High-yield abinogen bacillus subtilis strain and construction method thereof

Abstract

The invention discloses a high-yield abinogen bacillus subtilis strain and a construction method thereof, and the construction method comprises the following steps: 1) mutating a ComA protein binding site RE3 region sequence of a ComQXPA system targeted promoter PsrfA of the bacillus subtilis strain from TTTCAC to TTGCGG to obtain a mutated promoter PsrfA-RE3; (2) replacing a promoter Pveg of a gene cluster for synthesizing the abinogen in the bacillus subtilis CGF-P-02 with PsrfA-RE3 by utilizing a CRISPR (clustered regularly interspaced short palindromic repeats) / Cas9 (CRISPR associated protein 9) technology, so as to obtain a bacillus subtilis strain CGF-P-02-PsrfA-RE3 with high yield of the abinogen; the strain constructed by the invention has the advantages of high yield of abundant hormone, simple construction method and easiness in operation.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and particularly relates to a high-yield fengycin Bacillus subtilis strain and a construction method. Background Art

[0002] Fengyuansu is a cyclic lipopeptide synthesized by Bacillus via non-ribosomal peptide synthetase (NRPs). Structurally, it consists of a β-hydroxy fatty acyl chain of 15-19 carbon atoms and a decapeptide containing 8 amino acids. Fengyuansu has potent antifungal activity and can be used as a biological control agent. It is environmentally friendly compared to chemical synthetic products.

[0003] Low yield is the main factor limiting the industrial production of Fengyuansu. At present, the genetic engineering of strains is mainly static regulation, including promoter engineering; precursor engineering to increase the accumulation of amino acid and fatty acid precursors; knocking out biofilm and spore formation genes to extend the Fengyuansu production stage; knocking out or overexpressing related global regulatory factors, etc. However, the above measures may cause metabolic imbalance of strains, affect normal cell growth and lead to a decrease in yield.

[0004] The quorum sensing (QS) system is a natural dynamic regulatory system of microorganisms. It regulates cell density-dependent behavior by responding to bacterial density and has been increasingly used for dynamic metabolic engineering of strains. ComQXPA is a quorum sensing system in Bacillus subtilis. The signal molecule ComX is an autoinducing oligopeptide composed of 10 amino acid residues. Its precursor is processed and modified by the isoprenyl transferase ComQ and secreted into the extracellular space. As the cells grow and accumulate, when the critical concentration is reached, the transmembrane histidine kinase ComP responds to ComX for autophosphorylation, and the phosphate group is further transferred to the regulatory protein ComA. The phosphorylated ComA (ComA-P) binds to the target promoter and activates the transcription of downstream genes such as the surfactin srf operon. Promoter P srfA It is a quorum sensing targeted promoter. There is a ComA protein binding site in the upstream sequence of its RNAP binding site, which is composed of four recognition elements: RE1, RE2, RE3, and RE4 (-TTTCGC-4nt-CCGCAT-6nt-TTTCAC-5nt-TTTCGG-). However, the natural promoter P srfA Low intensity affects the expression of downstream genes and the yield of corresponding products. Therefore, it is necessary to study how to use the ComQXPA system in Bacillus subtilis to dynamically regulate the synthesis of fengycin and achieve high yield of fengycin. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a high-yield fengycin Bacillus subtilis strain;

[0006] The second object of the present invention is to provide a method for constructing a high-yield fengycin-producing Bacillus subtilis strain.

[0007] The third object of the present invention is to provide a method for producing Fengyuansu by fermenting a high-yield Fengyuansu Bacillus subtilis strain.

[0008] The fourth object of the present invention is to provide another high-yield fengycin Bacillus subtilis strain;

[0009] The fifth object of the present invention is to provide a method for constructing another high-yield fengin Bacillus subtilis strain.

[0010] The sixth object of the present invention is to provide another use of a high-yield fengyuansu Bacillus subtilis strain for producing fengyuansu by fermentation.

