Application of SerA and / or FliZ in regulating the production of bacillomycin L and the constructed genetically engineered bacterium

Abstract

The present invention discloses the application of SerA and / or FliZ in regulating the yield of bacillomycin L and the constructed genetically engineered bacterium. The present invention improves the yield of bacillomycin L in Bacillus velezensis by knocking out or inactivating the genes encoding SerA and / or FliZ in Bacillus velezensis. The genetically engineered bacterium is obtained by using Bacillus velezensis CPLK1314 as the starting strain and knocking out or inactivating the genes negatively regulating SerA and / or FliZ in the genome of the strain. The yield of bacillomycin L produced by the genetically engineered strain of the present invention through fermentation is 12 times that of the strain CPLK1314, and bacillomycin L can be stably produced in an optimized medium in a 5L fermenter for 48h, with a yield reaching 3.67 g / L. The high-yield genetically engineered bacterium of bacillomycin L and its construction method provide raw materials for accelerating the industrialization of bacillomycin L.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and particularly relates to the application of SerA and / or FliZ in regulating the yield of bacillomycin L and a genetically engineered bacterium constructed thereby. Background Art

[0002] Bacillomycin L is an amphiphilic lipopeptide compound composed of a polypeptide chain of seven amino acid residues (L-Asp-D-Tyr-D-Asn-L-Ser-L-Gln-D-Ser-L-Thr) and a 14-17 β-amino fatty acid chain, and belongs to the "Iturin" family together with Iturin A, C, D, E, Bacillomycin D, F, Lc and Mycosubtilin. Bacillomycin L has good antifungal activity against most yeasts and molds, and also has good hemolytic activity. In addition, Bacillomycin L can degrade the phospholipids on the fungal cell membrane and change the cell membrane permeability to inactivate and kill the fungi. Given the wide application prospects of Bacillomycin L in the fields of agricultural production, medicine and environmental governance, etc., but the yield of Bacillomycin L produced by Bacillus velezensis CPLK1314 under the microbial fermentation process is low, and it is of great value to select microbial resources that can efficiently produce Bacillomycin L.

[0003] Bacillomycin L is synthesized by a non-linear ribosomal pathway. Its gene cluster is about 39 kb in length and contains 4 open reading frames, which respectively encode four multifunctional composite enzymes, Bac D, Bac A, Bac B and Bac C, to catalyze and complete the synthesis process. Bacillomycin L has strong antifungal activity and is an antimicrobial agent with good application prospects. However, the low-yield characteristic in wild strains limits its further application. The applicant's previous research showed that the yield of Bacillomycin L in Bacillus velezensis CPLK1314 is extremely low, which is the biggest bottleneck restricting its application. Therefore, excavating the regulatory genes related to the synthesis of Bacillomycin L and clarifying its synthesis pathway can lay a foundation for increasing its yield, and then combining technical means such as homologous recombination to construct a high-yield engineering bacterium, which is of great significance for promoting its development and application. Summary of the Invention

[0004] Object of the Invention: Aiming at the small yield of Bacillomycin L produced by existing strains at present, which restricts its wide application, the present invention provides the application of SerA and / or FliZ in regulating the yield of Bacillomycin L. The present invention constructs a new genetically engineered bacterium with high yield of Bacillomycin L by knocking out or deleting the negative regulatory gene SerA and the FliZ gene in the genome of Bacillus velezensis CPLK1314.

[0005] The present invention also provides a genetically engineered bacterium for producing bacillomycin L, and a construction method and application thereof.

[0006] Technical solution: To achieve the above object, the present invention provides an application of the SerA and / or FliZ encoding gene in regulating the production of bacillomycin L by Bacillus velezensis.

[0007] Among them, the production of bacillomycin L by Bacillus velezensis is increased by knocking out or inactivating the SerA and / or FliZ encoding gene in Bacillus velezensis.

[0008] The present invention provides a genetically engineered bacterium for producing bacillomycin L, which is obtained by using Bacillus velezensis CPLK1314 as the starting strain and knocking out or inactivating the negative regulatory genes SerA and / or FliZ in the genome of the strain.

[0009] Among them, the nucleotide sequences of the negative regulatory genes SerA and FliZ are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

[0010] Among them, the Bacillus velezensis is Bacillus velezensis CPLK1314.

