Application of bacitene or bacitene combined surfactin in regulation and control of abelegen yield of bacillus velezensis strain HN-Q-8

Abstract

The invention provides application of bacitene or bacitene combined surfactin in regulating and controlling the yield of abelegen of a bacillus velezensis strain HN-Q-8, and belongs to the technical field of genetic engineering. The invention provides application of bacitracene or bacitracene and surfactin combined serving as a target spot in regulation and control of the yield of abinogen of a bacillus velezensis strain HN-Q-8, and provides application of bacitracene or bacitracene and surfactin combined serving as a target spot in regulation and control of the yield of abinogen of the bacillus velezensis strain HN-Q-8. The preservation number of the bacillus velezensis strain HN-Q-8 is CGMCC (China General Microbiological Culture Collection Center) No. 19554. Results of the embodiment of the invention show that after bacillene genes are knocked out or bacillene genes and surfactin genes are jointly knocked out in the bacillus velezensis, the yield and the antibacterial activity of the progen are remarkably improved compared with those of wild type bacillus velezensis; the results show that the bacitene or the bacitene combined surfactin can be used as a target spot to regulate and control the yield of the abinogen of the bacillus velezensis.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and particularly relates to the application of bacillusene or the bacillusene combined with surfactin in regulating the yield of fengycin in Bacillus velezensis strain HN-Q-8. Background Art

[0002] Bacillus sp. can produce a variety of lipopeptides with important applications, including fengyuansu. Fengyuansu is a lipopeptide compound with broad application prospects. Its unique molecular structure enables it to maintain high biological activity under harsh conditions such as high temperature and extreme pH. This compound has shown significant application value in pharmaceutical development, food preservation, forestry protection, agricultural disease control, and environmental remediation.

[0003] Currently, the production of fengyuansu relies on the fermentation of Bacillus sp., but fermentation efficiency and costs are limited by the strain's production capacity and fermentation conditions, resulting in high costs and low yields that have become bottlenecks for industrialization. Improving the fengyuansu biosynthesis capacity of Bacillus sp. through molecular engineering is an effective approach, but there are currently few reports on enhancing fengyuansu biosynthesis in Bacillus velez through molecular techniques. Summary of the Invention

[0004] In view of this, the present invention provides an application of a bacillusene gene and / or a surfactin gene in improving the fengycin production of Bacillus velezini, which can effectively improve the fengycin production of Bacillus velezini.

[0005] In order to achieve the above object, the present invention provides the following technical solutions:

[0006] The present invention provides the use of bacillusene or bacillusene combined with surfactin as a target in regulating the yield of fengycin in Bacillus velezensis strain HN-Q-8;

[0007] The deposit number of the Bacillus Velez strain HN-Q-8 is CGMCC No.19554.

[0008] Preferably, the bacillusene or bacillusene combined with surfactin negatively regulates the production of fengycin in the Bacillus velezensis strain HN-Q-8.

[0009] The present invention provides the use of bacillus cereus and / or surfactin as targets in regulating at least one of the antibacterial activity, spore production ability and biofilm formation of the Bacillus velezensis strain HN-Q-8.

[0010] Preferably, the bacillus cereus and / or surfactin negatively regulate the antibacterial activity and / or biofilm formation of Bacillus velezensis strain HN-Q-8;

[0011] The bacillus cereus and / or surfactin positively regulate the spore production ability of the Bacillus velezensis strain HN-Q-8.

[0012] The present invention provides the use of knocking out the bacillusene gene and the surfactin gene to increase the yield of fengycin and / or the antibacterial activity of the Bacillus velez strain HN-Q-8, reduce the spore production ability of the Bacillus velez strain HN-Q-8, and promote the formation of biofilm by the Bacillus velez strain HN-Q-8;

[0013] The deposit number of the Bacillus Velez strain HN-Q-8 is CGMCC No.19554.

[0014] Preferably, the reagent for knocking out the expression of the bacillusene gene includes a bacillusene knockout vector; the bacillusene knockout vector includes a first upstream homology arm, a first resistance gene fragment and a first downstream homology arm;

[0015] The nucleotide sequence of the upstream homology arm of the bacillusene vector is shown in SEQ ID NO: 1; the nucleotide sequence of the downstream homology arm of the bacillusene gene vector is shown in SEQ ID NO: 2.

[0016] The reagent for knocking out the expression of surfactin includes a surfactin knockout vector; the surfactin knockout vector includes a second upstream homology arm, a second resistance gene fragment and a second downstream homology arm;

[0017] The nucleotide sequence of the upstream homology arm of the surfactin vector is shown in SEQ ID NO: 3; the nucleotide sequence of the downstream homology arm of the surfactin gene vector is shown in SEQ ID NO: 4.

[0018] The present invention provides a mutant strain of Bacillus velez strain HN-Q-8, which is a Bacillus velez strain HN-Q-8 with the bacillusene gene and the surfactin gene knocked out;

[0019] The deposit number of the Bacillus Velez strain HN-Q-8 is CGMCC No.19554.

[0020] The present invention provides the use of a mutant strain of the Bacillus velezensis strain HN-Q-8 in producing fengycin and / or preventing and controlling potato diseases.

[0021] The present invention provides a method for producing Fengyuansu by fermentation, comprising culturing the mutant strain of Bacillus velezensis and separating a lipopeptide extract from the culture solution;

[0022] The nitrogen source in the culture medium includes 3-7 g / L sodium glutamate and 8-12 g / L peptone;

[0023] The deposit number of the Bacillus Velez strain HN-Q-8 is CGMCC No.19554.

[0024] Compared with the prior art, the present invention has the following advantages:

[0025] The present invention provides an application of bacillusene or bacillusene combined with surfactin as a target in regulating the yield of fengycin in Bacillus velezensis strain HN-Q-8; the deposit number of the Bacillus velezensis strain HN-Q-8 is CGMCC No.19554. In the present invention, both bacillusene and surfactin competitively consume key substrates such as amino acids and fatty acids, thereby limiting the synthesis efficiency of fengycin. The present invention can reduce the competitive consumption of substrate resources by knocking out the bacillusene gene and / or the surfactin gene, thereby increasing the yield of fengycin. The results of the examples of the present invention show that after knocking out the bacillusene gene or knocking out the bacillusene gene and the surfactin gene in Bacillus velezensis, the yield and antibacterial activity of fengycin are significantly improved compared with the wild type, which indicates that bacillusene or the bacillusene combined with surfactin can be used as a target to regulate the yield of fengycin in Bacillus velezensis.