[0011] The technical solution of the present invention is summarized as follows:

[0012] A method for constructing a high-yield fengycin-producing Bacillus subtilis strain comprises the following steps:

[0013] 1) Targeting the ComQXPA system of Bacillus subtilis strains to promoter P srfA The sequence of the ComA protein binding site RE3 region of the mutated promoter was mutated from TTTCAC to TTGCGG. srfA-RE3 ;

[0014] 2) Using CRISPR / Cas9 technology, the promoter of the fengycin synthesis gene cluster in Bacillus subtilis CGF-P-02 was transformed into veg Replace with P srfA-RE3 , a high-yield fengin Bacillus subtilis strain CGF-P-02-PsrfA-RE3 was obtained;

[0015] The promoter P srfA The nucleotide sequence is shown in SEQ ID NO.1;

[0016] The mutated promoter P srfA-RE3 The nucleotide sequence is shown in SEQ ID NO.2;

[0017] The promoter P veg The nucleotide sequence is shown in SEQ ID NO.3.

[0018] The above method constructs a high-yield sporeforming Bacillus subtilis strain CGF-P-02-PsrfA-RE3.

[0019] The use of the above-mentioned high-yield Fengyuansu Bacillus subtilis strain CGF-P-02-PsrfA-RE3 in fermentation to produce Fengyuansu.

[0020] Another method for constructing a high-yield fengycin Bacillus subtilis strain comprises the following steps: knocking out the rapJ gene and the rapE gene in a high-yield fengycin Bacillus subtilis strain CGF-P-02-PsrfA-RE3 to obtain a high-yield fengycin Bacillus subtilis strain CGF-P-02-PsrfA-RE3-ΔrapJE;

[0021] The nucleotide sequence of the rapJ gene is shown in SEQ ID NO.4;

[0022] The nucleotide sequence of the rapE gene is shown in SEQ ID NO.5.

[0023] A high-yield sporeforming Bacillus subtilis strain CGF-P-02-PsrfA-RE3-ΔrapJE constructed by the above another method.

[0024] The use of the above-mentioned another high-yield Fengyuansu Bacillus subtilis strain CGF-P-02-PsrfA-RE3-ΔrapJE in fermentation to produce Fengyuansu.

[0025] Advantages of the present invention:

[0026] The strain constructed by the invention has high Fengyuansu yield, and the construction method is simple and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The promoter P srfA With P srfA-RE3 Corresponding to the relative expression level of green fluorescent protein (eGFP).

[0028] Figure 2 It is the yield of sucrose from Bacillus subtilis CGF-P-02 and CGF-P-02-PsrfA-RE3 in Cal18 medium.

[0029] Figure 3 The yield of fengycin by Bacillus subtilis CGF-P-02-PsrfA-RE3 in different culture media.

[0030] Figure 4 The yield of abundant genotype by Bacillus subtilis CGF-P-02-PsrfA-RE3-ΔrapJE in Cal18 optimized medium. DETAILED DESCRIPTION

[0031] The present invention will be further described below in conjunction with specific examples. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, without departing from the essence and scope of the present invention, various changes or modifications of the material composition and dosage in these embodiments also belong to the protection scope of the present invention. The present invention will be described in more detail below by specific examples.

[0032] The original Bacillus subtilis 168 was purchased in 2021.5.: https: / / www.atcc.org / .

[0033] The Bacillus subtilis CGF-P-02 used in the present invention is also called a Bacillus subtilis strain with enhanced proline transport, named CGF-P-02 strain; the construction of this strain refers to the invention patent applied for by Tianjin University in April 2023, the patent application number is: 202310455512.6, and the name of the invention is: A method for producing fengycin by co-culturing recombinant Bacillus subtilis CGF-PG and recombinant Corynebacterium glutamicum.

[0034] Example 1: Construction of promoter P srfA Mutant P srfA-RE3

[0035] 1. Construction of plasmid pHT-PsrfA-eGFP (plasmid pHT is the abbreviation of plasmid pHT43, a commercial product)

[0036] The green fluorescent protein (eGFP) gene (SEQ ID NO. 6) was synthesized by Beijing Qingke Company.

[0037] The eGFP fragment was recombined with the linearized vector pHT double-digested with BamH I and Xba I using 2×MultiF Seamless Assembly Mix reagent (Table 1). The prepared system was placed in a 50°C metal bath for 60 min, and then ice-bathed for 5 min before being transformed into Escherichia coli DH5α competent cells by chemical transformation. Positive transformants were screened, plasmid strains were preserved, and plasmid pHT-eGFP was extracted.