[0011] The construction method of the genetically engineered bacterium for producing bacillomycin L according to the present invention includes the following steps:

[0012] (1) Design primers using the SerA and FliZ encoding genes as templates, and use the genomic DNA of Bacillus velezensis CPLK1314 as a template to amplify partial SerA and FliZ gene fragments;

[0013] (2) Construction of the homologous recombination plasmid vector pMUTINSerA: The amplified SerA gene fragment is digested with double enzymes, and then ligated into the plasmid vector pMUTINLoc digested with the same double enzymes to construct the homologous recombination integration plasmid vector pMUTINSerA;

[0014] (3) Construction of the homologous recombination plasmid vector pUCSCFliZ: The amplified FliZ gene fragment is digested with double enzymes, and then ligated into the plasmid vector pUCSCSrf digested with the same double enzymes to construct the homologous recombination integration plasmid vector pUCSCFliZ;

[0015] (4) Construction of the SerA gene inactivated mutant strain ΔSerA: The constructed recombinant plasmid pMUTINSerA is transformed into Bacillus velezensis CPLK1314 to obtain the SerA gene inactivated mutant strain ΔSerA with high production of bacillomycin L;

[0016] (5) Construction of the FliZ gene inactivated mutant strain ΔFliZ: The constructed recombinant plasmid pUCSCFliZ was transformed into Bacillus velezensis CPLK1314 to obtain the FliZ gene inactivated mutant strain ΔFliZ with high bacilysin L production;

[0017] (6) Construction of the SerA and FliZ double gene inactivated mutant strain ΔSerA+FliZ: The constructed recombinant plasmid pMUTINSerA was transformed into the Bacillus velezensis CPLK1314 mutant strain ΔFliZ to obtain the SerA and FliZ double gene inactivated mutant strain ΔSerA+FliZ with high bacilysin L production.

[0018] Preferably, the primers described in step (1) are SerA-F (5′-TTTAAGCTTCTCAGATAAGATGAGCAATG-3′) and SerA-R (5′-TTTGGATCCTTCCACTTCAAACACGTCAA-3′) for amplifying the SerA gene fragment; FliZ-F (5’-TTTGGATCCTCTGCATCTTCTGTCTCCGC-3’) and FliZ-R (5’-TTTAAGCTTCTGATATCGCATAAGCGGG-3’) for amplifying the FliZ gene fragment.

[0019] Preferably, the method for constructing the genetically engineered bacterium with high bacilysin L production of the present invention comprises the following steps:

[0020] (1) Design primers using the SerA and FliZ coding genes as templates, and use the genomic DNA of Bacillus velezensis CPLK1314 as a template to amplify partial SerA and FliZ gene fragments;

[0021] (2) Primers SerA-F and SerA-R were designed based on the SerA coding gene sequence in Bacillus velezensis CPLK1314. Using the genome of Bacillus velezensis CPLK1314 as a template, a partial SerA gene was amplified to obtain a 757 bp gene fragment. The amplified SerA gene fragment was double-digested with HindIII and BamHI, and then ligated into the plasmid vector pUCSCSrf that had been digested with the same two enzymes by T4 DNA ligase to construct a homologous recombination integration plasmid vector pUCSCSerA. This was then transformed into Bacillus velezensis CPLK1314 to obtain a genetically engineered strain, the SerA gene mutant strain ΔSerA, which has high yields of bacillomycin L. Primers FliZ-F and FliZ-R were designed based on the FliZ coding gene sequence in Bacillus velezensis CPLK1314. Using the genome of Bacillus velezensis CPLK1314 as a template, a partial FliZ gene was amplified to obtain a 921 bp gene fragment. The amplified FliZ gene fragment was double-digested with BamHI and HindIII, and then ligated into the plasmid vector pMUTINLoc that had been digested with the same two enzymes by T4 DNA ligase to construct a homologous recombination integration plasmid vector pMUTINFliZ. This was then transformed into Bacillus velezensis CPLK1314 to obtain a genetically engineered strain, the FliZ gene mutant strain ΔFliZ, which has high yields of bacillomycin L.

[0022] (3) Construction of the ΔSerA mutant strain

[0023] Competent cells were prepared using Bacillus velezensis CPLK1314 as the starting strain. By means of chemical transformation, the constructed SerA gene mutant vector pMUTINSerA was transformed into Bacillus velezensis CPLK1314. Among them, the recombinant plasmid vector pMUTINSerA contains homologous double exchange arms of the SerA gene and an erythromycin resistance gene, and can specifically inactivate the SerA gene in Bacillus velezensis CPLK1314.

[0024] (4) Construction of the ΔFliZ mutant strain

[0025] Competent cells were prepared using Bacillus velezensis CPLK1314 as the starting strain. By means of chemical transformation, the constructed FliZ gene mutant vector pUCSCFliZ was transformed into Bacillus velezensis CPLK1314. Among them, the recombinant plasmid vector pUCSCFliZ contains homologous double exchange arms of the FliZ gene and a spectinomycin resistance gene, and can specifically inactivate the FliZ gene in Bacillus velezensis CPLK1314.

[0026] (5) Construction of the ΔSerA+FliZ double gene inactivated mutant strain

[0027] Using the mutant strain ΔFliZ as the starting strain to prepare competent cells, and by means of chemical transformation, the constructed SerA gene mutant vector pMUTINSerA was transformed into the mutant strain ΔFliZ. Among them, the recombinant plasmid vector pMUTINSerA contains a SerA gene homologous double exchange arm and an erythromycin resistance gene, and thus a double-gene inactivated mutant strain ΔSerA+FliZ with erythromycin and spectinomycin resistance can be obtained.