[0026] The present invention provides a method for producing fengycin by fermentation, comprising culturing a mutant strain of Bacillus velezinii and isolating a lipopeptide extract from the culture broth; the nitrogen source in the culture medium comprises 3-7 g / L sodium glutamate and 8-12 g / L peptone; the deposit number of the Bacillus velezinii strain HN-Q-8 is CGMCC No. 19554. In an embodiment of the present invention, to increase the yield of fengycin in a ΔsrfAAΔbaeBE double knockout mutant strain, sodium glutamate and peptone were added during the preparation of the fermentation medium. The yield was increased by approximately 0.57 times compared to fermentation using sodium glutamate alone, indicating that the addition of sodium glutamate and peptone to the culture medium can significantly increase the yield of fengycin. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 This is the standard curve of Fengyuan;

[0028] Figure 2 This is a graph showing the production levels of Fengyuansu from different Bacillus strains;

[0029] Figure 3 The results of gene cluster analysis of HN-Q-8 strain are shown in Figure 1. A: HN-Q-8 complete genome; B: surfactin, bacilliden, and fengycin synthesis gene clusters;

[0030] Figure 4Electrophoresis diagram for the design of the baeBE knockout vector; A: Required gene for the baeBE knockout vector, MDL2000 marker, 1 upstream homology arm, 2 erythromycin resistance gene, 3 downstream homology arm; B: Double enzyme digestion of the pYC127 plasmid, MDL5000 marker, 1 cut plasmid;

[0031] Figure 5 Colony PCR verification of the baeBE knockout vector; A: baeBE knockout vector, B: colony PCR verification, MM DL5000 marker, lanes 1 to 6: colony PCR amplification electrophoresis detection;

[0032] Figure 6 Figure 2 is the PCR validation result of the ΔbaeBE mutant; M DL5000 marker, 1 negative control HN-Q-8 wild-type strain, 2 bacillus ΔbaeBE strain, 3 positive control;

[0033] Figure 7 Figure 2 shows the PCR verification results of the ΔsrfAAΔbaeBE double knockout mutant. A: srfAA gene PCR verification, MDL5000 marker, 1 negative control WT strain, 2 ΔsrfAAΔbaeBE double knockout mutant, 3 positive control pYC127-ΔsrfAA plasmid amplification; B: baeBE gene PCR verification, MDL5000 marker, 1 negative control WT strain, 2 ΔsrfAAΔbaeBE double knockout mutant, 3 positive control pYC127-ΔbaeBE plasmid amplification;

[0034] Figure 8 This is the statistical result of Fengyuansu production of mutant strains;

[0035] Figure 9 This is the result diagram of the effect of amino acids on Fengyuansu production, MSG is monosodium glutamate;

[0036] Figure 10 This is the result diagram of the effect of adding nitrogen source on Fengyuansu production. A shows the result of adding yeast extract powder, and B shows the result of adding peptone. MSG is monosodium glutamate, and YE is yeast extract powder.

[0037] Figure 11 Figure 2 is the result of the antibacterial activity test of the mutant strain;

[0038] Figure 12 This is a diagram showing the antibacterial effect of the mutant strain on a flat plate;

[0039] Figure 13The figure shows the effect of Fengyuansu on the pathogenicity of potato early blight; A: The growth of potato early blight on leaves; B: The statistical results of the area of ​​lesions caused by potato early blight on leaves;

[0040] Figure 14 The figure shows the effect of Fengyuansu on the pathogenicity of potato black mole bacteria; A: The growth of potato black mole bacteria on potato chips; B: The statistical results of the lesion area caused by potato black mole bacteria on potato chips;

[0041] Figure 15 This is a statistical chart of surfactin production in mutant strains;

[0042] Figure 16 The results of the effects of srfAA and baeBE on the volatile gases produced by the strain are shown in the figure;

[0043] Figure 17 is the morphological picture of the mutant colony;

[0044] Figure 18 Results of the biofilm formation ability test of the mutant strain; A: appearance of biofilm formation and color change after staining; B: statistical results of biofilm formation amount;

[0045] Figure 19 This is the result diagram showing the effects of srfAA and baeBE genes on the growth of HN-Q-8 strain;

[0046] Figure 20 The results of the effects of srfAA and baeBE genes on the conidia production rate of the HN-Q-8 strain are shown in Figure 1. A: Conidia production rate counting plate results; B: Conidia production rate statistics of the strain;

[0047] Figure 21 The results of the effects of srfAA and baeBE genes on the adsorption capacity of HN-Q-8 strain; A: the results of the adsorption capacity of different mutant strains to Congo red; B: the statistical results of the adsorption capacity of srfAA and baeBE genes on HN-Q-8 strain. DETAILED DESCRIPTION

[0048] The present invention provides an application of bacillusene or bacillusene combined with surfactin as a target in regulating the yield of fengycin in Bacillus velezensis strain HN-Q-8;

[0049] The deposit number of the Bacillus Velez strain HN-Q-8 is CGMCC No.19554.

[0050] In the present invention, the bacillusene or bacillusene combined with surfactin negatively regulates the fengycin production of the Bacillus velezensis strain HN-Q-8.

[0051] In the present invention, the Bacillus velezensis HN-Q-8 strain has certain advantages in synthesizing the antibacterial substance fengyogenin. Therefore, genetic modification of the HN-Q-8 strain further improves the content and antibacterial properties of fengyogenin. Both bacillusene and surfactin competitively consume key substrates such as amino acids and fatty acids, thereby limiting the efficiency of fengyogenin synthesis. Therefore, by knocking out bacillusene and / or surfactin, the synthesis pathway of bacillusene and / or surfactin can be blocked, reducing competitive consumption of substrate resources, releasing more precursor substances for fengyogenin biosynthesis, and thus increasing fengyogenin production. In the present invention, the effects of the bacillusene gene and the surfactant gene deletion on the fengin production of the HN-Q-8 strain were determined using the surfactin knockout mutant ΔsrfAA, the bacillusene knockout mutant ΔbaeBE, and the double knockout mutant ΔsrfAAΔbaeBE as materials. The results showed that the fengin production of the bacillusene knockout mutant ΔbaeBE was 1.6 times that of the WT, and the fengin production of the double knockout mutant ΔsrfAAΔbaeBE was 2.4 times that of the WT. This indicates that the surfactin synthesis pathway involved in the srfAA and baeBE genes and the bacillusene synthesis pathway compete with the fengin synthesis pathway, and bacillusene or the bacillusene combined with surfactin can be used as a target to regulate the fengin production of Bacillus velezinis.

[0052] In the present invention, the bacillusene includes the bacillusene gene and / or the protein encoded by the bacillusene gene; the surfactin includes the surfactin gene and / or the protein encoded by the surfactin gene;

[0053] The bacillusene gene preferably includes at least one of baeB, baeC, baeD, and baeE, and more preferably includes baeB, baeC, baeD, and baeE. The gene number and starting position of the baeB gene are: CP045711.1_gene_1715[locus_tag=GG619_08575][location=1712598..1713275]; the gene number and starting position of the baeE gene are: CP045711.1_gene_1718[locus_tag=GG619_08590][location=1715572..1717812].