[0038] Table 1 2×MultiF Seamless Assembly Mix seamless cloning reaction system

[0039]

[0040] Using the CGF-P-02 genome as a template, PsrfA-F (pHT) (SEQ ID NO.7) / PsrfA-R (pHT) (SEQ ID NO.8) as upstream and downstream primers, and Phanta Max Super-Fidelity DNA Polymerase, PCR was performed to amplify the ComQXPA system targeting promoter P of Bacillus subtilis strain. srfA (SEQ ID NO.1);

[0041] Using plasmid pHT-eGFP as template and pHT-F (SEQ ID NO.9) / pHT-R (SEQ ID NO.10) as upstream and downstream primers, PCR reaction was performed to amplify linearized vector fragment 1. The promoter P was recombined using seamless cloning technology. srfA Fragment and linearized vector fragment 1 were used to transform Escherichia coli DH5α competent cells, screen positive transformants, preserve plasmid strains, and extract plasmid pHT-PsrfA-eGFP.

[0042] The PCR reaction system is shown in Table 2.

[0043] Table 2 Phanta enzyme PCR reaction system

[0044]

[0045] The PCR reaction program settings are shown in Table 3.

[0046] Table 3 Phanta enzyme PCR reaction program

[0047]

[0048] 2. Construction of Plasmid pHT-PsrfA-RE3

[0049] The plasmid construction method is the same as step 1. Using the plasmid pHT-PsrfA-eGFP as a template and PsrfA-RE3-F (SEQ ID NO.11) / PsrfA-R (pHT) (SEQ ID NO.8) as upstream and downstream primers, PCR amplification was performed to obtain the mutated promoter P srfA-RE3 (SEQ ID NO.2) (Targeting the ComQXPA system of Bacillus subtilis strain to promoter P srfA The sequence of the ComA protein binding site RE3 region mutated from TTTCAC to TTGCGG);

[0050] Using plasmid pHT-eGFP as template and pHT-F (SEQ ID NO.9) / pHT-RE3 / 4-R (SEQ ID NO.12) as upstream and downstream primers, PCR reaction was performed to amplify linearized vector fragment 2. The promoter P after mutation was recombined using seamless cloning technology. srfA-RE3 The fragment and linearized vector fragment 2 were transformed into E. coli DH5α competent cells, positive transformants were screened, and plasmid pHT-PsrfA-RE3 was extracted. Sanger sequencing of the plasmid confirmed the promoter P srfA-RE3 sequence.

[0051] 3. Promoter P srfA and its mutated promoter P srfA-RE3 Expression intensity determination

[0052] (1) Plasmids pHT-PsrfA-eGFP and pHT-PsrfA-RE3 were transformed into Bacillus subtilis CGF-P-02 respectively:

[0053] Pick a single colony of activated Bacillus subtilis CGF-P-02 into 5 mL GMI medium (Table 4), and culture it at 37 ° C, 220 rpm for 18 h; draw 500 μL of bacterial solution and add it to fresh 4.5 mL GMI, and culture it at 37 ° C, 220 rpm for 4.5 h; draw 750 μL of bacterial solution and add it to fresh 4.25 mL GMII (Table 5), and culture it at 37 ° C, 220 rpm for 1.5 h to obtain the final Bacillus subtilis competent cells.

[0054] Pipette 1 mL of GMII culture medium, add about 500 ng of plasmid, incubate at 37°C for 45 min, place in a 30°C shaker, 200 rpm, incubate for 2.5 h, centrifuge at 5000 rpm for 3 min, and spread on a 10 μg / mL chloramphenicol resistance plate to screen for positive transformants.

[0055] Table 4 GMI medium

[0056]

[0057] Table 5 GMII culture medium

[0058]

[0059] 10×Spizizen basic salt solution: 12.0g / L sodium citrate, 183.0g / L K2HPO4, 60.0g / LKH2PO4, 20.0g / L (NH4)2SO4. (NH4)2SO4 must be prepared into a mother solution and sterilized by filtering with a 0.22μm sterile syringe filter, and the remaining ingredients are prepared into a mother solution and sterilized at 121℃ for 20min.