[0028] Application of the genetically engineered bacterium with high yield of bacilysin L in the fermentation production of bacilysin L according to the present invention.

[0029] Among them, the fermentation is carried out by fermenter fermentation using the genetically engineered bacterium: after activating the strain to prepare a seed liquid, inoculating it into fresh fermentation medium at an inoculation amount of 10-20% by volume ratio to start fermentation. During the fermentation process, the pH is controlled to be stable at 6.8-7.2, the temperature is maintained at 35-38 °C, and the dissolved oxygen is between 25-35%; the fermentation period is 40-50 h.

[0030] Preferably, the composition of the fermentation medium is: sucrose 20 g / L, malt syrup 40-50 g / L, corn steep liquor 15-20 g / L, urea 0.6-0.8 g / L, K 2 HPO 4 7 g / L, MgSO 4 ·7H 2 O 0.35 g / L, FeSO 4 ·7H 2 O 10 mg / L, (NH 4 ) 2 SO 4 3 g / L, MnSO 4 ·H 2 O 0.05 g / L, VB1, VB3, VB5, VB12, VH each 1-3 mg / L, pH 7.0-7.2.

[0031] Preferably, under the above fermentation medium and fermentation conditions, the yield of bacilysin L in the high-yield engineering bacterium can be increased to 3.67 g / L.

[0032] Application of the SerA and / or FliZ coding gene according to the present invention in regulating the yield of bacilysin L of Bacillus velezensis.

[0033] The existing understanding of the regulatory mechanism of bacillomycin L is unclear, resulting in slow progress in the research on bacillomycin L, and the yield of bacillomycin L produced by strains is low, affecting its subsequent commercial production and application. Through basic research, the present invention discovers the negative regulatory effects of two regulatory factors, SerA and FliZ, on bacillomycin L, initially breaks through the bottleneck in the research on bacillomycin L, improves the yield of bacillomycin L, and makes the commercial production of bacillomycin L possible.

[0034] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0035] The present invention discovers for the first time that knocking out or inactivating the genes encoding SerA and / or FliZ in Bacillus velezensis can improve the yield of bacillomycin L in Bacillus velezensis. By using the method of homologous recombination, a strain ΔSerA+FliZ with improved bacillomycin L yield is successfully constructed. Compared with the original strain CPLK1314, the ability of the strain ΔSerA+FliZ after strain improvement to produce bacillomycin L is significantly improved, and the yield is 12 times that of the original strain CPLK1314, far exceeding the yields of other existing wild-type strains or engineered strains of bacillomycin L.

[0036] In addition, the present invention also reveals that the SerA and FliZ genes have an inhibitory effect on the production of bacillomycin L. By using the pUCSCFliZ and pMUTINSerA vectors to genetically modify any heritable transformable Bacillus velezensis to obtain the corresponding improved strains, the purpose of improving the yield of bacillomycin L can be achieved. 3.67 g of pure bacillomycin L can be prepared per liter of fermentation broth in the optimized medium. The genetically engineered bacteria of the present invention and its construction method bring the possibility and hope for accelerating the industrialization of bacillomycin L, and also provide a feasible idea and solution for the construction and industrialization of other high-yield strains of lipopeptide antibiotics. Description of the Drawings

[0037] Figure 1 Flow chart for the construction of partial gene mutation vectors of SerA and FliZ;

[0038] Figure 2 Verification electrophoresis diagram of the homologous recombination integration plasmid vector of SerA and FliZ;

[0039] Figure 3 Schematic diagram for the determination of the antibacterial activity of Bacillus velezensis CPLK1314, mutant strain ΔSerA, mutant strain ΔFliZ, and mutant strain ΔSerA+FliZ against the spore germination of Fusarium;

[0040] Figure 4Schematic diagram of high performance liquid chromatography analysis of bacillomycin L produced by Bacillus velezensis CPLK1314, mutant strain ΔSerA, mutant strain ΔFliZ, and mutant strain ΔSerA+FliZ;

[0041] Figure 5 Mass spectrometry analysis chart of bacillomycin L. Detailed implementation manners

[0042] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0043] The materials, reagents, etc. used in the embodiments of the present invention can be obtained from commercial channels without special instructions. The experimental methods without specific conditions in the embodiments usually follow conventional conditions or the conditions recommended by the manufacturer.

[0044] Among them, Bacillus velezensis CPLK1314, with the preservation number CCTCC NO: M2017658, has been reported in many prior applications such as CN112522169A (originally named Bacillus amyloliquefaciens CPLK1314, now uniformly renamed Bacillus velezensis CPLK1314), provided by Huaiyin Institute of Technology, High-efficiency preparation methods and biological activities of four lipopeptide antibiotics, surfactin, bacillomycin L, bacillomycin L and pantocin, Southwest China Journal of Agricultural Sciences, Vol. 31, No. 11, 2018.