[0054] The surfactin gene preferably includes at least one of srfAA, srfAB, srfAC and srfAD, and more preferably includes srfAA. The gene number and starting position of the srfAA are: CP045711.1_gene_366[gene=srfAA][locus_tag=GG619_01830][location=341553..352307].

[0055] The present invention provides the use of bacillus cereus and / or surfactin as targets in regulating at least one of the antibacterial activity, spore production ability and biofilm formation of the Bacillus velezensis strain HN-Q-8.

[0056] In the present invention, the bacillus cereus and / or surfactin negatively regulate the antibacterial activity and / or biofilm formation of Bacillus velezensis strain HN-Q-8; the bacillus cereus and / or surfactin positively regulate the spore production ability of Bacillus velezensis strain HN-Q-8.

[0057] In the present invention, the bacillusene negatively regulates the antibacterial activity of Bacillus velezensis strain HN-Q-8 against Fusarium sambucinum (F. sambucinum) and / or Rhizoctonia solani (R. solani); the surfactin negatively regulates the antibacterial activity of Bacillus velezensis strain HN-Q-8 against Fusarium sambucinum (F. sambucinum) and / or Rhizoctonia solani (R. solani); the bacillusene and surfactin negatively regulate the antibacterial activity of Bacillus velezensis strain HN-Q-8 against Fusarium sambucinum, Fusarium oxysporum (F. oxysporum), Alternaria solani, Rhizoctonia solani, potato early blight and potato black spot pathogen.

[0058] In the embodiment of the present invention, the ΔsrfAA mutant strain, the ΔbaeBE mutant strain and the ΔsrfAAΔbaeBE double knockout mutant strain were used as materials to determine the effects of srfAA and baeBE deletion on the antibacterial activity, secondary metabolites, growth and spore production of the HN-Q-8 strain. The results of the plate standoff experiment on the antibacterial activity showed that compared with the wild-type control, the ΔsrfAAΔbaeBE double knockout mutant strain had an antibacterial effect on four fungi, namely, Fusarium sambucinum, Fusarium oxysporum, Alternaria solani and Rhizoctonia solani. The ΔsrfAA mutant strain had an antibacterial effect on Fusarium sambucinum, Alternaria solani and Rhizoctonia solani, and the ΔbaeBE mutant strain had an antibacterial effect on Fusarium sambucinum and Rhizoctonia solani. The results of in vitro antibacterial activity determination showed that compared with the wild-type control, the lesion area of ​​potato leaves or tubers treated with lipopeptides of the ΔsrfAAΔbaeBE double knockout mutant was significantly reduced, and the prevention effect against potato early blight and potato black mole was significantly improved.

[0059] In the present invention, the bacillusene negatively regulates the synthesis of at least one of 2,4-di-tert-butylphenol, acetoin, methoxyphenyloxime, pentadecanoic acid, and 2,5-dimethylpyrazine in the Bacillus velez strain HN-Q-8; the surfactin negatively regulates the synthesis of at least one of 2,4-di-tert-butylphenol, acetoin, methoxyphenyloxime, pentadecanoic acid, 2,5-dimethylpyrazine, benzaldehyde, and phenylethanol in the Bacillus velez strain HN-Q-8; the bacillusene and surfactin negatively regulate the synthesis of at least one of 2,4-di-tert-butylphenol, acetoin, and 2,5-dimethylpyrazine in the Bacillus velez strain HN-Q-8.

[0060] The secondary metabolites of each mutant strain were determined in the present invention. The results showed that knocking out the bacillusene and surfactin genes promoted the synthesis of volatile substances with antibacterial activity, including 2,4-di-tert-butylphenol, acetoin, and 2,5-dimethylpyrazine. Knocking out the bacillusene and surfactin genes promoted the synthesis of 2,4-di-tert-butylphenol, acetoin, methoxyphenyloxime, pentadecanoic acid, and 2,5-dimethylpyrazine, while inhibiting the synthesis of 2-dodecanone and dodecanal. Knocking out the surfactin gene promoted the synthesis of benzaldehyde and phenylethanol, while knocking out the bacillusene gene inhibited their synthesis. Growth and sporulation measurements showed that knocking out the bacillusene and / or surfactin genes increased biofilm formation and reduced sporulation.

[0061] The present invention provides an application of knocking out a bacillusene gene and a surfactin gene in increasing the yield of fengycin and / or the antibacterial activity of a Bacillus velez strain HN-Q-8, reducing the spore production ability of the Bacillus velez strain HN-Q-8, and promoting the formation of a biofilm by the Bacillus velez strain HN-Q-8; the deposit number of the Bacillus velez strain HN-Q-8 is CGMCC No. 19554.

[0062] In the present invention, the reagent for knocking out the expression of the bacillusene gene includes a bacillusene knockout vector; the bacillusene knockout vector includes a first upstream homology arm, a first resistance gene fragment and a first downstream homology arm;

[0063] The nucleotide sequence of the upstream homology arm of the bacillusene vector is as shown in SEQ ID NO: 1

[0064]

[0065] As shown; the lower portion of the bacillus ene gene vector The nucleotide sequence of the homology arm is as shown in SEQ ID NO: 2

[0066] shown.

[0067] The reagent for knocking out the expression of surfactin includes a surfactin knockout vector; the surfactin knockout vector includes a second upstream homology arm, a second resistance gene fragment and a second downstream homology arm;

[0068] The nucleotide sequence of the upstream homology arm of the surfactin vector is as shown in SEQ ID NO: 3

[0069] As shown; the surfactant group The nucleotide sequence of the downstream homology arm of the vector is as shown in SEQ ID NO: 4

[0070] shown.

[0071] In the present invention, the first upstream homology arm of the bacillus ene knockout vector is preferably amplified using the whole genomic DNA of the HN-Q-8 strain as a template by the primer pair bae-up-F (SEQ ID NO: 7) / bae-up-R (SEQ ID NO: 8). The first downstream homology arm of the bacillus ene knockout vector is preferably amplified using the whole genomic DNA of the HN-Q-8 strain as a template by the primer pair bae-down-F (SEQ ID NO: 11) / bae-down-R (SEQ ID NO: 12). The nucleotide sequence of the first resistance gene fragment is

[0072] The first resistance gene fragment is preferably amplified using plasmid PHT315 as a template using primer pair ErR-F (SEQ ID NO: 9) / ErR-R (SEQ ID NO: 10). The bacillusene knockout vector can knock out the bacillusene synthase genes baeB, baeC, baeD, and baeE to block the bacillusene biosynthesis pathway.