[0060] (2) Detection of green fluorescent protein: A single colony of the positive transformant obtained in step 3 of this example was picked up and placed in 10 mL of LB medium (containing 10 μg / ml chloramphenicol) and cultured at 37°C, 200 rpm for 12 h. The obtained bacterial solution was measured by OD 600 = 0.2 was inoculated into fresh 10 mL LB medium (containing 10 ug / ml chloramphenicol), cultured at 37°C, 200 rpm for 12 h, and the biomass (OD 600 ). Take 1 mL of bacterial solution and centrifuge at 8000 rpm for 2 min, resuspend in 1 mL of distilled water, place in a black 96-well plate, and use a microplate reader (Varioskan LUX) to detect the fluorescence intensity, with an excitation wavelength of 485 nm and an emission wavelength of 533 nm. The final fluorescence intensity is expressed as the fluorescence intensity per unit bacterial biomass (RFU / OD 600 ) indicates. Figure 1 As shown. After mutation, promoter P srfA-RE3 The expression intensity has increased by 5 times.

[0061] Example 2: Construction of a high-yield fengyuanxin Bacillus subtilis strain CGF-P-02-PsrfA-RE3

[0062] 1. Construction of plasmids pJOE-PppsA-PPS, pJOE-PsrfA-RE3-PPS

[0063] (1) Reference website( CRISPR RGEN Tools(rgenome.net) ) designed pps-sgRNA-F-1 (SEQ ID NO.13) / R-1 (SEQ ID NO.14) primers, annealed and connected to obtain the product pps-sgRNA-1, which was connected with the linearized vector pJOE8999 (commercial product) cut with Bsa I using T4 DNA Ligase, and the enzyme ligation system (Table 6) was placed in a 26°C water bath for 30 min, and then transformed into Escherichia coli DH5α competent cells after an ice bath for 5 min, and positive transformants were screened to extract the plasmid pJOE-PPS-sgRNA-1.

[0064] Table 6 T4 DNA Ligase reaction system

[0065]

[0066] Using the B. subtilis 168 genome as a template, PPS-UF (SEQ ID NO.15) / PPS-UR-1 (SEQ ID NO.16) as the upstream and downstream primers, PCR amplified the PPS upstream homology arm PPS-U-1; using PPS-DF-1 (SEQ ID NO.17) / PPS-DR (SEQ ID NO.18) as the upstream and downstream primers, PCR amplified the PPS downstream homology arm PPS-D-1. The PPS-U-1, PPS-D-1 fragments and the Sfi I-cut linearized vector pJOE-PPS-sgRNA-1 were recombined by seamless cloning, and the system and reaction conditions were the same as step 1 in Example 1. After the reaction of the recombination system was completed, the Escherichia coli DH5α competent cells were transformed, the positive transformants were screened, and the plasmid pJOE-PppsA-PPS was extracted.

[0067] (2) The same method was used to construct plasmid pJOE-PsrfA-RE3-PPS: refer to the website ( CRISPR RGEN Tools (rgenome.net) ) designed PPS-sgRNA-F-2 (SEQ ID NO.19) / PPS-sgRNA-R-2 (SEQ ID NO.20) primers, annealed and connected to obtain the product pps-sgRNA-2, which was ligated with the Bsa I-digested linearized vector pJOE8999 to construct plasmid pJOE-PPS-sgRNA-2.

[0068] Using the B. subtilis 168 genome as a template and PPS-UF (SEQ ID NO.15) / PPS-UR-2 (SEQ ID NO.21) as the upstream and downstream primers, PCR amplified the PPS upstream homology arm PPS-U-2; using PPS-DF-2 (SEQ ID NO.22) / PPS-DR (SEQ ID NO.18) as the upstream and downstream primers, PCR amplified the PPS downstream homology arm PPS-D-2.

[0069] Using plasmid pHT-PsrfA-RE3 as template and PsrfA-F (pJOE) (SEQ ID NO.23) / PsrfA-R (pJOE) (SEQ ID NO.24) as upstream and downstream primers, PCR amplified promoter P srfA-RE3 .