[0045] Among them, Bacillus subtilis Bs916, with the preservation number CGMCC No. 0808, has been reported in many prior applications such as CN103524600A, provided by Huaiyin Institute of Technology.

[0046] The Fusarium oxysporum f. sp. niveum used for measuring the antibacterial activity is a wild-type Fusarium oxysporum f. sp. niveum, provided by Huaiyin Institute of Technology.

[0047] Among them, the pMUTINLoc plasmid is based on pMUTIN4, and an exogenous DNA fragment Loc (using LocDF (5’-TTTAAGCTTTCAGGTACCAACGATGAACA-3’) and LocDR (5’-TTTGGATCCTTGTCCATTACAGCTACGGT-3’) as primers, using the genomic DNA of Bacillus subtilis Bs916 as a template, PCR amplified a fragment of 812bp as Loc, SEQ ID NO.3) was inserted at the multiple cloning sites HindIII and BamHI. pMUTIN4 was purchased from the Bacillus Genetic Stock Center in the United States, and the plasmid number is ECE139.

[0048] The pUCSCSrf plasmid is based on pUCSC, and an exogenous DNA fragment Srf (using SrfA-AF (5’-TTTAAGCTTACACAGATATCAGGCAAGC-3’) and SrfA-AR (5’-TTT GGATCC GTCCCATCGTTCCTTCACA-3’) as primers, and the genomic DNA of Bacillus subtilis Bs916 as a template, a 908bp fragment was amplified by PCR as Srf, SEQ ID NO.4). The pUCSC plasmid contains the Escherichia coli replication origin and ampicillin resistance gene of pUC19 (TransGen Biotech), and the spectinomycin resistance gene from the pDG1728 plasmid. Among them, pDG1728 was purchased from the Bacillus Genetic Stock Center in the United States, and the plasmid number is ECE1728.

[0049] Using SpecF (5’-TTTGGATCCCTGCAGCCCTGGCGAATG-3’) and SpecR (5’-TTTGAATTCAGATCCCCCTATGCAAGG-3’) as primers, and the pDG1728 plasmid as a template, a 1182bp fragment containing the spectinomycin expression cassette was amplified by PCR; after double digestion of the expression cassette with BamHI and EcoRI, it was cloned into the pUC19 vector to construct the recombinant vector pUCSC.

[0050] References for plasmid construction: Luo Chuping. Structural identification, synthetic pathway and biological function of bacillomycin L, surfactin, bacillomycin and fengycin produced by Bacillus subtilis Bs916 [D]. Nanjing Agricultural University, 2014.

[0051] SP salt: 0.2% (NH 4 ) 2 SO 4 , 1.4% K 2 HPO 4 , 0.6% KH 2 PO 4 , 0.02% MgSO 4 ·7H 2 O, 0.1% sodium citrate.

[0052] CAYE (100×): 2% Casamino acid, 10% yeast extract.

[0053] SpI medium: Add 1% of a 50% glucose solution by volume and 1% of a 100× CAYE solution to the SP salt solution.

[0054] SpII Medium: SPI medium is added with 1% by volume of 50 mmol / L CaCl 2 solution and 1% by volume of 250 mmol / L MgCl 2 solution.

[0055] Example 1

[0056] Construction of SerA and FliZ gene mutation vectors

[0057] Primers SerA-F (5′-TTTAAGCTTCTCAGATAAGATGAGCAATG-3′) and SerA-R (5′-TTTGGATCCTTCCACTTCAAACACGTCAA-3′) were designed based on the genomic SerA gene sequence in Bacillus velezensis CPLK1314. Using the genome of Bacillus velezensis CPLK1314 as a template, a partial SerA gene sequence was amplified to obtain a 757 bp gene fragment. Taq DNA polymerase Mix (Tsingke Biotechnology Co., Ltd.) was used, and the PCR program was: 94°C for 5 min; 35×(94°C for 30 s; 55°C for 30 s; 72°C for 1.5 min); 72°C for 10 min. As Figure 2 shown in

[0058] c, lane 4 is the amplified SerA fragment. Figure 2 Primers FliZ-F (5’-TTTGGATCCTCTGCATCTTCTGTCTCCGC-3’) and FliZ-R (5’-TTTAAGCTTCTGATATCGCATAAGCGGG-3’) were designed based on the FliZ gene sequence in the genome of Bacillus velezensis CPLK1314. Using the genome of Bacillus velezensis CPLK1314 as a template, a partial FliZ gene sequence was amplified to obtain a 921 bp gene fragment. Taq DNA polymerase Mix (Tsingke Biotechnology Co., Ltd.) was used, and the PCR program was: 94°C for 5 min; 35×(94°C for 30 s; 52°C for 30 s; 72°C for 1.5 min); 72°C for 10 min. As

