Capsaicin-tolerant bacillus subtilis strain with high yield of abinogen and construction method of capsaicin-tolerant bacillus subtilis strain

Abstract

The invention discloses a capsaicin-tolerant bacillus subtilis strain with high yield of abinogen and a construction method thereof, and the construction method comprises the following steps: replacing an lmrA gene of a bacillus subtilis strain CGF-P-02 with a P43 promoter by using a CRISPR / Cas9 technology to obtain a capsaicin-tolerant bacillus subtilis strain BS02 with high yield of abinogen; and replacing the ytrA gene of the bacillus subtilis strain BS02 with a second P43 promoter to obtain the capsaicin-tolerant bacillus subtilis strain BS03 with high yield of abinogen. The capsaicin-tolerant bacillus subtilis strain with high yield of the abinogen is high in fermentation and abinogen yield, can tolerate 84mg / L capsaicin, and is good in strain stability and high in repeatability. The used obtaining method is simple and easy to operate.

Description

Technical Field

[0001] The invention belongs to the field of Bacillus subtilis genetic engineering, and particularly relates to a capsaicin-tolerant Bacillus subtilis strain with high capsaicin production, a construction method and uses. Background Art

[0002] Fengyuansu is a lipopeptide synthesized by Bacillus subtilis through non-ribosomal peptide synthetase (NRPS), which exhibits multiple biological activities, including antibacterial and antifungal activities; at the same time, it is also safe, non-toxic, and easily degradable, and is a new green antibacterial agent. However, the low yield and high production cost of Fengyuansu in the existing technology limit its application.

[0003] Low-quality biomass, such as kitchen waste, has high organic content, rich nutrition, low price, and is a potential substrate for the synthesis of fengyuan. The successful use of kitchen waste in the synthesis of fengyuan has broad economic benefits. It is worth noting that capsaicinoids (hereinafter referred to as "capsaicin") are widely present in kitchen waste. Capsaicin includes capsaicin (CPC) and dihydrocapsaicin (DCPC), the ratio of which is 2:1, accounting for more than 90% of the total amount of capsaicin. Due to its strong irritation and broad-spectrum antibacterial effect, it can effectively inhibit the growth and metabolism of various pathogenic microorganisms such as Staphylococcus aureus, Escherichia coli, Bacillus subtilis, yeast and mold. Therefore, capsaicin is one of the main unfavorable factors for Bacillus subtilis to synthesize fengyuan using kitchen waste. Under capsaicin stress, Bacillus subtilis will make a series of stress responses. Bacillus subtilis regulates gene expression to maintain protein stability and cell membrane fluidity by mobilizing global regulatory factors; the compatible solute regulatory system balances the osmotic pressure difference between the inside and outside of the cell; the efflux pump (EP) system, such as ABC transporters, effluxes harmful substances to reduce the cytotoxicity of harmful substances and improve bacterial tolerance. Therefore, there is an urgent need for genetically engineered strains that can tolerate capsaicin and efficiently synthesize lipopeptide products. Summary of the invention

[0004] The purpose of the present invention is to overcome the deficiencies of the prior art and provide a capsaicin-tolerant Bacillus subtilis strain with high yield of capsaicin.

[0005] The second object of the present invention is to provide a method for constructing the capsaicin-tolerant Bacillus subtilis strain with high capsaicin production.

[0006] The third object of the present invention is to provide the use of the capsaicin-tolerant Bacillus subtilis strain with high capsaicin production.

[0007] The fourth object of the present invention is to provide a second capsaicin-tolerant Bacillus subtilis strain with high capsaicin production.

[0008] The fifth object of the present invention is to provide a method for constructing a second capsaicin-tolerant Bacillus subtilis strain with high capsaicin production.

[0009] The sixth object of the present invention is to provide a second use of a capsaicin-tolerant Bacillus subtilis strain with high capsaicin production.

