Pepper-olefin-tolerant bacillus subtilis strain with high production of formosin and construction method thereof

Abstract

This invention discloses a high-capsaicin-tolerant Bacillus subtilis strain and its construction method. The construction method is as follows: using CRISPR / Cas9 technology, the lmrA gene of Bacillus subtilis strain CGF-P-02 is replaced with a P43 promoter to obtain the high-capsaicin-tolerant Bacillus subtilis strain BS02; a second P43 promoter is used to replace the ytrA gene of Bacillus subtilis strain BS02 to obtain the high-capsaicin-tolerant Bacillus subtilis strain BS03. The high-capsaicin-tolerant Bacillus subtilis strain constructed in this invention exhibits high capsaicin yield during fermentation, can tolerate 84 mg / L capsaicin, and shows good stability and reproducibility. The acquisition method is simple and easy to operate.

Description

Technical Field

[0001] This invention belongs to the field of Bacillus subtilis genetic engineering, specifically involving a capsaicin-tolerant Bacillus subtilis strain with high capsaicin production, its construction method, and its uses. Background Technology

[0002] Fengyuanxin is a lipopeptide synthesized by Bacillus subtilis through non-ribosomal peptide synthase (NRPS), exhibiting various biological activities, including antibacterial and antifungal activities. It is also safe, non-toxic, and easily degradable, making it a novel, green antibacterial agent. However, the low yield and high production cost of existing Fengyuanxin technologies limit its application.

[0003] Low-quality biomass, such as kitchen waste, is rich in organic matter, nutrients, and inexpensive, making it a potential substrate for the synthesis of essential nutrients. The successful utilization of kitchen waste in nutrient synthesis has broad economic benefits. Notably, capsaicin compounds (hereinafter referred to as "capsaicin") are widely present in kitchen waste. Capsaicin includes capsaicin (CPC) and dihydrocapsaicin (DCPC) in a 2:1 ratio, accounting for over 90% of the total capsaicin. Due to its strong irritant properties and broad-spectrum antibacterial effects, 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 adverse factors for Bacillus subtilis in synthesizing essential nutrients from kitchen waste. Under capsaicin stress, Bacillus subtilis will exhibit a series of stress responses. Bacillus subtilis maintains protein stability and cell membrane fluidity by mobilizing global regulatory factors to regulate gene expression; its compatibility solute regulation system balances the osmotic pressure difference between the inside and outside of the cell; and its efflux pump (EP) system, such as ABC transporters, removes harmful substances, reducing cytotoxicity and improving bacterial tolerance. Therefore, there is an urgent need for genetically engineered strains that are tolerant to capsaicin and efficiently synthesize lipopeptide products. Summary of the Invention

[0004] The purpose of this invention is to overcome the shortcomings of the prior art and provide a capsaicin-tolerant Bacillus subtilis strain that produces a high amount of capsaicin.

[0005] The second objective of this invention is to provide a method for constructing a capsaicin-tolerant Bacillus subtilis strain that produces a high amount of capsaicin.

[0006] A third objective of this invention is to provide the use of the above-mentioned capsaicin-tolerant Bacillus subtilis strain that produces a high amount of capsaicin.

[0007] The fourth objective of this invention is to provide a second capsaicin-tolerant Bacillus subtilis strain that produces a high amount of capsaicin.

[0008] The fifth objective of this invention is to provide a method for constructing a second capsaicin-tolerant Bacillus subtilis strain that produces abundant capsaicin.

[0009] The sixth object of the present invention is to provide the use of a second capsaicin-tolerant Bacillus subtilis strain that produces abundant capsaicin.

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

[0011] The method for constructing a capsaicin-tolerant Bacillus subtilis strain with high capsaicin production includes the following steps: using CRISPR / Cas9 technology, the lmrA gene of Bacillus subtilis strain CGF-P-02 is replaced with the P43 promoter to enhance the expression of the lmrB gene, thereby obtaining a capsaicin-tolerant Bacillus subtilis strain with high capsaicin production, 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 above method was used to construct a capsaicin-tolerant Bacillus subtilis strain BS02 that produces abundant capsaicin.

[0016] Applications of the fermentation production of nutrient by strain BS02.

[0017] The preferred culture medium for fermentation is kitchen waste culture medium, but other culture media suitable for Bacillus subtilis strains can also be selected.

[0018] The second method for constructing a capsaicin-tolerant Bacillus subtilis strain with high capsaicin production includes the following steps: using CRISPR / Cas9 technology, the ytrA gene of strain BS02 is replaced with the second P43 promoter to enhance the expression of the ytrBCDEF gene, thereby obtaining a capsaicin-tolerant Bacillus subtilis strain with high capsaicin production, 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 second method described above was used to construct a capsaicin-tolerant Bacillus subtilis strain BS03 that produces abundant capsaicin.

