Brevibacillus parabrevis and application thereof

Abstract

The invention discloses brevibacillus parabrevis and application thereof, and belongs to the technical field of biology. Wild brevibacillus parabrevis is used as a starting strain, grsA gene high expression transformation is firstly carried out, multiple times of UV mutation and ARTP mutation are further carried out, fluorophenylalanine resistance screening is combined, and the brevibacillus parabrevis strain is obtained and is preserved in the China General Microbiological Culture Collection Center on February 20, 2025, and the preservation number is CGMCC No.33579. The GS fermentation process impurity proportion of the strain is obviously lower than that of other control strains, and the shake flask fermentation level is greatly improved to 5.8 g / L and is 3.2 times of that under similar conditions in the prior art. The strain is beneficial to greatly reducing the preparation efficiency of the GS, so that the industrial production cost of the GS is greatly reduced.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to Brevibacillus parabrevis and applications thereof. Background Art

[0002] Gramicidin S (GS) is a cyclic peptide antibiotic composed of 10 amino acids produced by the fermentation of Bacillus brevis. It was first discovered in 1944 during isolation and research from farm soil samples. GS has strong antimicrobial activity, particularly against Gram-positive bacteria and some pathogenic fungi. There is considerable interest in the design and synthesis of new GS analogs, and its clinical application as an antibiotic is promising.

[0003] Currently, there are two main GS-producing strains reported. One is the Bacillus brevis Migula strain with a US deposit number of ATCC9999. The strain has a shake flask fermentation level of approximately 1.8 g / L in synthetic culture medium (Erick J. Vandamme et al., 1981). After additional regulation and optimization with arginine and ornithine, the fermentation level reaches a maximum of approximately 2.8 g / L (AGNES POIRIER et al., 1981). The other is the Bacillus brevis Migula strain with a deposit number of VKPM B-10212 from Valenta Pharmaceuticals Co., Ltd., which has an optimized fermentation level of approximately 2 g / L (CN 111172225 A, EP19218434, etc.). According to the existing technology, there is still room for technical improvement in GS-producing strains in terms of improving fermentation levels and controlling fermentation impurities. Summary of the Invention

[0004] The present invention aims to provide a Bacillus parabrevicus and improve the fermentation efficiency of GS production strains through its application, including improving the fermentation level and reducing process impurities, and ultimately reducing the cost of industrial production of GS.

[0005] The object of the present invention is achieved by the following technical solutions: As a first aspect, a Brevibacillus parabrevis is provided, which is deposited in the General Microbiology Center of the China Culture Collection Administration Committee of Microorganisms, the deposit address of which is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, the deposit date is February 20, 2025, and the deposit number is: CGMCC No. 33579; the classification name is: Brevibacillus parabrevis; its 16S rRNA has the gene sequence shown in SEQ ID No. 1.

[0006] As a second aspect, a method for constructing a gramicidin S high-producing strain is provided, wherein wild Bacillus parabrevicaulis is used as a starting strain, the grsA gene is highly expressed, and screening is performed using progressive traditional physical and chemical mutagenesis.

[0007] In some embodiments, the grsA gene high expression modification includes: amplifying a grsA gene fragment from wild Bacillus parabrevis, connecting it to a pET28a vector, and then transforming Escherichia coli to obtain a grsA-pET28a plasmid; after enzyme digestion, connecting the grsA gene fragment to a pNW33N vector, transforming Escherichia coli again to obtain a pNW33N-grsA plasmid, and introducing it into wild Bacillus parabrevis competent cells.

[0008] Furthermore, the progressive traditional physical and chemical mutation includes multiple UV mutations and ARTP mutations.

[0009] Furthermore, the construction method also includes using L-phenylalanine structural analogs for resistance screening.

[0010] Furthermore, the L-phenylalanine structural analogue is p-fluorophenylalanine.

[0011] As a third aspect, a method for constructing the gramicidin S high-producing strain is provided, and the gramicidin S high-producing strain is obtained by constructing the gramicidin S high-producing strain.

[0012] As a fourth aspect, provided is a use of the Brevibacillus parabrevis described in the first aspect or the gramicidin S high-yielding strain described in the third aspect in the preparation of gramicidin S.

[0013] The beneficial effects of the present invention are as follows:

[0014] (1) Using the wild Bacillus parabrevis obtained by isolation and screening as the starting strain, the grsA gene was highly expressed in it, which increased its GS shake flask fermentation level by 123%.