[0073] In the present invention, the second upstream homology arm of the surfactin knockout vector is preferably amplified using the whole genomic DNA of the HN-Q-8 strain as a template by the primer pair SrfFront-F (SEQ ID NO: 13) / SrfFront-R (SEQ ID NO: 14), and the second downstream homology arm of the surfactin knockout vector is preferably amplified using the whole genomic DNA of the HN-Q-8 strain as a template by the primer pair SrfBack-F (SEQ ID NO: 15) / SrfBack-R (SEQ ID NO: 16). The nucleotide sequence of the second resistance gene fragment is The second resistance gene segment is preferably the chloramphenicol resistance gene CmR carried by the backbone vector pYC127. The surfactin knockout vector can knock out the surfactin srfAA gene to block the surfactin synthesis pathway.

[0074] In the present invention, the backbone vector of the bacillusene knockout vector or the surfactin knockout vector includes pYC127. The cloning sites of the exogenous gene in the bacillusene knockout vector are AatII and EcoRV; the cloning sites of the upstream homology arm in the surfactin knockout vector are AatII and BamHI, and the cloning sites of the downstream homology arm are SphⅠ and EcoRV.

[0075] The present invention provides a mutant strain of Bacillus velez strain HN-Q-8, which is a Bacillus velez strain HN-Q-8 with the bacillusene gene and the surfactin gene knocked out;

[0076] The deposit number of the Bacillus Velez strain HN-Q-8 is CGMCC No.19554.

[0077] In the present invention, the method for constructing the mutant strain (double knockout mutant ΔsrfAAΔbaeBE) of the Velez Bacillus strain HN-Q-8 preferably comprises transforming the surfactin knockout vector into the Velez Bacillus strain HN-Q-8 to obtain the surfactin knockout mutant ΔsrfAA. The knockout mutant ΔsrfAA is transformed with the bacillus ene gene homologous recombination vector to obtain the double knockout mutant ΔsrfAAΔbaeBE. The transformation method preferably comprises a chemical transformation method. The knockout mutant ΔsrfAA is identified by the primer pair SrfFront-F (SEQ ID NO: 13) / SrfBack-R (SEQ ID NO: 14). The double knockout mutant strain ΔsrfAAΔbaeBE was identified by primer pair bae-up-F (SEQ ID NO: 7) / bae-down-R (SEQ ID NO: 8) and primer pair SrfFront-F (SEQ ID NO: 13) / SrfBack-R (SEQ ID NO: 14). Primer pairs were used to identify the authenticity of the strain species using gyrB-F (SEQ ID NO: 17) and gyrB-R (SEQ ID NO: 18). In the embodiment of the present invention, a surfactin knockout mutant strain ΔsrfAA, a bacillus ene knockout mutant strain ΔbaeBE and a double knockout mutant strain ΔsrfAAΔbaeBE were constructed by the method described. The antibacterial activity, fengycin production and biofilm formation of the bacillus ene knockout mutant strain ΔbaeBE and the double knockout mutant strain ΔsrfAAΔbaeBE were significantly improved, and the spore production ability was significantly reduced.

[0078] Based on the characteristics of the mutant strain of the Bacillus velez strain HN-Q-8 having high fengycin production and high antibacterial activity, the present invention provides an application of the mutant strain of the Bacillus velez strain HN-Q-8 in producing fengycin and / or preventing and controlling potato diseases.

[0079] The present invention provides a method for producing Fengyuansu by fermentation, comprising culturing the mutant strain of Bacillus velezensis and separating a lipopeptide extract from the culture solution;

[0080] The nitrogen source in the culture medium includes 3-7 g / L sodium glutamate and 8-12 g / L peptone;

[0081] The deposit number of the Bacillus Velez strain HN-Q-8 is CGMCC No.19554.

[0082] In the present invention, the mutant strain of Bacillus velez is cultured. The nitrogen source in the culture medium preferably includes 4-6 g / L sodium glutamate and 9-11 g / L peptone, more preferably 5 g / L sodium glutamate and 10 g / L peptone. The culture medium also includes the following components: KCl 0.5 g / L, KH2PO4 1 g / L, MgSO4 0.5 g / L, MnSO4 5 mg / L, CuSO4 0.16 mg / L, FeSO4·7H2O 0.15 mg / L and glucose 20 g / L. The culture temperature is preferably 35-39°C, more preferably 37°C. The culture time is preferably 36-60 h, more preferably 42-54 h, and most preferably 48 h. The culture can increase the content of fengyrin in Bacillus velez and improve the antibacterial activity of Bacillus velez. In an embodiment of the present invention, in order to increase the yield of Fengyuan in the ΔsrfAAΔbaeBE double knockout mutant, sodium glutamate was replaced with proline, valine, tyrosine, threonine or alanine when preparing the fermentation medium. The results showed that the Fengyuan yield in each group was lower than that when sodium glutamate was used alone. After further supplementation with yeast extract powder and protein commonly used in bacterial culture media as nitrogen sources, the Fengyuan yield in each group was still lower than that when sodium glutamate was used alone. Finally, after supplementation with peptone as a nitrogen source, the Fengyuan yield increased significantly, among which the fermentation group containing sodium glutamate and peptone increased by about 0.57 times compared with the fermentation using sodium glutamate alone, indicating that the addition of sodium glutamate and peptone in the culture medium can significantly increase the yield of Fengyuan, thereby improving the antibacterial activity.

[0083] In order to further illustrate the present invention, the solutions provided by the present invention are described in detail below with reference to the accompanying drawings and embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0084] Materials and methods

[0085] 1. Test materials

[0086] 1.1 Strains and plasmids

[0087] The pYC127 expression vector is consistent with that in the prior art (Chai Y, Chu F, Kolter R, Losick R. Bistability and biofilm formation in Bacillus subtilis. Mol. Microbiol. 2008; 67: 254-263. doi: 10.1111 / j.1365-2958.2007.06040.x.).

[0088] Table 1 Test strains and plasmids

[0089] plasmids Features and uses source PHT315 expression vector Baosai Bio Cloning host Escherichia coli (E. coli) DH5α Competent cells Tiangen Biochemical Technology (Beijing) Co., Ltd.