[0059] shown in

[0060] Table 1 PCR reaction system

[0061]

[0062] The amplified partial SerA fragment and the pMUTINLoc plasmid were digested with BamHI and HindIII at 37°C for 3 h, and then the gel was recovered by cutting. The recovered product was ligated using a rapid ligation kit (T4 ligase) ( Figure 1 ). The obtained ligation product was directly transformed into competent Escherichia coli DH5α cells, and positive clones were screened by LB solid plates containing ampicillin. Further, the correct transformants were identified by colony PCR. The upper and lower primers designed in step (1) were used for amplification, with the colonies of the correct transformants as templates. The amplification conditions were the same as in step (1). The PCR products were detected by agarose gel electrophoresis, and the results are shown in Figure 2 lane 6 of gel c, showing a band of 757 bp in size. The results were verified to be correct. By extracting the plasmid and verifying by enzyme digestion, as shown in Figure 2 a, lane 2 was the pMUTINLoc plasmid digested with HindIII and BamHI. The recombinant plasmid pMUTINSerA ( Figure 2 a) was digested with double enzymes and verified by 1% agarose gel electrophoresis to obtain two bands of 757 bp and 8610 bp in size, which was consistent with the expected results. The successfully verified vector can be used for the transformation of Bacillus subtilis.

[0063] The amplified partial FliZ fragment and the pUCSCSrf plasmid were digested with HindIII and BamHI at 37°C for 3 h, and then the gel was recovered by cutting. The recovered product was ligated using a rapid ligation kit (T4 ligase) ( Figure 1 ). The obtained ligation product was directly transformed into competent Escherichia coli DH5α cells, and positive clones were screened by LB solid plates containing ampicillin. Further, the correct transformants were identified by colony PCR. The upper and lower primers designed in step (1) were used for amplification, with the colonies of the correct transformants as templates. The amplification conditions were the same as in step (1). The PCR products were detected by agarose gel electrophoresis, and the results are shown in Figure 2 lane 5 of gel c, showing a band of 921 bp in size. The results were verified to be correct. By extracting the plasmid and verifying by enzyme digestion, as shown in Figure 2 b, lane 3 was the pUCSCSrf plasmid digested with HindIII and BamHI. The recombinant plasmid pUCSCFliZ ( Figure 2 b) was digested with double enzymes and verified by 1% agarose gel electrophoresis to obtain two bands of 921 bp and 4705 bp in size, which was consistent with the expected results. The successfully verified vector can be used for the transformation of Bacillus subtilis.

[0064] Example 2

[0065] Construction of gene mutation Bacillus subtilis ΔSerA, ΔFliZ, ΔSerA+FliZ

[0066] The constructed pMUTINSerA mutant vector was transformed into Bacillus velezensis CPLK1314 and integrated into the genome of Bacillus velezensis CPLK1314 by homologous recombination. Double crossover occurred at the SerA locus of the CPLK1314 genome. Erythromycin-resistant strains were screened on resistant plates and verified by PCR. The correct transformants were named ΔSerA. The SerA gene of this strain was inserted and mutated by the erythromycin resistance gene, becoming a mutant strain with a mutated SerA gene, that is, the improved strain ΔSerA.

[0067] The constructed pUCSCFliZ mutant vector was transformed into Bacillus velezensis CPLK1314 and integrated into the genome of Bacillus velezensis CPLK1314 by homologous recombination. Double crossover occurred at the FliZ locus of the CPLK1314 genome. Spectinomycin-resistant strains were screened on resistant plates and verified by PCR. The correct transformants were named ΔFliZ. The FliZ gene of this strain was inserted and mutated by the spectinomycin resistance gene, becoming a mutant strain with a mutated FliZ gene, that is, the improved strain ΔFliZ.

[0068] The specific process was as follows: A single colony of Bacillus velezensis CPLK1314 was picked and inoculated into 25 mL of SpI medium, and cultured overnight at 30 °C with shaking at 180 r / min. It was inoculated into fresh SpI medium at a volume ratio of 1:25 and cultured at 30 °C with shaking at 250 r / min until the OD600 reached 2.0, which took about 3.5 h. Then it was inoculated into SpII medium at a volume ratio of 1:10 and cultured at 37 °C with shaking at 150 r / min for 1.5 h, and then collected by centrifugation at 5000 r / min. The cells were suspended in 1 / 10 volume of the supernatant and used as the competent cells of Bacillus velezensis CPLK1314. 1 mmol / L EGTA was added to 250 μL of the competent cells, and cultured at 37 °C with shaking at 150 r / min. After 10 min, 1 μg of pMUTINSerA or pUCSCFliZ plasmid was added, and cultured at 37 °C with shaking at 150 r / min for 1 h, and then continued to be cultured at 37 °C with shaking at 250 r / min for 30 min. Finally, it was spread on LB resistant plates containing erythromycin (1 μg / mL) or spectinomycin (50 μg / mL).