[0010] The technical solution of the present invention is summarized as follows:

[0011] The method for constructing a capsaicin-tolerant Bacillus subtilis strain with high yield of fengycin comprises the following steps: using CRISPR / Cas9 technology, using a P43 promoter to replace the lmrA gene of the Bacillus subtilis strain CGF-P-02, achieving enhanced expression of the lmrB gene, and obtaining a capsaicin-tolerant Bacillus subtilis strain with high yield of fengycin, named BS02;

[0012] The nucleotide sequence of the P43 promoter is shown in SEQ ID NO.12;

[0013] The nucleotide sequence of the lmrA gene is shown in SEQ ID NO.5;

[0014] The nucleotide sequence of the lmrB gene is shown in SEQ ID NO.13.

[0015] The capsaicin-tolerant Bacillus subtilis strain BS02 with high capsaicin production was constructed using the above method.

[0016] The use of strain BS02 in fermentation to produce Fengyuansu.

[0017] The culture medium used for fermentation is preferably kitchen waste culture medium, and other culture medium suitable for the production of Bacillus subtilis strains can also be selected.

[0018] The second method for constructing a capsaicin-tolerant Bacillus subtilis strain with high yield of fengycin comprises the following steps: using CRISPR / Cas9 technology, using a second P43 promoter to replace the ytrA gene of strain BS02, thereby enhancing the expression of the ytrBCDEF gene, and obtaining a capsaicin-tolerant Bacillus subtilis strain with high yield of fengycin, which is named BS03;

[0019] The nucleotide sequence of the second P43 promoter is shown in SEQ ID NO.25;

[0020] The nucleotide sequence of the ytrA gene is shown in SEQ ID NO.18;

[0021] The nucleotide sequence of the ytrBCDEF gene is shown in SEQ ID NO.26;

[0022] The capsaicin-tolerant Bacillus subtilis strain BS03 with high capsaicin production constructed by the second method mentioned above.

[0023] The use of strain BS03 in fermentation to produce Fengyuansu.

[0024] The culture medium used for fermentation is preferably kitchen waste culture medium, and other culture medium suitable for the production of Bacillus subtilis strains can also be selected.

[0025] Advantages of the present invention:

[0026] The capsaicin-tolerant Bacillus subtilis strain with high yield of fengyuanin constructed by the present invention has high yield of fengyuanin during fermentation, can tolerate 84 mg / L capsaicin, has good strain stability and strong repeatability, and the acquisition method used is simple and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The capsaicin production of Bacillus subtilis CGF-P-02, BS02 and BS03 after fermentation for 72 hours in Cal-18 medium containing capsaicin.

[0028] Figure 2 This is the yield of fengycin produced by Bacillus subtilis CGF-P-02, BS02 and BS03 in kitchen waste medium after fermentation for 120 hours. DETAILED DESCRIPTION

[0029] The invention enhances the expression of ABC transporter lmrB gene and ytrBCDEF gene in Bacillus subtilis, thereby improving the tolerance of Bacillus subtilis to capsaicin and significantly improving the capsaicin yield of Bacillus subtilis (cultured in a culture medium containing capsaicin).

[0030] The present invention will be further described below in conjunction with specific embodiments:

[0031] The Bacillus subtilis CGF-P-02 used in the present invention is a laboratory-constructed Bacillus subtilis strain that enhances proline transport. See the patent application of Tianjin University, "Method for producing Fengyuansu by co-culturing recombinant Bacillus subtilis CGF-PG and recombinant Corynebacterium glutamicum" CN202310455512.6[P]:2024-10-25.)

[0032] Preparation of GM I and GMII culture medium.

[0033] 10×Spizizen basic salt solution: 12.0g / L sodium citrate, 183.0g / L K2HPO4, 60.0g / L KH2PO4, 20.0g / L (NH4)2SO4. (NH4)2SO4 must be prepared into a mother solution and sterilized by filtration using a 0.22μm sterile syringe filter, and the remaining ingredients must be prepared into a mother solution and sterilized at 121℃ for 20min.

[0034] GM I medium (100.0 mL): 10.0 mL 10× Spizizen basic salt solution, 2.0 mL 2% casein hydrolyzate stock solution, 2.0 mL 5% yeast extract stock solution, 2.0 mL 40% glucose stock solution, 1.0 mL 0.5% L-tryptophan stock solution, 100.0 μL 20% MgSO4·7H2O stock solution, and finally add 82.9 mL sterile water.