[0023] Applications of the fermentation production of nutrient by strain BS03.

[0024] The preferred culture medium for fermentation is kitchen waste culture medium, but other culture media suitable for Bacillus subtilis strains can also be selected.

[0025] Advantages of this invention:

[0026] The high-capsaicin-tolerant Bacillus subtilis strain constructed in this invention exhibits high capsaicin yield during fermentation, can tolerate 84 mg / L capsaicin, and demonstrates good strain stability and reproducibility. The acquisition method used is simple and easy to operate. Attached Figure Description

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

[0028] Figure 2 The yield of Bacillus subtilis CGF-P-02, BS02 and BS03 increased after 120 hours of fermentation in kitchen waste culture medium. Detailed Implementation

[0029] This invention enhances the tolerance of Bacillus subtilis to capsaicin by strengthening the expression of the ABC transporter genes lmrB and ytrBCDEF in Bacillus subtilis, and significantly increases the capsaicin production of Bacillus subtilis (cultured in a capsaicin-containing medium).

[0030] The present invention will be further described below with reference to specific embodiments:

[0031] The Bacillus subtilis CGF-P-02 strain used in this invention is a laboratory-constructed strain of Bacillus subtilis with enhanced proline transport. (See the patent application filed by Tianjin University, "Method for producing fertility-enhancing agents 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 media.

[0033] 10×Spizizen basic salt solution: 12.0 g / L sodium citrate, 183.0 g / L K₂HPO₄, 60.0 g / L KH₂PO₄, 20.0 g / L (NH₄)₂SO₄. The (NH₄)₂SO₄ must be prepared as a stock solution and sterilized by filtration through a 0.22 μm sterile syringe filter. The remaining components should be prepared as a stock solution and sterilized at 121°C for 20 min.

[0034] GM I medium (100.0 mL): 10.0 mL 10×Spizizen basic salt solution, 2.0 mL 2% casein hydrolysate 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 (100.0 mL): 10.0 mL 10×Spizizen basic salt solution, 1.0 mL 2.0% casein hydrolysate stock solution, 2.0 mL 40.0% glucose stock solution, 800.0 μL 20.0% MgSO4·7H2O stock solution, and finally add 86.2 mL sterile water.

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

[0037] 1. Constructing the pJOE8999a-P43-lmrB plasmid

[0038] (1) Primers lmrA-sgRNA-S (SEQ ID NO.1) and lmrA-sgRNA-A (SEQ ID NO.2) were designed using the reference website (CRISPRRGEN Tools (rgenome.net / cas-designer / )). Annealing and ligating lmrA-sgRNA-S / A yielded the ligation product lmrA-sgRNA. Using a seamless cloning enzyme, lmrA-sgRNA was ligated into the BsaI-linearized pJOE8999a plasmid (Hou ZJ, Cao CY, Gao GR, et al. Enhanced Iturin: A Production of Engineered Bacillus amyloliquefaciens by Knockout of Endogenous Plasmid and Rap Phosphatase Genes[J]. Journal of Agricultural and Food Chemistry, 2024.) to obtain the pJOE8999a-lmrA-sgRNA plasmid. This plasmid was transformed into *E. coli* DH5α and plated on a 25 μg / mL kanamycin-resistant plate for screening for positive transformants. The pJOE8999a-lmrA-sgRNA plasmid was extracted using a plasmid miniprep kit and set aside for later use.

[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 amplified by PCR 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 amplified by PCR using primers lmrA-DF (SEQ ID NO.7) and lmrA-DR (SEQ ID NO.8); and the P43 promoter (SEQ ID NO.12) was amplified by PCR using primers lmrB-P43F (SEQ ID NO.10) and lmrB-P43R (SEQ ID NO.11).

[0040] Overlap PCR was used to sequentially ligate lmrA-U, the P43 promoter, and lmrA-D to obtain the lmrAU-P43-lmrAD sequence. lmrAU-P43-lmrAD was then ligated into the Sfi I-linearized pJOE8999a-lmrA-sgRNA plasmid to obtain the pJOE8999a-P43-lmrB plasmid, which was transformed into *E. coli* DH5α and screened for positive transformants on 25 μg / mL kanamycin-resistant plates. The pJOE8999a-P43-lmrB plasmid was extracted using a plasmid miniprep kit and prepared for later use.

[0041] (2) PCR reaction system. The total system volume 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, and sterile water to bring the total volume to 50 μL; the PCR reaction program is as follows: 95℃ for 3 min, 95℃ for 15 s, 55℃ for 15 s, 72℃ for 60 s / kb, 72℃ for 5 min, and incubation at 16℃, with a cycle count of 35.