[0069] The constructed pMUTINSerA mutant vector was transformed into the mutant strain ΔFliZ and integrated into the genome of the mutant strain ΔFliZ by homologous recombination. Double exchange occurred at the SerA locus of the genome of the mutant strain ΔFliZ, and strains with erythromycin and spectinomycin resistance were screened on the resistance plate and verified by PCR. The correct transformants were named ΔSerA+FliZ. The SerA gene of this strain was insertion-mutated by the erythromycin and spectinomycin resistance genes, becoming a mutant strain with mutated SerA gene and FliZ gene, that is, the improved strain was ΔSerA+FliZ.

[0070] The specific process was as follows: Pick a single colony of the mutant strain ΔFliZ and inoculate it into 25 ml of SpI medium, and culture it overnight at 30 °C with shaking at 180 r / min. Inoculate it into fresh SpI medium at a volume ratio of 1:25, and culture it at 30 °C with shaking at 250 r / min until the OD600 reached 2.0, about 3.5 h; inoculate it into SpII medium at a volume ratio of 1:10, and culture it at 37 °C with shaking at 150 r / min for 1.5 h, then centrifuge and collect it at 5000 r / min. The cells were suspended with 1 / 10 volume of the supernatant and used as competent cells of the mutant strain ΔFliZ. Add EGTA with a final concentration of 1 mmol / L to 250 μL of competent cells, culture it at 37 °C with shaking at 150 r / min, add 1 μg of pMUTINSerA plasmid after 10 min, culture it at 37 °C with shaking at 150 r / min for 1 h, and then continue to culture it at 37 °C with shaking at 250 r / min for 30 min. Finally, coat it on an LB double-resistant plate containing erythromycin (1 μg / mL) and spectinomycin (50 μg / mL).

[0071] Example 3

[0072] Determination of the antibacterial activity of Bacillus velezensis CPLK1314, the SerA gene-inactivated mutant strain ΔSerA, the FliZ gene-inactivated mutant strain ΔFliZ, and the mutant strain ΔSerA+FliZ against the spore germination of Fusarium

[0073] The specific steps included: Activate Bacillus velezensis CPLK1314, the mutant strain ΔSerA, the mutant strain ΔFliZ, and the mutant strain ΔSerA+FliZ. Pick a single colony of each mutant strain and culture it in 5 mL of LB liquid medium at 180 rpm / min in a constant temperature incubator at 37 °C for 48 h. Cool 100 mL of LB solid medium to 46 °C and pour it into a sterile petri dish, and label the petri dish. Dilute the Fusarium spores to 10 6Take 500 μL and coat it on an LB plate. Let it dry, then punch holes (about 6 mm in diameter), and drop 50 μL of the above-mentioned Bacillus velezensis CPLK1314, mutant strain ΔSerA, mutant strain ΔFliZ, and mutant strain ΔSerA+FliZ that have completed cultivation respectively. Observe after culturing at 28°C for 1 - 3 days.

[0074] As Figure 3 , on the 3rd day, the inhibition zone radius of Bacillus velezensis CPLK1314 was 5.5 mm, the inhibition zone radius of ΔSerA was 9.6 mm, the inhibition zone radius of ΔFliZ was 8 mm, and the inhibition zone radius of the mutant strain ΔSerA+FliZ was 11.0 mm. The inhibition zone areas were 3 times, 2.11 times, and 4 times that of the wild-type strain respectively.

[0075] In summary, the strain ΔSerA+FliZ has a good inhibitory effect on the spore germination of Fusarium, and has a significant synergistic effect compared with the original strain and the first two mutant strains.

[0076] Example 4

[0077] Qualitative and quantitative analysis of bacitracin L produced by Bacillus velezensis CPLK1314, mutant strain ΔSerA, mutant strain ΔFliZ, and mutant strain ΔSerA+FliZ

[0078] Activate Bacillus velezensis CPLK1314, mutant strain ΔSerA, mutant strain ΔFliZ, and mutant strain ΔSerA+FliZ. Pick single colonies and transfer them to 5 ml of LB liquid medium respectively, culture at 37°C and 180 r / min for 12 h to prepare seed solutions. Inoculate the seed solutions into 500 mL flasks containing 200 ml of LB culture medium according to an inoculation amount of 1% by volume ratio, culture at 37°C and 180 rpm for 48 h. Centrifuge the bacterial solution (10000 r / 5 min), take the supernatant and acid precipitate it. Adjust the pH value to 2.8 with hydrochloric acid, let it stand in a 4°C refrigerator for 24 h, centrifuge again (12000 r / 15 min), take the precipitate and extract it with 100% methanol. The extract is filtered through a 0.22 μm filter membrane to obtain the crude extract of bacitracin L.