[0015] (2) The modified strain was further subjected to multiple UV and ARTP mutations, combined with screening for resistance to fluorophenylalanine, to ultimately obtain a high-performance strain (original number: HDCC00294). The GS shake flask fermentation level of this strain was significantly improved to 5.8 g / L, which is 3.2 times that of the existing technology under similar conditions (Erick J. Vandamme et al., 1981). More importantly, the proportion of impurities in the fermentation process of this strain was significantly lower than that of other control strains, which is conducive to significantly reducing the industrial production cost of GS. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 This is the shake flask fermentation curve of HDCC00294 strain;

[0017] Figure 2 Comparison of HPLC (High-Performance Liquid Chromatography) spectra of fermentation broths of different strains; Figure 2 (a) is the HPLC spectrum of the fermentation broth of strain 263UV&AR-083#, Figure 2 (b) is the HPLC spectrum of the fermentation broth of strain 263UV-166#, Figure 2 (c) in the figure is the HPLC spectrum of the fermentation broth of strain HDCC00294. DETAILED DESCRIPTION

[0018] Unless otherwise specified, the materials and reagents used in the following examples are all common commercial products and can be purchased on the market.

[0019] The present invention will be described in more detail by the following examples, which are merely illustrative and are not intended to limit the present invention.

[0020] The Bacillus parabrevis HDCC00294 of the present invention has been deposited in the General Microbiology Center of the China Culture Collection Administration, a depository designated by the Patent Office of China. The depository address is: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing; the deposit date is February 20, 2025, the deposit number is: CGMCC No. 33579; the classification name is: Brevibacillus parabrevis.

[0021] Example 1: Isolation and screening of wild strains

[0022] Forty-seven samples of forest soil and streamside mud from Hangzhou, Zhejiang, and Suzhou, Anhui, were collected. After being treated in an 80°C water bath and then serially diluted, the samples were plated onto beef extract peptone medium for isolation. A total of 2,357 bacterial strains were isolated. Among the 34 strains screened by colony morphology and microscopic morphology, one clostridial, spore-forming bacterium showed 98.92% similarity to Brevibacillus parabrevis by 16S rRNA sequencing. After isolation and purification, this bacterium was fermented using the synthetic medium described by (Erick J. Vandamme et al., 1981). GS was successfully detected in the fermentation broth, with a retention time identical to that of a GS control. This strain, originally designated BH-132, originated from a sample of mud from the Bianhe River in Suzhou. Based on its morphological, cultural, and physiological and biochemical characteristics, and in accordance with Bergey's Handbook of Bacterial Identification, it was confirmed as Brevibacillus parabrevis.

[0023] The morphological, cultural, physiological, and biochemical characteristics of strain BH-132 are as follows: The bacteria are rod-shaped (fusiform), arranged singly or in a V-shaped pattern, with meso-spores and G-positive results. It is weakly positive for acid production upon assimilating glycerol and fructose, but negative for acid production upon assimilating glucose, arabinose, xylose, and mannitol. It is positive for nitrate reduction to nitrite, hydrolyzes gelatin, and is catalase-negative. It grows at 55°C and pH 5.0 and is resistant to 7% NaCl. It utilizes nitrate, citrate, and propionate.

[0024] Example 2: Construction of GrsA high-expression strain

[0025] The main reagents include: restriction endonucleases (XbaI, BamHI), T4 DNA ligase, plasmid extraction kit, PCR purification kit, electroporation buffer (0.5 M mannitol, 10% v / v glycerol), SOC medium, LB medium, SMMS medium (for Bacillus transformation), kanamycin (25 μg / mL), and chloramphenicol (10 μg / mL).

[0026] The main experimental steps are as follows:

[0027] 1. Obtaining the target gene grsA

[0028] (1) Primers

[0029] Forward primer (SEQ ID No. 2): 5′-GCTCTAGAATGGCAAAGAAAATCAAC-3′;

[0030] Reverse primer (SEQ ID No. 3): 5′-CGGGATCCTTATTTCAGCCGTTTTC-3′.