[0042] (3) Overlap PCR reaction system. The total system volume 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 (1mrA-U) 100 ng, template 2 (P43 promoter) 100 ng, template 3 (1mrA-D) 100 ng, and sterile water to bring the total volume to 50 μL. The PCR reaction program is as follows: 95℃ for 3 min, 95℃ for 15 s, 60℃ for 15 s, 72℃ for 60 s / kb, 95℃ for 15 s, 55℃ for 15 s, 72℃ for 60 s / kb, 72℃ for 5 min, incubate at 16℃, cycle number 1:10 (decreasing by 0.5℃ per cycle), cycle number 2:25.

[0043] 2. Construction of Bacillus subtilis BS02

[0044] (1) Preparation of Bacillus subtilis competent cells. A single colony of activated Bacillus subtilis CGF-P-02 was picked and cultured in 5 mL of GM I medium at 37℃ and 220 rpm for 18 h. 500 μL of the bacterial culture was added to 4.5 mL of fresh GM I medium and cultured at 37℃ and 220 rpm for 4.5 h. 750 μL of the bacterial culture was added to 4.25 mL of fresh GMII medium and cultured at 37℃ and 220 rpm for 1.5 h to obtain the final Bacillus subtilis competent cells.

[0045] (2) The pJOE8999a-P43-lmrB plasmid from step 1 was transformed into Bacillus subtilis competent cells using chemical transformation. The cells were incubated at 37°C for 45 min, then placed on a 30°C shaker at 200 rpm for 2.5 h. After centrifugation at 5000 rpm for 3 min, the cells were plated onto a kanamycin-resistant plate containing 0.2% mannose and 25 μg / mL to screen for positive transformants. The correct transformants were streaked onto antibiotic-free plates and incubated at 50°C to lose the plasmid, ultimately yielding Bacillus subtilis BS02. This demonstrated the use of the P43 promoter to replace the lmrA gene, the repressor of the lmrB gene, thereby enhancing the expression of the lmrB gene (SEQ ID NO. 13).

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

[0047] 1. Constructing the pJOE8999a-second P43-ytrBCEDF plasmid

[0048] (1) Primers for ytrA-sgRNA-S (SEQ ID NO.14) and ytrA-sgRNA-A (SEQ ID NO.15) were designed using the reference website (CRISPRRGEN Tools (rgenome.net / cas-designer / )). The ligation product ytrA-sgRNA was obtained by annealing ytrA-sgRNA-S / A. Using a seamless cloning enzyme, ytrA-sgRNA was ligated into the Bsa I-linearized pJOE8999a plasmid to obtain the pJOE8999a-ytrA-sgRNA plasmid. This plasmid was transformed into *E. coli* DH5α and screened for positive transformants by plasmidizing it in 25 μg / mL kanamycin-resistant plates. The pJOE8999a-ytrA-sgRNA plasmid was extracted using a plasmid miniprep kit and set aside 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 amplified by PCR 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 amplified by PCR using primers ytrA-DF (SEQ ID NO.20) and ytrA-DR (SEQ ID NO.21); and the second P43 promoter (SEQ ID NO.25) was amplified by PCR using primers ytrBCDEF-second P43F (SEQ ID NO.23) and ytrBCDEF–second P43R (SEQ ID NO.24).

[0050] Overlap PCR was used to sequentially ligate ytrA-U, the second P43 promoter, and ytrA-D to obtain the ytrAU-second P43-ytrAD sequence. ytrAU-second P43-ytrAD was then ligated into the Sfi I-linearized pJOE8999a-ytrA-sgRNA plasmid to obtain the pJOE8999a-second P43-ytrBCDEF plasmid, which was transformed into *E. coli* DH5α and screened for positive transformants on 25 μg / mL kanamycin-resistant plates. The pJOE8999a-second P43-ytrBCDEF plasmid was extracted using a plasmid miniprep kit and prepared for later use.

[0051] (2) PCR reaction system. The total system volume 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, and sterile water to bring the total volume to 50 μL; the PCR reaction program is as follows: 95℃ for 3 min, 95℃ for 15 s, 55℃ for 15 s, 72℃ for 60 s / kb, 72℃ for 5 min, and incubation at 16℃, with a cycle count of 35.

[0052] (3) Overlap PCR reaction system. The total volume 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 bring the volume to 50 μL; the PCR reaction program is: 95℃ for 3 min, 95℃ for 15 s, 60℃ for 15 s, 72℃ for 60 s / kb, 95℃

[0053] 15s, 55℃ for 15s, 72℃ for 60s / kb, 72℃ for 5min, 16℃ for holding, cycle number 1:10 (each cycle decreases by 0.5℃), cycle number 2:25.

