgccttgaag aaagcatgaa tgtgatcatg 180
gaccggtacg atgtatttcg taccgtgttc attcacgaaa aagtaaaaag gccggtccaa 240
gtcgtattga aaaaacggca gtttcagata gaagaaatcg atctgacaca cttaacgggc 300
agcgagcaag catccaaaat taatgaatac aaagaacagg ataagatcaa gggctttgat 360
ttgacgcggg atattccgat gcgggcagcc atctttaaaa aatcggaaga aagctttgaa 420
tgggtgtgga gctaccacca catcattttg gacggctggt gcttcggcat cgtcgtgcag 480
gatctgttta aggtatacaa tgccctgcga gaacaaaagc cgtacagcct gccgccggtc 540
aaaccgtata aagactatat caagtggctt gaaaagcagg ataaacaagc atcactgcat 600
tactggcgcg ggtacttaga agattttgaa ggacaaacga catttgcgga gcaaagaaag 660
aaacaagaga acggctatga gccgaaagag ctgctcttct cactgccgga ggcggaaaca 720
aaagcattta ccgagcttgc aaaatcgcag cataccactt tgagtacggc gctgcaggcg 780
gtttggagtg tattaatcag ccgctaccag cagtccggcg atttgatctt cggcacagtc 840
gtttccgggc gtcccgcaga aatcaaaggc gttgaacata tggtcgggct gtttatcaat 900
gctgttccga ggcgggtgaa gctgtctgag gatactacat ttaacggctt gctcaagcag 960
ctgcaggagc aatcgctgca gtctgagcct catcaatatg tgccgctcta tgacatccaa 1020
agccaggccg atcagccaaa gctgatt 1047
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<211> 666
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cgatcggtga atggacggaa aacggcaaag tcggcatcaa cttagaagga cacggccggg 60
aagaaatcat accgaatgtc aatatttcac ggacggtcgg ctggtttacg gcccaatatc 120
cgttgattct gcagatcagc aaggaagacg gcgtctcttc cgtcattaaa acggtaaaag 180
agacagtgcg gcgcgttcca gctaaaggtg taggatacgg gattctccgg tatctgtcat 240
ccgatgaaac agaaaaaggc gccgcgcctg aaatcagttt taactacttg gggcagtttg 300
acaatgaagt gaaaacggaa tggtttgagc cgtctccata tgatatggga cgtcaagtca 360
gcgaagagtc agaggcgtta tacgcactga gcttcagcgg gatggttaca ggcggccgct 420
tcgtcatttc ctgctcatac aatcaggaag aatatgaaag aagcaccgtc gaaacacaga 480
tgcagcggtt taaagacaat cttttaatga tcatccgcca ttgcacggcc aaagaggaga 540
aagaatttac gccgagcgat ttcagcgcgc aggatcttga gatggatgaa atgggagaca 600
tctttgacat gcttgaggag aatttaacgt gacaaaaaca gttaacagaa ggggggagcg 660
gagcag 666
<210> 3
<211> 698
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
gatcatatca ttgtgtttga aaattatccg cttcaggatg caaaaaatga agaaaacagt 60
gaaaacggct ttgatatgga ggacgtccat gtttttgaga aatcgaatta tgatctcaac 120
ctgatggctt ctccaggtga tgagatgctg attaagcttg cctataacgg gaatgtgttt 180
gatgaggcgt ttatcctgcg cttaaaatct cagcttctca cagcgattca gcagctcatc 240
cagaagccgg atcaccctgt caatacgatc agacttgttg atgaaaagga aagagagctt 300
ctgcttaccg gcttaaaccc gccggctgag actcatcagg cgaagcctct gacggattgg 360
ttcaaggaag cggtgaatgt aaatcctgat gcaccggcgc ttacgtattc cggccagact 420
ctttcctatc gcgaattaga tgaggaagcg aaccgtcttg cgcgccgttt gcaaaaacaa 480
ggtgcgggta aagacaccgt tgtcgcgttg tacacgaagc gctcgcttga actggtgatc 540
ggcattctcg gcgtattaaa agcgggagcg gcttatctgc cggttgatcc gaagctgccg 600
gaggaccgaa tctcgtatat gctgactgac agtgcggctg cctgtctgct gacacatcag 660
gagatgaaag aaaaagcggc tcagctgccg tatacagg 698
Claims (4)
1. A bacillus amyloliquefaciens engineering bacterium for efficiently inhibiting staphylococcus aureus is characterized in that the engineering bacterium is a knockout bacillus amyloliquefaciens (Bacillus amyloliquefaciens)Bacillus amyloliquefaciens) HZ-12 surfactin synthetic genesrfACThus, the compound was obtained.
2. A Bacillus amyloliquefaciens engineered bacterium according to claim 1 wherein the gene that is knocked out issrfACThe nucleotide sequence of (A) is shown in SEQ ID NO. 1.
3. A construction method of a Bacillus amyloliquefaciens engineering bacterium as claimed in claim 1, comprising the following steps:
1) PCR amplification with genome DNA of Bacillus amyloliquefaciens HZ-12 as templatesrfACUpstream homology arms of genes andsrfACa downstream homology arm of a gene;
2) by overlap extension PCRsrfACUpstream homology arms of genes andsrfACthe downstream homology arms of the genes are connected together to form a homology arm fusion fragment;
3) by usingXbaI andBamHI restriction enzyme Dual-ligation of the homology arm fusion fragment and the plasmid T2(2) -ori
Enzyme cutting to obtain enzyme cutting gene fragment and linear plasmid fragment, and connecting with DNA ligase to obtain knock-out plasmid T2(2) -deltasrfAC(ii) a The knockout plasmid T2(2) - Δ was ligatedsrfACTransferring the bacillus amyloliquefaciens into HZ-12, and screening by taking kanamycin as a screening marker to obtain a positive transformant;
4) positive transformants were transferred to the culture medium at 45 ℃ based on the number of transfer culturesThen, carrying out colony PCR detection to obtainsrfACUpstream homology arms of genes orsrfACThe downstream homology arm of the gene and the genome DNA of the bacillus amyloliquefaciens HZ-12 generate a positive single-exchange conjugator strain with complete single exchange;
5) selecting positive single crossover binder strain, inoculating to 37 deg.C culture medium without kanamycin, transferring to culture medium for several times, and PCR screening to obtain the final productsrfACGenetically modified Bacillus amyloliquefaciens HZ deltasrfAC。
4. A Bacillus amyloliquefaciens engineering bacterium of claim 1 for inhibiting staphylococcus aureus.
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| WEI W.: "Bacillus amyloliquefaciens surfactin non-ribosomal peptide synthetase SrfAC", 《EMBL DATABASE》 * |
| 刘洪霞: "脂肽Plipastatin合成酶COM结构域的修饰及新型脂肽的合成", 《万方》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114606172A (en) * | 2022-03-30 | 2022-06-10 | 华中农业大学 | Bacillus amyloliquefaciens engineering bacterium for improving yield of heme and construction method thereof |
| CN114606172B (en) * | 2022-03-30 | 2023-09-22 | 华中农业大学 | Bacillus amyloliquefaciens engineering bacteria for improving heme yield and construction method thereof |
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| Publication number | Publication date |
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| CN111961634B (en) | 2022-08-09 |
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