[0031] (2) PCR amplification

[0032] High-fidelity PCR amplification was performed using Brevibacillus parabrevis genomic DNA as a template. The amplified product was verified by 1% agarose gel electrophoresis, with a 3.5 kb band. The amplified product was then ligated into the pET28a vector and transformed into Escherichia coli DH5α. Single colonies were picked for sequencing verification. The amplification reaction system consisted of: 5 μL of 10× high-fidelity buffer, 4 μL of 2.5 mM dNTPs, 1 μL each of the forward and reverse primers shown in SEQ ID No. 2-3 (10 μM), 1 μg of template DNA, 1 U of high-fidelity DNA polymerase, and ddH2O to 50 μL. The amplification reaction program was: 95°C for 5 min → 30 cycles (95°C for 30 s, 55°C for 30 s, 72°C for 2 min) → 72°C for 10 min.

[0033] 2. Expression vector construction

[0034] The grsA-pET28a plasmid and the pNW33N vector were double-digested with the restriction endonucleases XbaI and BamHI. The purified grsA fragment was then ligated with the linearized vector at a 3:1 insert / vector ratio (16°C overnight). The ligation product was then transformed into Escherichia coli DH5α and plated on LB plates containing chloramphenicol. Positive clones were selected for plasmid extraction, and the recombinant plasmid, pNW33N-grsA, was verified by double-enzyme digestion and PCR.

[0035] 3. Conversion

[0036] Inoculate Bacillus parabrevicaulis into SMMS medium (containing 0.5% w / v glucose) and culture at 37°C until OD 600 =0.6. Ice bath for 30 min, centrifuge at 4°C (5000 rpm, 10 min), wash 3 times with pre-cooled electroporation buffer, and resuspend to a final concentration of about 10 8 CFU / mL. Mix 50 μL of competent cell suspension with 1 μg of pNW33N-grsA plasmid, incubate on ice for 5 minutes, transfer to a pre-chilled electroporation cup, immediately add 1 mL of SOC medium, and recover at 37°C for 2 hours.

[0037] 4. Screening and verification of recombinant strains

[0038] Take the revived bacterial solution and spread it on the SMMS plate containing chloramphenicol, and culture it at 37°C for 48 hours. Pick a single colony and subculture it 3 times to ensure the stability of the plasmid. Extract the genomic DNA of the recombinant bacteria, amplify it with grsA-specific primers (this embodiment uses primers as shown in SEQ ID No. 2-3), and confirm the target band by electrophoresis for verification. Take the successfully verified strain and the wild strain (control) and inoculate them into the synthetic culture medium reported by (Erick J. Vandamme et al., 1981) for fermentation, ferment at 37°C for 48 hours, and use HPLC to determine the fermentation liquid after treatment. The results showed that the GS yield of the obtained pNW33N-grsA-16# strain was increased by 123% compared with the wild strain.

[0039] Example 3: HDCC00294 strain mutation screening

[0040] The pNW33N-grsA-16# strain, which was verified to be qualified and genetically stable, was used as the initial starting strain for a series of mutations. The starting bacteria used in each mutation were inoculated into LB slant medium, cultured at 37°C for 24 hours, and then the bacterial lawn was washed with sterile saline and shaken to obtain a bacterial suspension (the initial bacterial concentration was controlled to be approximately 10 5CFU / ml) for mutation treatment. The mutant bacterial suspension was gradiently diluted and spread on LB screening plate culture medium supplemented with 0.1% w / v p-fluorophenylalanine, cultured at 37°C for about 48h, and 1 / 2 of each colony was picked and inoculated into shake flask liquid fermentation medium. The remaining colonies were refrigerated in a refrigerator at 2-8°C for selection. The shake flask fermentation was placed at 37°C and 250rpm for shaking culture for about 40h to end the fermentation. The fermentation broth was soaked in anhydrous ethanol, diluted, and then the titer and impurity ratio were detected by HPLC. Colonies with excellent performance (high titer and low impurity ratio) were selected (refrigerated at 2-8°C), subcultured on LB slant culture medium, cultured at 37°C for 24h, and washed with sterile glycerol aqueous solution to prepare the original strain library of excellent strains. The superior strain was used as the starting strain for further mutation, and further mutation screening finally obtained the strain originally numbered HDCC00294. The initial screening titer of the strain in the shake flask reached 5.8 g / L, and the impurity ratio was significantly lower than that of the pNW33N-grsA-16# starting strain.