[0070] (3) PPS-U-2, PPS-D-2, and the mutated promoter P obtained by amplification in step 1 (2) of this embodiment srfA-RE3The vector pJOE-PPS-sgRNA-2 was linearized with Sfi I and recombined by seamless cloning. After the reaction of the recombination system was completed, the competent cells of E. coli DH5α were transformed, the positive transformants were screened, and the plasmid pJOE-PsrfA-RE3-PPS was extracted.

[0071] 2. Construction of strain CGF-P-02-PsrfA-RE3

[0072] The preparation method of the competent state of Bacillus subtilis CGF-P-02 is the same as step 3 in Example 1. 1 mL of GMII culture medium was taken, and about 500 ng of plasmid pJOE-PppsA-PPS was added, incubated at 37°C for 45 min, placed in a 30°C shaker, 200 rpm, incubated for 2.5 h, centrifuged at 5000 rpm for 3 min, and coated on a kanamycin resistance plate containing 0.2% mannose and 50 μg / mL to screen positive transformants. The correct transformant was streaked on a non-resistant plate and placed in a 50°C incubator to lose the plasmid. The intermediate strain CGF-P-02-PppsA was obtained. The intermediate strain CGF-P-02-PppsA was prepared in the same way, and the plasmid pJOE-PsrfA-RE3-PPS was transformed to obtain the recombinant strain CGF-P-02-PsrfA-RE3, and Sanger sequencing confirmed the promoter P srfA-RE3 The sequence is correct.

[0073] Using CRISPR / Cas9 technology to clone the promoter of the fengycin biosynthesis gene cluster in Bacillus subtilis CGF-P-02 veg (SEQ ID NO.3) is replaced by P srfA-RE3 , and obtained a high-yield sporeforming Bacillus subtilis strain CGF-P-02-PsrfA-RE3.

[0074] Example 3: Fengyuansu yield of high-yield Fengyuansu Bacillus subtilis strain CGF-P-02-PsrfA-RE3 in Cal18 medium

[0075] 1. Fermentation culture: Pick single colonies of Bacillus subtilis CGF-P-02 and CGF-P-02-PsrfA-RE3 respectively in 10 mL of LB medium and culture at 37°C and 200 rpm for 12 h. 600 =0.2 was inoculated into 50 mL seed culture medium and cultured at 37°C, 200 rpm for 12 h. The seed culture solution was measured by OD 600 =0.2 was inoculated into 80 mL Cal18 medium and cultured at 30°C and 180 rpm for 72 h.

[0076] LB medium: 10 g / L peptone, 5 g / L yeast extract powder, 10 g / L NaCl.

[0077] Seed culture medium: 30 g / L glucose, 5 g / L beef extract, 10 g / L peptone, 5 g / L yeast extract powder, 5 g / L NaCl;

[0078] Cal18 culture medium: 50g / L maltodextrin, 40g / L yeast extract powder, 20g / L Na2HPO4·12H2O, 6.7g / LNa2MoO4·2H2O, 1.3g / L MgSO4·7H2O.

[0079] 2. Fengyuansu detection: The content of Fengyuansu was detected by high performance liquid chromatography. After the fermentation, 1 mL of fermentation liquid was centrifuged at 12000 rpm for 2 min, 400 ul of the obtained supernatant was taken, 400 ul of chromatographic grade methanol was added for dilution, and oscillation was performed to mix. Fengyuansu was analyzed by high performance liquid chromatography using a C18 reverse chromatographic column (4.6 mm × 150 mm, 5 μm) (Agilent Technologies, Santa Clara, CA, USA), and the mobile phase was 50.0% acetonitrile and 50.0% purified water (containing 1.0% trifluoroacetic acid). Determination conditions: UV detection wavelength was 210 nm, column temperature was 30 ° C, total flow rate was 0.8 mL / min, injection volume was 10.0 μL, and detection time was 45 min.

[0080] like Figure 2 The production of fengycin by the Bacillus subtilis strain CGF-P-02-PsrfA-RE3 in Cal18 medium was 1832 mg / L, which was 3.74 times higher than that of the strain CGF-P-02.