[0090] 1.2 Main test reagents

[0091] Table 2 Main test reagents

[0092] Main test reagents source Restriction endonuclease AatII New England Biotechnology (Beijing) Co., Ltd. Restriction enzyme EcoRV New England Biotechnology (Beijing) Co., Ltd. Restriction endonuclease BamHI New England Biotechnology (Beijing) Co., Ltd. Restriction endonuclease SphⅠ New England Biotechnology (Beijing) Co., Ltd. T4 DNA ligase New England Biotechnology (Beijing) Co., Ltd. SuperKfxDNAPolymerase Kangwei Century Biotechnology Co., Ltd. Bacterial DNA extraction kit Quanshijin Biotechnology Co., Ltd. Standard agarose gel DNA recovery kit Shanghai Shenggong Bioengineering Co., Ltd. Plasmid extraction kit Tiangen Biochemical Technology Co., Ltd. Homologous Recombination Seamless Cloning Kit Quanshijin Biotechnology Co., Ltd. Ampicillin Beijing Solebow Technology Co., Ltd. Chloramphenicol Beijing Solebow Technology Co., Ltd. Erythromycin Beijing Solebow Technology Co., Ltd.

[0093] 1.3 Culture medium

[0094] (1) Potato dextrose agar (PDA): Peel 200 g of potatoes, cut into small pieces, and boil in boiling water for 20 min. Filter through gauze, add 20.0 g of glucose and 20.0 g of agar to the filtrate, dilute to 1 L with distilled water, and sterilize at 115°C for 30 min.

[0095] (2) Oatmeal agar medium (OMA): 30 g of oat grains / oat flakes, 20 g of agar, and distilled water to 1000 mL.

[0096] (3) Water agar medium: 10 g agar, distilled water to 1000 mL.

[0097] (4) Seed culture medium: beef extract 5 g, peptone 10 g, yeast powder 5 g, NaCl 5 g, glucose 10 g, distilled water to 1 L, pH = 7.0;

[0098] (5) Fermentation medium: 5 g sodium L-glutamate, 0.5 g KCl, 1 g KH2PO4, 0.5 g MgSO4, 5 mg MnSO4, 0.16 mg CuSO4, 0.15 mg FeSO4·7H2O, 20 g glucose, distilled water to 1 L, pH = 7.0, sterilized at 115°C for 30 min;

[0099] (6) LB medium: 10 g of sodium chloride, 5 g of yeast extract, and 10 g of tryptone, dilute to 1 L with distilled water, and sterilize at 121°C for 20 min. Solid LB medium contains 1.5% agar powder;

[0100] (7) LBGM medium: LB medium supplemented with 1% glycerol and 100 μM MnCl2

[0101] (8) Transformation medium

[0102] GCHE: glucose 10 g / L, potassium L-glutamate 2 g / L, 100 mM potassium phosphate buffer (pH 7.0), trisodium citrate 0.88 g / L, MgSO4 0.36 g / L, ammonium ferric citrate 0.022 g / L, L-tryptophan 0.05 g / L, hydrolyzed casein 2 g / L.

[0103] GC: glucose 10 g / L, 100 mM potassium phosphate buffer (pH 7.0), trisodium citrate 0.88 g / L, MgSO4 0.36 g / L, ammonium ferric citrate 0.022 g / L, L-tryptophan 0.05 g / L.

[0104] Conversion buffer: (NH4)2SO41.98g / L, K2HPO313.92g / L, KH2PO46.12g / L, MgCl22.85g / L, EGTA0.38g / L, glucose 4.5g / L and trisodium citrate 10.29g / L.

[0105] 1.4 Instruments and equipment used in the test

[0106] Table 3 Main instruments and equipment

[0107] Instrument name Manufacturer High-pressure steam sterilizer JapanHirayama Constant temperature oscillator Shanghai Youyi Instrument Spectrophotometer Shenzhen Leidu High-speed centrifuge Thermo Fisher Scientific Clean bench Suzhou Antai Constant temperature incubator Ningbo Jiangnan Instrument Factory PCR instrument Thermo Fisher Scientific electrophoresis apparatus Bio-Rad Life Sciences Products Co., Ltd. Gel imaging system Bio-Rad Life Sciences Products Co., Ltd. Agilent HPLC 1260 Agilent Technologies

[0108] 2. Test methods

[0109] 2.1 Crude extraction method of Bacillus velezinoffe

[0110] (1) A single colony was picked from the LB plate and inoculated into a seed culture medium (250 mL shake flask, 100 mL of liquid) and cultured on a shaker (37°C, 180 rpm) for 24 h.

[0111] (2) Inoculate 2% of the seed solution into a fermentation medium (250 mL shake flask, 100 mL of liquid) and culture on a shaker (37°C, 200 rpm) for 48 h to obtain a Bacillus fermentation broth;

[0112] (3) The fermentation broth was divided into 50 mL centrifuge tubes, balanced, and centrifuged at 5000 rpm for 100 min at 4°C to collect the supernatant and remove the bacterial cells.

[0113] (4) The supernatant was adjusted to pH 2.0 with 6 M HCl and placed in a refrigerator at low temperature overnight;

[0114] (5) All the solution was transferred to a 50 mL centrifuge tube and centrifuged at 5000 rpm for 10 min at 4°C in a low-temperature refrigerated centrifuge. The supernatant was discarded and the precipitate was repeatedly extracted with methanol.

[0115] (6) The resulting solution was transferred to a 50 mL centrifuge tube and centrifuged in a low-temperature refrigerated centrifuge at 4°C and 8000 rpm for 10 min. The resulting supernatant was the crude lipopeptide extract.

[0116] (7) The crude lipopeptide extract was filtered through a 0.22 μm organic filter membrane to obtain the lipopeptide extract for HPLC quantitative analysis.

[0117] 2.2 HPLC quantitative detection method of fengycin and surfactin content

[0118] The chromatographic column was Agilent C18 column, the column temperature was 30 °C, and the injection volume was 10 μg

[0119] Fengycin content determination: 60% acetonitrile aqueous solution (containing 0.1% trifluoroacetic acid); flow rate 1 mL / min, 210 nm UV detection. For details, refer to the prior art (Zhu Hongyuan. Research on the effective antibacterial components of Bacillus amyloliquefaciens B15 [D]. Xinjiang University, 2015.) for fengycin determination.

[0120] Surfactin content determination: 0-9 min: acetonitrile (0.1% TFA) 60-93%, water (0.1% TFA) 40-7%; 9-20 min: acetonitrile (0.1% TFA) 93%, water (0.1% TFA) 7%; flow rate 0.84 mL / min; UV detection at 210 nm. For details, refer to the liquid phase method for detecting surfactin content in the existing technology (Zhou Yingjun. Breeding of biocontrol bacteria for potato scab and research on their disease resistance mechanism [D]. Jiangnan University, 2023. DOI: 10.27169 / d.cnki.gwqgu.2023.000090.).

[0121] 2.3 Establishment of fengyuan standard curve

[0122] The pure Fengyuansu was diluted to different concentrations and analyzed under isocratic liquid phase conditions. Each sample was injected three times. The results showed that the peak time of Fengyuansu ranged from 5.7min to 16min. The peak area of ​​Fengyuansu obtained by integrating closely to the baseline was used to obtain a calibration curve of peak area and concentration through regression analysis. The equation was y = 0.0594x + 3.6371, and the correlation coefficient was 0.997 ( Figure 1 ).