[0035] GMⅡ medium (100.0mL): 10.0mL 10×Spizizen basic salt solution, 1.0mL 2.0% casein hydrolyzate stock solution, 2.0mL 40.0% glucose stock solution, 800.0μL 20.0% MgSO4·7H2O stock solution, and finally add 86.2mL sterile water.

[0036] Example 1: Construction of a capsaicin-tolerant Bacillus subtilis strain BS02 with high capsaicin production, comprising the following steps:

[0037] 1. Construction of pJOE8999a-P43-lmrB plasmid

[0038] (1) Design lmrA-sgRNA-S (SEQ ID NO. 1) and lmrA-sgRNA-A (SEQ ID NO. 2) primers according to the website (CRISPRRGEN Tools (rgenome.net / cas-designer / )). Annealing and connecting lmrA-sgRNA-S / A to obtain the ligation product lmrA-sgRNA, using seamless cloning enzyme to connect lmrA-sgRNA to pJOE8999a plasmid (Hou ZJ, Cao CY, Gao GR, et al. Enhanced Iturin A Production of Engineered Bacillus amyloliquefaciens by Knockout of Endogenous Plasmid and RapPhosphatase Genes [J]. Journal of Agricultural and Food Chemistry, 2024.) after Bsa I linearization to obtain pJOE8999a-lmrA-sgRNA plasmid, transformed into Escherichia coli DH5α, and coated on 25 μg / mL kanamycin resistance plates to screen positive transformants. The pJOE8999a-lmrA-sgRNA plasmid was extracted using a plasmid mini-extraction kit and set aside.

[0039] Using the Bacillus subtilis CGF-P-02 genome as a template, the upstream homologous arm lmrA-U (SEQ ID NO.6) of the lmrA gene (SEQ ID NO.5) was PCR amplified using primers lmrA-UF (SEQ ID NO.3) and lmrA-UR (SEQ ID NO.4); the downstream homologous arm lmrA-D (SEQ ID NO.9) of the lmrA gene was PCR amplified using primers lmrA-DF (SEQ ID NO.7) and lmrA-DR (SEQ ID NO.8); and the P43 promoter (SEQ ID NO.12) was PCR amplified using primers lmrB-P43F (SEQ ID NO.10) and lmrB-P43R (SEQ ID NO.11).

[0040] Overlap PCR was used to sequentially connect lmrA-U, P43 promoter and lmrA-D to obtain lmrAU-P43-lmrAD sequence. lmrAU-P43-lmrAD was connected to the pJOE8999a-lmrA-sgRNA plasmid linearized with Sfi I to obtain the pJOE8999a-P43-lmrB plasmid, which was transformed into Escherichia coli DH5α and coated on a 25 μg / mL kanamycin resistance plate to screen positive transformants. The pJOE8999a-P43-lmrB plasmid was extracted using a plasmid mini-extraction kit and set aside.

[0041] (2) PCR reaction system. The total system is 50 μL. Phanta Max Super Fidelity DNA Polymerase 1 μL, 2× Phanta Max Buffer 25 μL, dNTP Mix 1 μL, upstream primer 2 μL, downstream primer 2 μL, template 100 ng, sterile water to 50 μL; PCR reaction program: 95℃ 3 min, 95℃ 15 s, 55℃ 15 s, 72℃ 60 s / kb, 72℃ 5 min, 16℃ insulation, number of cycles: 35.

[0042] (3) Overlap PCR reaction system. The total system is 50 μL. Phanta Max Super Fidelity DNA Polymerase 1 μL, 2× Phanta Max Buffer 25 μL, dNTP Mix 1 μL, upstream primer 2 μL, downstream primer 2 μL, template 1 (lmrA-U) 100 ng, template 2 (P43 promoter) 100 ng, template 3 (lmrA-D) 100 ng, and sterile water to make up to 50 μL; PCR reaction program: 95℃ 3 min, 95℃ 15 s, 60℃ 15 s, 72℃ 60 s / kb, 95℃ 15 s, 55℃ 15 s, 72℃ 60 s / kb, 72℃ 5 min, 16℃ insulation, cycle number 1:10 (0.5℃ drop for each cycle), cycle number 2:25.