[0041] The HDCC00294 strain was initially derived from the pNW33N-grsA-16# strain. The specific mutation screening process is as follows:

[0042] pNW33N-grsA-16# strain → UV mutation → 263UV-166# strain → UV mutation → natural isolation → ARTP mutation → UV mutation → ARTP mutation → 263UV&AR-083# strain → ARTP mutation → natural isolation → HDCC00294 strain

[0043] The 16S rRNA gene sequence of strain HDCC00294 was determined to be as shown in SEQ ID No. 1. The strain was deposited in the General Microbiology Center of China Culture Collection of Microorganisms on February 20, 2025, with the deposit number: CGMCC No. 33579.

[0044] Example 4: Verification and comparison of shake flask fermentation performance of HDCC00294 strain

[0045] Take the frozen tubes of HDCC00294 strain and the other two control strains (263UV-166# strain and 263UV&AR-083# strain) and spread them onto LB plate culture medium supplemented with 0.1% w / v p-fluorophenylalanine after thawing. Incubate at 37°C for about 48 hours. Dip a small amount of colonies into the liquid seed culture medium of the shake flask and shake at 37°C and 250 rpm for about 16 hours to obtain mature seed liquid. The mature seed liquid was inoculated into the liquid fermentation medium of the shake flask at a 5% (v / v) inoculum and placed at 37°C and 250 rpm for shaking. The HDCC00294 strain was placed in the bottle at 8h, 16h, 24h, 28h, 32h, 36h, 40h, 44h, and 48h, and the OD and titer were tested to draw the fermentation curve. The other two control strains were placed in the bottle at 40h for comparison of the impurity ratio of the HDCC00294 strain.

[0046] The HPLC method was as follows: the chromatographic column was an Agilent ZORBAX SB-C8 (4.6×150 mm, 3.5 μm), the detection wavelength was 210 nm, the flow rate was 1.0 ml / min, the column temperature was 20° C., the injection volume was 5 μl, the run time was 15 min, and the mobile phase was 0.1% TFA aqueous solution:acetonitrile = 47:53.

[0047] Specific results such as Figure 1 、 Figure 2 As shown, after a long lag phase, the HDCC00294 strain entered a rapid titer production phase at 24 hours, reaching its peak titer at 40 hours. The titer then declined, reaching a peak of 5.82 g / L. At this point, the impurity content was significantly lower than that of the control strain fermentation broth. Furthermore, the HDCC00294 strain's titer was 3.2 times higher than that of the existing technology (1.8 g / L) under similar conditions, demonstrating the HDCC00294 strain's significant advantages.

[0048] It should be noted that although the above-mentioned embodiments have been described herein, this does not limit the scope of patent protection of the present invention. Therefore, based on the innovative concept of the present invention, changes and modifications to the embodiments described herein, or equivalent structural or process transformations made using the contents of the present invention specification, and direct or indirect application of the above technical solutions to other related technical fields are all included in the scope of patent protection of the present invention.

Claims

1. A Brevibacillus parabrevis, characterized in that It was deposited in the General Microbiology Center of China Culture Collection Administration, with the deposit date being February 20, 2025, and the deposit number being: CGMCC No.33579.

2. A method for constructing a gramicidin S high-yielding strain, characterized in that: include: Using wild Bacillus parabrevis as the starting strain, the grsA gene was transformed for high expression and screened using progressive traditional physical and chemical mutagenesis.

3. The method according to claim 2, characterized in that The grsA gene high expression transformation includes: amplifying a grsA gene fragment from wild Bacillus parabrevis, connecting it to a pET28a vector, and then transforming Escherichia coli to obtain a grsA-pET28a plasmid; after enzyme digestion, connecting the grsA gene fragment to a pNW33N vector, transforming Escherichia coli again to obtain a pNW33N-grsA plasmid, and introducing it into wild Bacillus parabrevis competent cells.

4. The method according to claim 2, characterized in that The progressive traditional physical and chemical mutation includes multiple UV mutations and ARTP mutations.

5. The method according to claim 2, characterized in that The construction method further comprises using L-phenylalanine structural analogs for resistance screening.

6. The method according to claim 5, characterized in that The L-phenylalanine structural analog is p-fluorophenylalanine.

7. A gramicidin S high-producing strain constructed by the method according to any one of claims 2 to 6.

8. Use of the Brevibacillus parabrevis according to claim 1 or the gramicidin S high-producing strain according to claim 7 in the preparation of gramicidin S.

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