[0079] The crude extract was diluted with deionized water to an aqueous solution containing 30% methanol, and the pH was adjusted to 7.0 with sodium hydroxide. It was passed through an NH2 solid-phase extraction column and gradient eluted with 50% methanol-water, 100% methanol, 0.5% formic acid methanol solution, 1% formic acid methanol solution, and 2% formic acid methanol solution. The target compound was contained in the 1% formic acid methanol elution solution. The pH of the 1% formic acid methanol elution solution was adjusted to 7.0, concentrated by nitrogen blowing and then diluted with deionized water to an aqueous solution containing 30% methanol, and then loaded onto a C18 solid-phase extraction column; gradient eluted with 30%, 50%, 70%, and 90% methanol aqueous solutions with a volume three times that of the column. The pure product of bacillomycin L was obtained in the 70% methanol water eluate. The 70% methanol water eluate was concentrated by nitrogen blowing and then vacuum dried to obtain the pure product of bacillomycin L.

[0080] The detection conditions of bacillomycin L for high performance liquid chromatography (HPLC) are as follows: using a C18 (5μm; 4.6×250mm; VYDAC218TP; VYDAC, Hesperia, CA) column; the mobile phase is acetonitrile: water: trifluoroacetic acid (40:60:0.5 vol / vol), and the flow rate is 0.5 mL / min -1 , and the ultraviolet detection wavelength is 210 nm.

[0081] The experimental results are as Figure 4 shown. The target compound was detected in the 1% formic acid methanol elution solution. The retention times of bacillomycin L homolog A were 12.2 min and that of bacillomycin L homolog B was 17.03 min. Among them, the peak area of the starting strain Bacillus velezensis CPLK1314 was 7026.3, the peak area of the mutant ΔFliZ was 13697.72, the peak area of the mutant ΔSerA was 48998.48, and the peak area of the mutant ΔSerA+FliZ was 78967.4. That is, the yield of the mutant ΔFliZ was about 2 times that of the wild type, the yield of the mutant ΔSerA was about 7 times that of the wild type, and the yield of the mutant ΔSerA+FliZ was about 12 times that of the wild type, indicating that knocking out or inactivating the negative regulatory genes SerA and FliZ in the strain genome of the mutant ΔSerA+FliZ significantly increased the efficiency. In addition, using the optimized medium of the present invention, the bacillomycin L can be further improved (see Example 5 for details).

[0082] The mass spectrometry detection operation of bacillomycin L is as follows: After dissolving the above-mentioned pure product of bacillomycin L in methanol, it was detected by electrospray quadrupole time-of-flight tandem mass spectrometry Q-TOF2. The spraying conditions were: capillary voltage 32 V, spraying voltage 20 kV, and capillary temperature 320 °C. Detection mode: positive ion mode. The results are as Figure 5Bacillomycin L was successfully prepared. Its mass spectrum contains two main mass-to-charge ratio peaks (m / z), with molecular weights of 1044.7 and 1058.7 respectively, which are homologues A and B of bacillomycin L, and the two differ by a methylene group (-CH2).

[0083] Example 5

[0084] Optimization of the fermentation medium for the high-yield production of bacillomycin L by the mutant strain ΔSerA+FliZ.

[0085] First, activate Bacillus velezensis CPLK1314, mutant strain ΔSerA, mutant strain ΔFliZ, and mutant strain ΔSerA+FliZ using LB medium and culture them at 37 °C and 180 rpm for 24 h.

[0086] Using this logarithmic-phase culture broth as the seed liquor, inoculate it into a 5 L fermenter containing 3 L of LB medium or optimized medium (20 g / L sucrose, 40 g / L malt syrup, 15 g / L corn steep liquor, 0.8 g / L urea, K 2 HPO 4 7 g / L, MgSO 4 ·7H 2 O 0.35 g / L, FeSO 4 ·7H 2 O 10 mg / L, (NH 4 ) 2 SO 4 3 g / L, MnSO 4 ·H 2 O 0.05 g / L, 2 mg / L each of VB1, VB3, VB5, VB12, and VH) and culture them. During the fermentation process, the pH is stabilized at around 7.0, the temperature is maintained at 37 °C, the dissolved oxygen is 30%, and the fermentation cycle is 48 h to obtain the seed liquor. The content of bacillomycin L produced by Bacillus velezensis CPLK1314, mutant strain ΔSerA, mutant strain ΔFliZ, and mutant strain ΔSerA+FliZ was determined by the same method as in Example 4. The results showed that the yield of bacillomycin L produced by Bacillus velezensis CPLK1314 in LB medium reached 0.21 g / L, the yield of bacillomycin L produced by mutant strain ΔSerA+FliZ in LB medium reached 2.55 g / L, and the yield of bacillomycin L produced by fermentation using the above optimized medium and fermentation conditions reached 3.67 g / L, as shown in Table 1 specifically. In addition, it can be seen that the effect of ΔSerA+FliZ is significantly better than the sum of mutant strain ΔSerA and mutant strain ΔFliZ, proving that the combined regulation of the two has a significant synergistic advantage.

