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CN-119799754-B - Capsaicin-tolerant bacillus subtilis strain with high yield of Feng Yuan elements and construction method

CN119799754BCN 119799754 BCN119799754 BCN 119799754BCN-119799754-B

Abstract

The invention discloses a capsaicin-tolerant bacillus subtilis strain with high yield of Feng Yuan elements and a construction method, wherein the construction method comprises the steps of replacing lmrA genes of bacillus subtilis strain CGF-P-02 by using a P43 promoter to obtain a capsaicin-tolerant bacillus subtilis strain BS02 with high yield of Feng Yuan elements, and replacing ytrA genes of bacillus subtilis strain BS02 by using a second P43 promoter to obtain a capsaicin-tolerant bacillus subtilis strain BS03 with high yield of Feng Yuan elements. The capsaicin-tolerant bacillus subtilis strain with high yield of Feng Yuan th element constructed by the invention has the advantages of high fermentation yield of the Fender original element, tolerance of 84mg/L capsaicin, good strain stability and strong repeatability. The acquisition method is simple and easy to operate.

Inventors

  • CHENG JINGSHENG
  • Duan Tianxu
  • SONG KAIGE

Assignees

  • 天津大学

Dates

Publication Date
20260505
Application Date
20250106

Claims (8)

  1. 1. The construction method of the capsaicin-tolerant bacillus subtilis (Bacillus subtilis) strain with high yield of Feng Yuan elements is characterized by comprising the following steps of using a CRISPR/Cas9 technology to replace lmrA genes of bacillus subtilis strain CGF-P-02 by using a P43 promoter to obtain the capsaicin-tolerant bacillus subtilis strain with high yield of Feng Yuan elements, which is named as BS02; the nucleotide sequence of the P43 promoter is shown in SEQ ID NO. 12; The nucleotide sequence of lmrA gene is shown in SEQ ID NO. 5.
  2. 2. A capsaicin-tolerant bacillus subtilis strain BS02 with high Feng Yuan-fold production constructed by the method of claim 1.
  3. 3. Use of the strain BS02 according to claim 2 for the fermentative production of Fengyuan.
  4. 4. Use according to claim 3, characterized in that the medium used for fermentation is kitchen waste medium.
  5. 5. The construction method of the capsaicin-tolerant bacillus subtilis strain with high yield of Feng Yuan elements is characterized by comprising the following steps of replacing ytrA gene of the strain BS02 in claim 2 by using a second P43 promoter by using a CRISPR/Cas9 technology to obtain the capsaicin-tolerant bacillus subtilis strain with high yield of Feng Yuan elements, which is named as BS03; The nucleotide sequence of the second P43 promoter is shown in SEQ ID NO. 25; the nucleotide sequence of ytrA gene is shown as SEQ ID NO. 18.
  6. 6. A capsaicin-tolerant Bacillus subtilis strain BS03 with high Feng Yuan-element yield constructed by the method of claim 5.
  7. 7. Use of the strain BS03 according to claim 6 for the fermentative production of Fengyuan.
  8. 8. The method according to claim 7, wherein the medium used for fermentation is kitchen waste medium.

Description

Capsaicin-tolerant bacillus subtilis strain with high yield of Feng Yuan elements and construction method Technical Field The invention belongs to the field of bacillus subtilis genetic engineering, and particularly relates to a capsaicin-tolerant bacillus subtilis strain with high yield of Feng Yuan elements, a construction method and application. Background The Fengyuan is lipopeptide synthesized by bacillus subtilis through non-ribosomal peptide synthetase (NRPS), shows various biological activities including antibacterial and antifungal activities, and simultaneously has the characteristics of safety, no toxicity and easy degradation, and is a novel green antibacterial agent. However, the low yield of Feng Yuan elements of the prior art is costly to produce, limiting its use. Poor biomass, such as kitchen waste, has high organic content, rich nutrition and low price, and is a potential substrate for Feng Yuan element synthesis. The successful utilization of kitchen waste in the synthesis of Fengyuan has wide economic benefit. It is noted that capsaicin compounds (hereinafter referred to as "capsaicin") are widely present in kitchen waste. The capsaicin comprises capsaicin (capsaicin, CPC) and dihydrocapsaicin (dihydrocapsaicin, DCPC) in a ratio of 2:1, and accounts for more than 90% of the total capsaicin. Because of strong irritation and broad-spectrum antibacterial effect, the capsaicin can effectively inhibit the growth and metabolism of various pathogenic microorganisms such as staphylococcus aureus, escherichia coli, bacillus subtilis, saccharomycetes, mould and the like, so that the capsaicin is one of the main adverse factors of the bacillus subtilis for synthesizing the Fengyuan by utilizing kitchen waste. Under capsaicin stress, bacillus subtilis can respond to a series of stresses. The bacillus subtilis regulates gene expression to maintain protein stability and cell membrane fluidity by regulating global regulatory factors, a compatible solute regulating system balances the difference of intracellular and extracellular osmotic pressures, and an Efflux Pump (EP) system, such as ABC transporter, can be used for efflux of harmful substances, so that the cytotoxicity of the harmful substances can be reduced, and the bacterial tolerance can be improved. Thus, there is a need for genetically engineered strains that are resistant to capsaicin and that efficiently synthesize lipopeptide products. Disclosure of Invention The invention aims to overcome the defects of the prior art and provide a capsaicin-tolerant bacillus subtilis strain with high yield of Feng Yuan th element. The second object of the invention is to provide a construction method of the capsaicin-tolerant bacillus subtilis strain with high yield of Feng Yuan. A third object of the present invention is to provide the use of a capsaicin-tolerant bacillus subtilis strain with high Feng Yuan-th element yield as described above. It is a fourth object of the present invention to provide a second, high Feng Yuan-element producing capsaicin-tolerant strain of Bacillus subtilis. A fifth object of the present invention is to provide a method for constructing a second capsaicin-tolerant Bacillus subtilis strain with high Feng Yuan-th element yield. It is a sixth object of the present invention to provide the use of a second, high-Feng Yuan-element-producing capsaicin-tolerant strain of Bacillus subtilis. The technical scheme of the invention is summarized as follows: The construction method of the capsaicin-tolerant bacillus subtilis strain with high yield of Feng Yuan elements comprises the following steps of replacing lmrA gene of bacillus subtilis strain CGF-P-02 by using a P43 promoter by using CRISPR/Cas9 technology, realizing enhancement of lmrB gene expression, and obtaining the capsaicin-tolerant bacillus subtilis strain with high yield of Feng Yuan elements, which is named as BS02; the nucleotide sequence of the P43 promoter is shown in SEQ ID NO. 12; the nucleotide sequence of lmrA gene is shown in SEQ ID NO. 5; the nucleotide sequence of lmrB gene is shown in SEQ ID NO. 13. Capsaicin-tolerant bacillus subtilis strain BS02 with high yield of Feng Yuan elements constructed by the method. The application of the strain BS02 in fermentation production of Fengyuansu. The culture medium used for fermentation is preferably kitchen waste culture medium, and other culture mediums suitable for bacillus subtilis strain production can be selected. The construction method of the second capsaicin-tolerant bacillus subtilis strain with high yield of Feng Yuan elements comprises the following steps of replacing ytrA gene of a strain BS02 by using a CRISPR/Cas9 technology and using a second P43 promoter to realize enhancement of ytrBCDEF gene expression so as to obtain the capsaicin-tolerant bacillus subtilis strain with high yield of Feng Yuan elements, which is named as BS03; The nucleotide sequence of the second P43 promoter is shown in SEQ ID NO. 2