[0043] 2. Construction of Bacillus subtilis BS02

[0044] (1) Preparation of Bacillus subtilis competent cells. Pick a single colony of activated Bacillus subtilis CGF-P-02 and place it in 5 mL of GM I medium, culture it at 37°C, 220 rpm for 18 h, take 500 μL of the bacterial solution and add it to fresh 4.5 mL of GM I, culture it at 37°C, 220 rpm for 4.5 h, take 750 μL of the bacterial solution and add it to fresh 4.25 mL of GMII, culture it at 37°C, 220 rpm for 1.5 h, and obtain the final Bacillus subtilis competent cells.

[0045] (2) The pJOE8999a-P43-lmrB plasmid in step 1 was transformed into Bacillus subtilis competent cells by chemical transformation, 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 25 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 and finally obtain Bacillus subtilis BS02, which achieved the use of the P43 promoter to replace the lmrA gene, the inhibitor of the lmrB gene, and enhance the expression of the lmrB gene (SEQ ID NO.13).

[0046] Example 2: Construction of capsaicin-tolerant Bacillus subtilis strain BS03 with high capsaicin production, comprising the following steps:

[0047] 1. Construction of pJOE8999a-second P43-ytrBCEDF plasmid

[0048] (1) Refer to the website (CRISPRRGEN Tools (rgenome.net / cas-designer / )) to design ytrA-sgRNA-S (SEQ ID NO.14) and ytrA-sgRNA-A (SEQ ID NO.15) primers. Annealing and connecting ytrA-sgRNA-S / A to obtain the ligation product ytrA-sgRNA, use seamless cloning enzyme to connect ytrA-sgRNA to Bsa I linearized pJOE8999a plasmid to obtain pJOE8999a-ytrA-sgRNA plasmid, transform into Escherichia coli DH5α, and spread on 25 μg / mL kanamycin resistance plate to screen positive transformants. Use plasmid mini-extraction kit to extract pJOE8999a-ytrA-sgRNA plasmid for later use.

[0049] Using the Bacillus subtilis BS02 genome as a template, the upstream homologous arm ytrA-U (SEQ ID NO.19) of the ytrA gene (SEQ ID NO.18) was PCR amplified using primers ytrA-UF (SEQ ID NO.16) and ytrA-UR (SEQ ID NO.17); the downstream homologous arm ytrA-D (SEQ ID NO.22) of the ytrA gene was PCR amplified using primers ytrA-DF (SEQ ID NO.20) and ytrA-DR (SEQ ID NO.21); the second P43 promoter (SEQ ID NO.25) was PCR amplified using primers ytrBCDEF-second P43F (SEQ ID NO.23) and ytrBCDEF-second P43R (SEQ ID NO.24).

[0050] Overlap PCR sequentially connected ytrA-U, the second P43 promoter and ytrA-D to obtain the ytrAU-second P43-ytrAD sequence. The ytrAU-second P43-ytrAD was connected to the pJOE8999a-ytrA-sgRNA plasmid linearized with Sfi I to obtain the pJOE8999a-second P43-ytrBCDEF plasmid, which was transformed into Escherichia coli DH5α and coated on a 25 μg / mL kanamycin resistance plate to screen positive transformants. The pJOE8999a-second P43-ytrBCDEF plasmid was extracted using a plasmid mini-extraction kit and set aside.

[0051] (2) PCR reaction system. The total system is 50 μL. Phanta Max Super Fidelity DNA Polymerase 1 μL, 2× Phanta Max Buffer 25 μL, dNTP Mix 1 μL, upstream primer 2 μL, downstream primer 2 μL, template 100 ng, sterile water to 50 μL; PCR reaction program: 95℃ 3 min, 95℃ 15 s, 55℃ 15 s, 72℃ 60 s / kb, 72℃ 5 min, 16℃ insulation, number of cycles: 35.