[0054] 2. Construction of Bacillus subtilis BS03

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

[0056] (2) The pJOE8999a-second P43-ytrBCDEG plasmid from step 1 was transformed into freshly prepared Bacillus subtilis competent cells using chemical transformation. The cells were incubated at 37°C for 45 min, then placed on a 30°C shaker at 200 rpm for 2.5 h. After centrifugation at 5000 rpm for 3 min, the cells were plated onto a kanamycin-resistant plate containing 0.2% mannose and 25 μg / mL to screen for positive transformants. The correct transformants were streaked onto antibiotic-free plates and incubated at 50°C to lose the plasmid, ultimately yielding Bacillus subtilis BS02. This demonstrated the use of the second P43 promoter to replace the repressor ytrA gene of the ytrBCDEF gene, thereby enhancing 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, comprising 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 medium and cultured at 37℃ and 200 rpm for 12 h.

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

[0060] (2) The bacterial solution obtained in step (1) is adjusted according to OD 600 =0.2% was inoculated into 50mL of Cal-18 medium and incubated at 37℃ and 200rpm for 12h.

[0061] Cal-18 medium composition: maltodextrin 50 g / L, yeast extract 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 Cal-18 medium to a final concentration of 75 mg / L, resulting in Cal-18 medium containing capsaicin.

[0063] (3) The bacterial solution obtained in step (2) is divided according to OD 600 =0.2% was inoculated into Cal-18 medium containing capsaicin and cultured at 30℃ and 160rpm for 72h.

[0064] (4) Extraction of nutrient. After fermentation, 40 mL of fermentation broth was centrifuged, acid-precipitated, freeze-dried under vacuum, and extracted with methanol. (5) Detection of nutrient. The content of nutrient was detected by high performance liquid chromatography. A C18 reversed-phase column (ZORBAX Eclipse XDB-C18, Agilent, 4.6 mm × 150 mm) was used. The mobile phase was 50% acetonitrile and 50% pure water containing 1% trifluoroacetic acid. The detection wavelength was 210 nm, and the column temperature was 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 kitchen waste culture medium (capsaicin content 84.38 mg / L), including 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 medium and cultured at 37℃ and 200 rpm for 12 h.

[0068] (2) The bacterial solution obtained in step (1) is adjusted according to OD 600 =0.2% was inoculated into 50mL of Cal-18 medium and incubated at 37℃ and 200rpm for 12h.

[0069] (3) The bacterial solution obtained in step (2) is divided according to OD 600 =0.2% added to kitchen waste culture medium and incubated at 30℃ and 160rpm for 120h.

[0070] The food waste culture medium consisted of: maltodextrin 30 g / L, yeast extract 15 g / L, tryptone 10 g / L, food waste (from the No. 1 student canteen of Tianjin University Beiyangyuan Campus) 200 g / L, MgSO4·7H2O 1.0 g / L, Na2HPO4·12H2O 2.0 g / L, Na2MoO4·2H2O 2.0 g / L, and pH 6.4.

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

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

[0073] (5) Extraction of nutrient. After fermentation in step (3), take 40 mL of fermentation broth, centrifuge, acid precipitate, freeze dry under vacuum, and extract with methanol.

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

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

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

Claims

1. Capsaicin-tolerant Bacillus subtilis with high capsaicin production (Bacillus subtilis) The method for constructing the strain is characterized by: The steps include: utilizing CRISPR / Cas9 technology, using... P43 The promoter replaces the Bacillus subtilis strain CGF-P-02 lmrA Genes were used to obtain a capsaicin-tolerant Bacillus subtilis strain that produces a high amount of capsaicin, which was named BS02. The P43 The nucleotide sequence of the promoter is shown in SEQ ID NO.12; The lmrA The nucleotide sequence of the gene is shown in SEQ ID NO.

5.

2. The capsaicin-tolerant Bacillus subtilis strain BS02, which is produced by the method of claim 1, is high in capsaicin.

3. Use of the strain BS02 described in claim 2 for fermentation to produce nutrient.

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 that produces high levels of capsaicin, characterized by: The steps include: utilizing CRISPR / Cas9 technology, using a second... P43 The promoter replaces the strain BS02 of claim 2. ytrA Genes were used to obtain a capsaicin-tolerant Bacillus subtilis strain that produces a high amount of capsaicin, which was named BS03. The second P43 The nucleotide sequence of the promoter is shown in SEQ ID NO.25; The ytrA The nucleotide sequence of the gene is shown in SEQ ID NO.

18.

6. The capsaicin-tolerant Bacillus subtilis strain BS03, which is produced by the method of claim 5, is high in capsaicin.

7. Use of the strain BS03 of claim 6 for fermentation to produce nutrient.

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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