[0081] Example 4: Production of fengycin by Bacillus subtilis CGF-P-02-PsrfA-RE3 in different fermentation media

[0082] 1. Fermentation culture: The fermentation seed culture process of strain CGF-P-02-PsrfA-RE3 was the same as step 1 in Example 3. The seed culture solution was heated to OD 600 =0.2 were inoculated into 80 mL Cal18 and 80 mL Cal18 optimized medium respectively. Cal18 medium was the same as step 1 in Example 3.

[0083] Cal18 optimized culture medium: 50g / L maltodextrin, 40g / L yeast extract powder, 10g / L peptone, 20g / LNa2HPO4·12H2O, 6.7g / L Na2MoO4·2H2O, 1.3g / L MgSO4·7H2O.

[0084] 2. Fengyuansu detection: the same as step 2 in Example 3. Figure 3As shown, the fengycin production of strain CGF-P-02-PsrfA-RE3 in Cal18 optimized medium was increased to 2850 mg / L.

[0085] Example 5: Construction of Bacillus subtilis CGF-P-02-PsrfA-RE3-ΔrapJE

[0086] 1. Transcriptomic analysis of the genomes of strains CGF-P-02 and CGF-P-02-PsrfA-RE3

[0087] (1) Sample preparation and analysis: The strains CGF-P-02 and CGF-P-02-PsrfA-RE3 were cultured in Cal18 medium for 12 h, 36 h, and 60 h. 10 mL of fermentation broth was centrifuged at 5000 rpm for 10 min at 4 °C, the precipitate was washed with 10 mL of PBS buffer and centrifuged again, the supernatant was discarded, the strains in the fermentation broth were collected, and frozen in liquid nitrogen for 30 min. Transcriptome analysis was performed at Biozeron (Shanghai, China) using the Illumina PE150 sequencing platform.

[0088] (2) Transcriptome analysis: According to the results of transcriptional analysis, the expression levels of genes involved in the quorum sensing regulatory network of the two strains were compared. Compared with strain CGF-P-02, the expression of RapJ and RapE phosphatases in strain CGF-P-02-PsrfA-RE3 at 12h was downregulated.

[0089] 2. Construction of plasmids pJOE-ΔrapJ, pJOE-ΔrapE

[0090] (1) Same as step 1 in Example 2. Reference website ( CRISPR RGEN Tools(rgenome.net) ) designed ΔrapJ-sgRNA-F (SEQ ID NO.25) / ΔrapJ-sgRNA-R (SEQ ID NO.26) primers, annealed and connected to obtain the product ΔrapJ-sgRNA, which was ligated with the Bsa I-digested linearized vector pJOE8999 to construct the plasmid pJOE-ΔrapJ-sgRNA.

[0091] Using the B. subtilis 168 genome as a template and ΔrapJ-UF (SEQ ID NO.27) / ΔrapJ-UR (SEQ ID NO.28) as the upstream and downstream primers, PCR amplified the upstream homology arm ΔrapJ-U of the rapJ gene (SEQ ID NO.4);

[0092] Using ΔrapJ-DF (SEQ ID NO.29) / ΔrapJ-DR (SEQ ID NO.30) as upstream and downstream primers, PCR amplified the downstream homology arm ΔrapJ-D of the rapJ gene. The ΔrapJ-U and ΔrapJ-D fragments were recombined with the Sfi I-cut linearized vector pJOE-ΔrapJ-sgRNA by seamless cloning. After the completion of the recombination system reaction, the competent cells of Escherichia coli DH5α were transformed, the positive transformants were screened, and the plasmid pJOE-ΔrapJ was extracted.

[0093] (2) Same as step 1 in Example 2. Reference website ( CRISPRRGENTools (rgenome.net) ) designed ΔrapE-sgRNA-F (SEQ ID NO.31) / ΔrapE-sgRNA-R (SEQ ID NO.32) primers, annealed and connected to obtain the product ΔrapE-sgRNA, which was ligated with the Bsa I-digested linearized vector pJOE8999 to construct the plasmid pJOE-ΔrapE-sgRNA.