[0123] 2.4 Method for determining the level of fengycin production in strains

[0124] The antimicrobial lipopeptide fengyrin was extracted from the fermentation broth using acid precipitation and quantitatively analyzed by high-performance liquid chromatography (HPLC). The fengyrin production of each strain was calculated by substituting the peak area corresponding to the retention time (5.7 min to 16 min) into the standard curve, thereby evaluating the fengyrin production capacity of the strain. Figure 2 This is a graph showing the production levels of fengyuansu from different Bacillus strains.

[0125] Example 1

[0126] Construction of the ΔsrfAAΔbaeBE double knockout mutant

[0127] 1. HN-Q-8 strain gene cluster analysis and HN-Q-8 strain transformation strategy

[0128] Through antiSMASH online prediction analysis of the gene cluster of the HN-Q-8 strain, the non-ribosomal synthetase genes fenC, fenD, fenE, fenA, and fenB on the 1.6 gene cluster have the complete function of fengycin synthesis; bacillaene on the 1.5 gene cluster contains multiple key genes, such as baeA, baeB, baeC, baeD, and baeE, which are involved in the biosynthesis of bacillaene; the non-ribosomal synthetase genes srfAA, srfAB, srfAC, and srfAD on the 1.1 gene cluster are responsible for the synthesis of surfactin. By comparing with the whole genome of the HN-Q-8 strain, the baeB, baeC, baeD, and baeE genes were selected to be knocked out to hinder their synthesis.

[0129] Both bacillusene and surfactin competitively consume key substrates such as amino acids and fatty acids, limiting the efficiency of fengycin synthesis. Based on this, the genetic modification strategy for the HN-Q-8 strain involved: knocking out the srfAA gene to create a ΔsrfAA mutant that blocks surfactin synthesis; knocking out the baeB, baeC, baeD, and baeE genes (baeBE) to create a ΔbaeBE mutant that blocks bacillusene synthesis; and constructing a ΔsrfAAΔbaeBE double knockout mutant that simultaneously blocks the synthesis pathways of surfactin and bacillusene, reducing competitive consumption of substrate resources and releasing more precursors for fengycin biosynthesis.

[0130] The gene cluster online analysis website antiSMASH was used to analyze the fengycin and bacillaen gene cluster information of Bacillus velezensis strain HN-Q-8 (CP045711.1), and the desired target genes were found by comparing the whole genome of the HN-Q-8 strain.

[0131] The target gene required for the bacillusene knockout vector design consists of three parts: 1. The bacillusene baeB and baeC genes are amplified as upstream homology arms using the bae-up-F / bae-up-R primers; 2. The erythromycin resistance fragment is amplified using the ErR-F / ErR-R primers and the PHT315 plasmid as a template; 3. The downstream homology arm is amplified downstream of the baeE gene using the bae-down-F / bae-down-R primers. The bacillusene synthase genes baeB, baeC, baeD, and baeE are knocked out to block the bacillusene biosynthesis pathway.

[0132] Surfactin knockout vector design: Use primers Srf Front-F / Srf Front-R to amplify the upstream homology arm of srfAA, and primers Srf Back-F / Srf Back-R to amplify the downstream homology arm of srfAA. Knockout the surfactin srfAA gene to block the surfactin biosynthesis pathway. The nucleotide sequences of the primers are shown in Table 4.

[0133] Table 4 Primer names and sequences

[0134]

[0135] Upstream and downstream homology arm amplification method: The complete genome of Bacillus velezensis HN-Q-8 was extracted using the Complete Gold Bacteria Genomic DNA Purification Kit. Using the HN-Q-8 complete genomic DNA as a template, the bacillusene baeB gene was amplified using the bae-up-F / bae-up-R method as the upstream homology arm. The downstream homology arm was amplified downstream of the baeE gene using the bae-down-F / bae-down-R method. The upstream homology arm of srfAA was amplified using primers SrfFront-F / SrfFront-R, and the downstream homology arm of srfAA was amplified using primers SrfBack-F / SrfBack-R. The amplification reaction system is shown in Table 5, and the reaction procedure is shown in Table 6.

[0136] Table 5 PCR reaction system

[0137] Element content DNA template 2μL Primers 1 μL each SuperKfxDNAPolymerase 25 μL <![CDATA[ddH2O]]> Dilute to 50 μL

[0138] Table 6 PCR reaction program

[0139]

[0140]

[0141] After the PCR reaction was completed, the amplified product was detected by 1.0% agarose gel electrophoresis.

[0142] After agarose gel electrophoresis, the amplified product was excised and recovered using a common agarose gel DNA recovery kit.

[0143] The extraction methods of pYC127 plasmid and subsequent recombinant plasmids were based on the plasmid extraction kit (Tiangen Biochemical Technology Co., Ltd.).

[0144] The plasmid vector was double-digested with restriction endonucleases. After the double-digestion reaction at 37° C. for 1 h, the plasmid was separated by 1% 1.0% agarose gel electrophoresis. The target band was cut out and recovered from the gel to obtain a linearized vector.

[0145] Table 7 Double enzyme digestion reaction conditions

[0146] Element content plasmids 1 μL Restriction endonuclease AatII 1 μL Restriction endonuclease EcoRV 1 μL rCutSmart 5μL <![CDATA[ddH2O]]> Make up to 50 μL

[0147] Seamless cloning was performed using the full-strength gold seamless cloning kit method. The linearized vector and the amplified DNA fragment were ligated and reacted at 50°C for 15 minutes to obtain the recombinant product, which was then stored at -20°C.

[0148] Table 8 Seamless cloning reaction system

[0149] Element content 2×BasicAssemblyMix 5μL Linearized vector 1 μL Fragmented DNA 1:1:1 <![CDATA[ddH2O]]> Make up to 10 μL

[0150] Method for transforming DH5α competent cells:

[0151] Take the DH5α competent cells out of the -80℃ freezer and quickly place them on an ice bath to melt; add 2ml of the recombinant product to 50ml of cells, gently flick the wall of the centrifuge tube to mix, and place on ice for 30min; heat shock in a 42℃ water bath for 30s, then immediately transfer to ice to cool for 2min; add 450μL LB medium and culture at 37℃ and 250rpm for 1h; take 100μL of cells and evenly spread them on a plate containing 50μg / mL ampicillin, and culture in a 37℃ incubator overnight; the next day, select a single clone into 10μL of sterile water, pipette to mix, take 1μL of the mixture into a 25μL PCR system, and use primers bae-up-F / bae-down-R and / or primers SrfFront-F / SrfBack-R to identify positive clones.