[0052] (3) Overlap PCR reaction system. The total system is 50 μL. Phanta Max Super Fidelity DNA Polymerase 1 μL, 2× Phanta Max Buffer 25 μL, dNTP Mix 1 μL, upstream primer 2 μL, downstream primer 2 μL, template 1 (ytrA-U) 100 ng, template 2 (second P43 promoter) 100 ng, template 3 (ytrA-D) 100 ng, and sterile water to make up to 50 μL; PCR reaction program: 95℃ 3 min, 95℃ 15 s, 60℃ 15 s, 72℃ 60 s / kb, 95℃

[0053] 15s, 55℃15s, 72℃60s / kb, 72℃5min, keep warm at 16℃, cycle number 1:10 (0.5℃ decrease in each cycle), cycle number 2:25.

[0054] 2. Construction of Bacillus subtilis BS03

[0055] (1) Prepare Bacillus subtilis competent cells according to the method of step 2 of Example 1.

[0056] (2) The pJOE8999a-second P43-ytrBCDEG plasmid in step 1 was transformed into the newly prepared Bacillus subtilis competent cells by chemical transformation, 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 25 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 and finally obtain Bacillus subtilis BS02, realizing the use of the second P43 promoter to replace the ytrA gene, the inhibitor of the ytrBCDEF gene, and strengthen the expression of the ytrBCDEF gene (SEQ ID NO. 26).

[0057] Example 3: Fermentation of Bacillus subtilis CGF-P-02, BS02 and BS03 in Cal-18 medium containing capsaicin comprises the following steps:

[0058] (1) Single colonies of Bacillus subtilis GFP-PG-02, BS02 and BS03 were picked and placed in 10 mL of LB liquid culture medium and cultured at 37°C and 200 rpm for 12 h.

[0059] LB liquid medium components: NaCl 10g / L, tryptone 10g / L, yeast extract powder 5g / L, the balance is distilled water, steam sterilized at 115℃ for 15min;

[0060] (2) The bacterial solution obtained in step (1) was quantified by OD 600 =0.2 was inoculated into 50 mL Cal-18 medium and cultured at 37°C, 200 rpm for 12 h.

[0061] Cal-18 medium components: maltodextrin 50 g / L, yeast extract powder 40 g / L, MgSO4·7H2O 1.3 g / L, Na2HPO4·12H2O 20.0 g / L, Na2MoO4·2H2O 6.7 g / L, pH 7.0.

[0062] Capsaicin was added to the Cal-18 medium to a final concentration of 75 mg / L to obtain a Cal-18 medium containing capsaicin.

[0063] (3) The bacterial solution obtained in step (2) was quantified by OD 600 =0.2 was inoculated into Cal-18 medium containing capsaicin and cultured at 30°C and 160rpm for 72h.

[0064] (4) Extraction of fengyrin. After the fermentation is completed, 40 mL of the fermentation liquid is centrifuged, acid precipitated, vacuum freeze-dried, and extracted with methanol. (5) Detection of fengyrin. The content of fengyrin is detected by high performance liquid chromatography. A C18 reverse phase column (ZORBAX Eclipse XDB-C18, Agilent, 4.6 mm × 150 mm) is used, the mobile phase is 50% acetonitrile and 50% pure water containing 1% trifluoroacetic acid, the detection wavelength is 210 nm, and the column oven temperature is 30 ° C.

[0065] (6) Figure 1 As shown, the capsaicin production of Bacillus subtilis GFP-PG-02, BS02 and BS03 in Cal-18 medium containing capsaicin was 118.21 mg / L, 836.44 mg / L and 580.48 mg / L, respectively.

[0066] Example 4: Bacillus subtilis CGF-P-02, BS02 and BS03 were cultured in a kitchen waste medium (capsaicin content of 84.38 mg / L), comprising the following steps:

[0067] (1) Single colonies of Bacillus subtilis GFP-PG-02, BS02 and BS03 were picked and placed in 10 mL of LB liquid culture medium and cultured at 37°C and 200 rpm for 12 h.

[0068] (2) The bacterial solution obtained in step (1) was quantified by OD 600 =0.2 was inoculated into 50 mL Cal-18 medium and cultured at 37°C, 200 rpm for 12 h.