[0094] Using the B. subtilis 168 genome as a template, ΔrapE-UF (SEQ ID NO.33) / ΔrapE-UR (SEQ ID NO.34) as upstream and downstream primers, PCR amplified the upstream homology arm ΔrapE-U of the rapE gene (SEQ ID NO.5); ΔrapE-DF (SEQ ID NO.35) / ΔrapE-DR (SEQ ID NO.36) as upstream and downstream primers, PCR amplified the downstream homology arm ΔrapE-D of the rapE gene. The ΔrapE-U, ΔrapE-D fragments were recombined with the Sfi I-cut linearized vector pJOE-ΔrapE-sgRNA by seamless cloning. After the completion of the recombination system reaction, the Escherichia coli DH5α competent cells were transformed, the positive transformants were screened, and the plasmid pJOE-ΔrapE was extracted.

[0095] 3. Construction of a high-yield fengin Bacillus subtilis strain CGF-P-02-PsrfA-RE3-ΔrapJE

[0096] The same as step 2 in Example 2. Plasmid pJOE-ΔrapJ was used to transform strain CGF-P-02-PsrfA-RE3 to obtain intermediate strain CGF-P-02-PsrfA-RE3-ΔrapJ. Plasmid pJOE-ΔrapE was used to transform intermediate strain CGF-P-02-PsrfA-RE3-ΔrapJ to obtain recombinant strain CGF-P-02-PsrfA-RE3-ΔrapJE.

[0097] Example 6: Production of fengycin by Bacillus subtilis CGF-P-02-PsrfA-RE3-ΔrapJE in Cal18 optimized medium

[0098] 1. Fermentation culture: The fermentation seed culture process of strains CGF-P-02-PsrfA-RE3 and CGF-P-02-PsrfA-RE3-ΔrapJE was the same as step 1 in Example 3. The seed culture solution was respectively heated to OD 600 =0.2 inoculated into 80 mL Cal18 optimized medium.

[0099] 2. Fengyuansu detection: the same as step 2 in Example 3. Figure 4 As shown, compared with strain CGF-P-02-PsrfA-RE3, the fengycin production of strain CGF-P-02-PsrfA-RE3-ΔrapJE in Cal18 optimized medium was increased to 3190 mg / L.

Claims

1. A method for constructing a high-yield fengycin-producing Bacillus subtilis strain, characterized in that The steps include: 1) Targeting the ComQXPA system of Bacillus subtilis strains to promoter P srfA The sequence of the ComA protein binding site RE3 region of the mutated promoter was mutated from TTTCAC to TTGCGG. srfA-RE3 ; 2) Using CRISPR / Cas9 technology, the promoter of the fengycin synthesis gene cluster in Bacillus subtilis CGF-P-02 was transformed into veg Replace with P srfA-RE3 , a high-yield fengin Bacillus subtilis strain CGF-P-02-PsrfA-RE3 was obtained; The promoter P srfA The nucleotide sequence is shown in SEQ ID NO.1; The mutated promoter P srfA-RE3 The nucleotide sequence is shown in SEQ ID NO.2; The promoter P veg The nucleotide sequence is shown in SEQ ID NO.

3.

2. A high-yield sporeforming Bacillus subtilis strain CGF-P-02-PsrfA-RE3 constructed by the method of claim 1.

3. A method of producing Fengyuansu by fermentation of a high-yield Fengyuansu Bacillus subtilis strain CGF-P-02-PsrfA-RE3 according to claim 2 use.

4. A method for constructing a high-yield fengycin-producing Bacillus subtilis strain, characterized in that The method comprises the following steps: knocking out the rapJ gene and the rapE gene in a high-yield fengycin Bacillus subtilis strain CGF-P-02-PsrfA-RE3 to obtain a high-yield fengycin Bacillus subtilis strain CGF-P-02-PsrfA-RE3-ΔrapJE; The nucleotide sequence of the rapJ gene is shown in SEQ ID NO.4; The nucleotide sequence of the rapE gene is shown in SEQ ID NO.

5.

5. A high-yield fengin Bacillus subtilis strain CGF-P-02-PsrfA-RE3- constructed by the method of claim 4 ΔrapJE.

6. Use of the high-yield Fengyuansu Bacillus subtilis strain CGF-P-02-PsrfA-RE3-ΔrapJE of claim 5 in fermenting Fengyuansu.

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