[0087] Table 1 Bacillus velezensis CPLK1314 and mutant strain ΔSerA+FliZ fermentation production of bacilysin L

[0088]

Claims

1. Application of SerA and / or FliZ encoding genes in regulating the yield of bacillomycin L in Bacillus velezensis, improving the yield of bacillomycin L in Bacillus velezensis by knocking out or inactivating the SerA and / or FliZ encoding genes in Bacillus velezensis, and the Bacillus velezensis is Bacillus velezensis CPLK1314, with the deposit number CCTCC NO: M2017658.

2. A genetically engineered bacterium with high yield of bacillomycin L Characterized in that The genetically engineered bacterium uses Bacillus velezensis as the starting strain, and is obtained by knocking out or inactivating the negative regulatory genes SerA and FliZ, or FliZ in the genome of the strain, and the Bacillus velezensis is Bacillus velezensis CPLK1314, with the deposit number CCTCC NO: M2017658.

3. The genetically engineered bacterium with high yield of bacillomycin L according to claim 2 Characterized in that The nucleotide sequences of the negative regulatory genes SerA and FliZ are shown in SEQ ID NO.1 and SEQ ID NO.2 respectively.

4. A method for constructing the genetically engineered bacterium with high yield of bacillomycin L according to claim 3 Characterized in that It includes the following steps: (1) Design primers using the SerA and FliZ encoding genes as templates, and use the genomic DNA of Bacillus velezensis CPLK1314 as a template to amplify the SerA and FliZ gene fragments; (2) Construction of the homologous recombination plasmid vector pMUTINSerA: The amplified SerA gene fragment is double digested, and then ligated into the plasmid vector pMUTINLoc that has been double digested in the same way to construct the homologous recombination integration plasmid vector pMUTINSerA; (3) Construction of the homologous recombination plasmid vector pUCSCFliZ: The amplified FliZ gene fragment is double digested, and then ligated into the plasmid vector pUCSCSrf that has been double digested in the same way to construct the homologous recombination integration plasmid vector pUCSCFliZ; (4)Inactivated mutant strain of SerA gene ΔSerA Construction: The constructed recombinant plasmid pMUTINSerA was transformed into Bacillus velezensis CPLK1314 to obtain an inactivated mutant strain of SerA gene with high yield of bacilysin ΔSerA ; (5)Inactivated mutant strain of FliZ gene ΔFliZ Construction: The constructed recombinant plasmid pUCSCFliZ was transformed into Bacillus velezensis CPLK1314 to obtain an inactivated mutant strain of FliZ gene with high yield of bacilysin ΔFliZ ; (6)Construction of the double gene inactivated mutant strain of SerA and FliZ ΔSerA+FliZ : The constructed recombinant plasmid pMUTINSerA was transformed into the mutant strain of Bacillus velezensis CPLK1314 ΔFliZ to obtain the double gene inactivated mutant strain of SerA and FliZ with high yield of bacilysin L ΔSerA+FliZ ; The primers described in step (1) are SerA-F: 5′-TTTAAGCTTCTCAGATAAGATGAGCAATG-3′ and SerA-R: 5′-TTTGGATCCTTCCACTTCAAACACGTCAA-3′ for amplifying the SerA gene fragment; primers FliZ-F: 5’-TTTGGATCCTCTGCATCTTCTGTCTCCGC-3’ and FliZ-R: 5’-TTTAAGCTTCTGATATCGCATAAGCGGG-3’ for amplifying the FliZ gene fragment.

5. Application of the genetically engineered bacterium with high yield of bacillomycin L according to claim 3 in the fermentation production of bacillomycin L.

6. The application according to claim 5 Characterized in that The fermentation is carried out in a fermenter by a genetically engineered bacterium: after activating the strain, a seed solution is prepared and inoculated into fresh fermentation medium at an inoculation amount of 10-20% by volume to start fermentation. During the fermentation process, the pH is controlled to be stable at 6.8-7.2, the temperature is maintained at 35-38 °C, and the dissolved oxygen is between 25-35%; the fermentation period is 40-50 h.

7. According to the application described in claim 6, characterized in that The composition of the fermentation medium is as follows: sucrose 20 g / L, malt syrup 40 - 50 g / L, corn steep liquor 15 - 20 g / L, urea 0.6 - 0.8 g / L, K 2 HPO 4 7 g / L, MgSO 4 ·7H 2 O 0.35 g / L, FeSO 4 ·7H 2 O 10 mg / L, (NH 4 ) 2 SO 4 3 g / L, MnSO 4 ·H 2 O 0.05 g / L, VB1, VB3, VB5, VB12, VH each 1 - 3 mg / L, pH 7.0 - 7.2.

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