[0069] (3) The bacterial solution obtained in step (2) was quantified by OD 600 =0.2 was inoculated into the kitchen waste culture medium and cultured at 30°C and 160rpm for 120h.

[0070] The components of the kitchen waste culture medium: maltodextrin 30 g / L, yeast extract powder 15 g / L, tryptone 10 g / L, kitchen waste (the first student canteen of Beiyang Campus of Tianjin University) 200 g / L, MgSO4·7H2O 1.0 g / L, Na2HPO4·12H2O 2.0 g / L, Na2MoO4·2H2O 2.0 g / L, pH 6.4.

[0071] (4) Determination of capsaicin content in food waste culture medium. Capsaicin in food waste culture medium was extracted by solid phase extraction (SPE).

[0072] The kitchen waste culture medium was mixed with n-hexane in a volume ratio of 1:1 and shaken for 30 minutes, and the upper organic phase was discarded for degreasing. The lower aqueous phase was mixed with ethyl acetate in a volume ratio of 1:1 and shaken for 30 minutes to extract capsaicin. The upper organic phase was passed through a well-balanced silica SPE column to adsorb capsaicin. Finally, the sample of capsaicin in the kitchen waste culture medium was obtained by methanol elution. The capsaicin content was detected by high performance liquid chromatography. A C18 reverse phase column (ZORBAX SB-C18, Agilent, 4.6mm×150mm) was used, and the mobile phase was 80% methanol and 20% pure water. The UV detection wavelength, flow rate and column temperature were 280nm, 0.8mL / min and 30℃, respectively. The content of capsaicin in the kitchen waste culture medium was 84.38mg / L.

[0073] (5) Extraction of Fengyuansu. After the fermentation in step (3) is completed, 40 mL of the fermentation liquid is centrifuged, acid precipitated, vacuum freeze-dried, and extracted with methanol.

[0074] (7) Detection of fengyuan: The content of fengyuan was detected by high performance liquid chromatography.

[0075] (8) Figure 2 As shown, the fengycin production of Bacillus subtilis GFP-PG-02, BS02 and BS03 cultured in kitchen waste medium were 1048.79 mg / L, 2597.35 mg / L and 2072.08 mg / L, respectively.

[0076] Experiments have shown that culture media suitable for the production of Bacillus subtilis strains can be used for the fermentation culture of strain BS02 and strain BS03.

Claims

1. A method for constructing a capsaicin-tolerant Bacillus subtilis strain with high capsaicin production, characterized in that The method comprises the following steps: using CRISPR / Cas9 technology, using P43 promoter to replace lmrA gene of Bacillus subtilis strain CGF-P-02, and obtaining a capsaicin-tolerant Bacillus subtilis strain with high capsaicin production, named BS02; The nucleotide sequence of the P43 promoter is shown in SEQ ID NO.12; The nucleotide sequence of the lmrA gene is shown in SEQ ID NO.

5.

2. The capsaicin-tolerant Bacillus subtilis strain BS02 with high capsaicin production constructed by the method of claim 1.

3. Use of the strain BS02 described in claim 2 in fermenting and producing Fengyuansu.

4. The use according to claim 3, characterized in that The culture medium used for fermentation is kitchen waste culture medium.

5. A method for constructing a capsaicin-tolerant Bacillus subtilis strain with high capsaicin production, characterized in that The method comprises the following steps: using CRISPR / Cas9 technology to replace the ytrA gene of the strain BS02 according to claim 2 with a second P43 promoter, thereby obtaining a capsaicin-tolerant Bacillus subtilis strain with high capsaicin production, which is named BS03; The nucleotide sequence of the second P43 promoter is shown in SEQ ID NO.25; The nucleotide sequence of the ytrA gene is shown in SEQ ID NO.

18.

6. The capsaicin-tolerant Bacillus subtilis strain BS03 with high capsaicin production constructed by the method of claim 5.

7. Use of the strain BS03 described in claim 6 in fermenting and producing Fengyuansu.

8. The use according to claim 7, characterized in that The culture medium used for fermentation is kitchen waste culture medium.

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