[0152] Chemical transformation of HN-Q-8 strain:

[0153] (1) The HN-Q-8 strain suspension shaken overnight was diluted with GCHE to OD 600 = 0.3, 37 ° C, 200 rpm culture until OD 600 = about 1.4;

[0154] (2) Dilute with an equal volume of GC solution and incubate at 37°C, 200 rpm, for 1 h;

[0155] (3) Aliquot 3 mL into each tube, centrifuge at 6000 rpm for 5 min at 4°C, and resuspend in 200 μl of the supernatant;

[0156] (4) Add 1 μg of recombinant plasmid and 2 mL of transformation buffer and incubate at 37°C, 75 rpm for 30 min;

[0157] (5) Add 1 mL of liquid LB medium (containing 0.3 μg / mL erythromycin solution) and incubate at 37°C, 200 rpm, for 2 h;

[0158] (6) Each 200 μl of culture solution was spread on solid LB medium containing 3 μg / mL erythromycin solution and cultured at 37°C;

[0159] (7) The next day, single colonies were picked and placed in an equal concentration of antibiotics and cultured overnight at 37°C and 200 rpm.

[0160] (8) DNA was extracted from the strain cultured overnight, and primers bae-up-F / bae-down-R were used to identify bacillus-positive clones; primers Srf Front-F / Srf Back-R were used to identify surfactin-positive clones; primers gyrB-F (TGRCGGHRGYGGHTATAAAGT, SEQ ID NO: 17) and gyrB-R (TCCDCCSTCAGARTCWCCCTC, SEQ ID NO: 18) were used to identify the authenticity of the strain species. At the same time, the strain was cultured for three consecutive generations to observe its stability.

[0161] The ΔsrfAAΔbaeBE double knockout mutant was constructed by chemically transforming the ΔsrfAA and ΔbaeBE mutants into the baeBE / srfAA knockout vector. Primers bae-up-F / bae-down-R were used to identify the baeBE mutation site, and primers SrfFront-F / SrfBack-R were used to identify the srfAA mutation site.

[0162] 2. Methods for constructing the ΔbaeBE mutant

[0163] Using Bacillus velezensis HN-Q-8 as the research strain, a bacillusene gene deletion strain was constructed by DNA homologous recombination technology. Primers were designed based on the genome of Bacillus velezensis HN-Q-8 strain, and the 809bp baeB gene sequence was selected as the upstream homology arm and the 790bp baeB gene sequence was selected as the downstream homology arm. The knockout vector primers were designed to carry the erythromycin resistance gene, and the upstream and downstream homology arms of the target gene were amplified by PCR ( Figure 4 A). The plasmid vector was double-digested with restriction endonucleases AatII and EcoRV. After the circular plasmid was double-digested, the electrophoretic band migration rate was higher than that of the undigested position, indicating that the plasmid vector was successfully double-digested and can be used for seamless cloning ( Figure 4 Middle B).

[0164] The product was recovered by gel, and impurities such as primers and DNA were removed. The target gene recombinant fragment was connected to the linearized vector double enzyme digestion product through seamless cloning.

[0165] The seamless cloning reaction products were transformed into E. coli DH5α chemically competent cells and cultured at 37°C.

[0166] Subsequently, a preliminary colony PCR amplification was performed on the single colonies grown to verify whether they were correctly connected. Electrophoresis detection showed that the results were consistent with the expected fusion fragment size. Among them, colony No. 2 failed to amplify a band and was determined to be a false positive; the remaining colonies successfully amplified the correct target fragment ( Figure 5 This indicates that the transformation of the recombinant product was successfully completed, and the seamless cloning efficiency was high. Ultimately, the bacillus knockout vector pYC127-ΔbaeBE for the Bacillus velezensis HN-Q-8 strain was successfully constructed.

[0167] The knockout plasmid was introduced into the Bacillus velezensis HN-Q-8 strain by chemical transformation. After plate culture, the single colonies grown were verified by PCR using primers bae-up-F and bae-down-R. Figure 6 As shown, the fragment amplified from the WT strain was greater than 5000bp, while the fragment amplified from the selected transformants was approximately 3000bp, consistent with the positive control. Further sequencing and blast comparison of the amplified PCR products revealed that the sequence of the amplified product fragment shared 100% homology with the designed baeBE knockout vector, successfully generating the baeBE gene mutant strain ΔbaeBE.

[0168] 3. Method for constructing ΔsrfAA mutant

[0169] The upstream homology arm of srfAA was amplified using primers SrfFront-F / SrfFront-R, and the downstream homology arm of srfAA was amplified using primers SrfBack-F / SrfBack-R, carrying the chloramphenicol resistance gene. The amplified fragments were connected to the linearized vector after double digestion of pYC127 and transformed into Escherichia coli HD5α. The correct plasmid was extracted and verified to be the surfactin knockout vector pYC127-Δ sr fAA.

[0170] The obtained knockout vector pYC127-ΔsrfAA was chemically transformed into the HN-Q-8 strain and verified using primers srf Front-F / srfBack-R. The fragment size of the successfully transformed strain was approximately 4 kb, and the fragment amplified from the wild-type strain was approximately 10 kb. The successfully transformed strain was designated as the ΔsrfAA mutant strain.

[0171] 4. Method for constructing ΔsrfAAΔbaeBE double knockout mutant

[0172] The ΔsrfAA mutant strain and the ΔbaeBE mutant strain were used as chassis strains and transferred into the pYC127-baeBE knockout vector or the pYC127-srfAA knockout vector by chemical transformation. Finally, the ΔsrfAAΔbaeBE double knockout mutant strain with the ΔsrfAA mutant strain as the chassis strain was obtained.

[0173] The specific process is as follows: strain ΔsrfAA is used as the chassis strain, and the pYC127-baeBE knockout vector is transferred into it by chemical transformation. After plate culture, the single colony grown is verified by PCR using primers Srf Front-F / Srf Back-R and bae-up-F / bae-down-R. Figure 7 As shown, the srfAA and baeBE fragments amplified from the WT strain were both greater than 5000 bp, while the fragment amplified from the selected transformants was approximately 3000 bp, consistent with the positive control. Further sequencing and blast analysis of the amplified PCR products revealed 100% sequence homology between the amplified product fragment and the designed baeBE knockout vector. Thus, a ΔsrfAAΔbaeBE double knockout mutant was successfully generated.

[0174] Example 2

[0175] Detection of Fengyuansu content in mutant strains

[0176] 1. Determination of Fengyuansu Production in Mutant Strains

[0177] In order to explore the effect of srfAA and baeBE gene deletion on the yield of fengycin synthesized by fermentation of the strain, the ΔsrfAA mutant, ΔbaeBE mutant and ΔsrfAAΔbaeBE double knockout mutant constructed in Example 1 were compared with the wild-type strain (WT) in terms of fengycin yield. Single colonies were picked from the plates activated for 24 hours and inoculated into seed culture medium respectively, and cultured under shaking conditions of 37°C and 200rpm for 24 hours. Subsequently, the culture was inoculated into fermentation medium (250mL shake flask, liquid volume 100mL) at an inoculation ratio of 2%, and the culture was continued under shaking conditions for 48 hours. The relationship between the sample concentration and peak area of ​​the antibacterial lipopeptide fengycin obtained by fermentation was detected by HPLC high performance liquid chromatography. The sample peak area was calculated based on the established fengycin standard curve, and the fengycin yield of the three mutant strains was calculated.

[0178] Depend on Figure 8It can be seen that after the srfAA gene was knocked out, the fengycin production increased from 41.32 mg / L to 49.43 mg / L, but the fengycin production of the ΔsrfAA mutant was not significantly different from that of the WT. After the baeBE gene was knocked out, the fengycin production increased to 66.12 mg / L, a 0.6-fold increase compared to the WT. When both the srfAA and baeBE genes were knocked out simultaneously, the fengycin production further increased to 98.83 mg / L, a 1.4-fold increase compared to the WT.

[0179] These results suggest that the surfactin and bacillusene biosynthesis pathways, which are involved in the srfAA and baeBE genes, compete with the fengycin biosynthesis pathway. When these biosynthesis pathways are blocked, the synthesis substrate is more inclined to fengycin synthesis, resulting in increased fengycin production.

[0180] 2. Effect of culture medium component optimization on Fengyuansu production

[0181] In order to further improve the yield of Fengyuansu in the ΔsrfAAΔbaeBE double knockout mutant, the culture medium was optimized. When preparing the fermentation medium, sodium glutamate was replaced with proline, valine, tyrosine, threonine or alanine, and the amino acid dosage was 5g / L. Figure 9 As shown in the figure, the production of fengycin after adding different amino acids was 54.47 mg / L (proline), 13.03 mg / L (valine), 5.08 mg / L (tyrosine), 49.02 mg / L (threonine) and 40.65 mg / L (alanine). Among the added amino acids, the production of fengycin was the lowest when tyrosine was added, while the production was the highest when proline was added. However, there was still a large gap in production compared to the addition of monosodium glutamate. It is speculated that this may be because directly replacing monosodium glutamate with amino acids may lead to nutrient imbalance in the culture medium, or monosodium glutamate is a key substrate in the synthesis of fengycin.

[0182] In order to verify the above hypothesis, the fermentation medium or the fermentation medium containing sodium glutamate was replaced with proline, valine, tyrosine, threonine or alanine, and yeast extract powder and protein commonly used in bacterial culture medium were used as nitrogen sources. Figure 10 As shown in Figure A, after adding 5g / L yeast extract powder, the yields of Fengyuansu increased significantly, reaching 82.93mg / L, 81.56mg / L, 57.22mg / L, 43.00mg / L, and 74.07mg / L, respectively. On the contrary, the combination of monosodium glutamate and yeast extract powder had the lowest yield of only 33.69mg / L. The highest yield was proline + yeast extract powder, but it was still significantly lower than the yield of monosodium glutamate.

[0183] Further optimization of the nitrogen source revealed that when 10 g / L peptone was added as a nitrogen source supplement, the yield of fengyuan also increased significantly, increasing to 134.83 mg / L, 147.23 mg / L, 112.17 mg / L, 147.71 mg / L and 75.65 mg / L, respectively. The highest yield was obtained with sodium glutamate + peptone, with fengyuan further increased to 155.61 mg / L, which was about 0.57 times higher than that when sodium glutamate was used alone ( Figure 10 This indicates that optimizing the nitrogen source composition in the culture medium can significantly increase the yield of fengycin, providing a reference for subsequent fermentation culture optimization.

[0184] The final optimized fermentation medium was: 5 g / L sodium glutamate, 10 g / L peptone, 0.5 g / L KCl, 1 g / L KH2PO4, 0.5 g / L MgSO4, 5 mg / L MnSO4, 0.16 mg / L CuSO4, 0.15 mg / L FeSO4·7H2O and 20 g / L glucose.

[0185] Example 3

[0186] Effects of srfAA and baeBE genes on antibacterial effects

[0187] 1. Determination of antibacterial activity by plate standoff method

[0188] In order to clarify the changes in the antibacterial activity of the mutant strain, the plate confrontation method was used to determine the inhibitory effects of the lipopeptide extracts extracted from the mutant strain on four potato pathogens.

[0189] A bacterial cake approximately 5 mm in diameter was inoculated in the center of a potato dextrose agar (PDA) plate. 200 μL of the lipopeptide extract was added to an Oxford cup placed 25 cm from the center of the cake. For the control group, an equal amount of methanol was added. The PDA plates containing the indicator strains were incubated at 25°C for 5 days to evaluate the effects of changes in fengycin content in the different mutants on their antimicrobial activity.

[0190] The results are as follows Figure 11 As shown, the three mutant strains showed significant changes in their inhibitory effects on F. sambucinum. Among them, the strain ΔsrfAAΔbaeBE had the most significant inhibitory effect, with an inhibition bandwidth of 5.54 mm, which was 59.37% higher than the wild-type (WT) strain.

[0191] In the inhibitory effect test on Fusarium oxysporum (F. oxysporum), the inhibitory ability of the ΔsrfAA mutant strain decreased, while the ΔsrfAAΔbaeBE double knockout mutant strain showed significant inhibitory effect with an inhibition bandwidth of 9.87 mm, which was 39.80% higher than that of the WT strain.

[0192] For A. solani, both the ΔsrfAA mutant and the ΔsrfAAΔbaeBE double knockout mutant showed significant inhibitory ability, among which the inhibition bandwidth of the ΔsrfAAΔbaeBE double knockout mutant was 14.40 mm, which was 33.33% higher than that of the WT strain.

[0193] In the inhibition test against R. solani, all three mutant strains showed significantly improved inhibition ability, especially the ΔsrfAAΔbaeBE double knockout mutant, whose inhibition bandwidth was 11.03 mm, an increase of 113.76% compared with the WT strain.

[0194] The wild-type HN-Q-8 strain produced 41.32 mg / L of fengycin, while the ΔsrfAAΔbaeBE double-knockout mutant increased its yield to 98.83 mg / L. This significant increase in fengycin production in the HN-Q-8 strain also enhanced the antifungal activity of its lipopeptide solution, reaching levels comparable to those of B. amyloliquefaciens ZR against F. sambucinum and A. solani. This suggests that enhancing fengycin biosynthesis through genetic engineering can further enhance the antifungal activity of lipopeptides derived from the HN-Q-8 strain.

[0195] 2. In vitro assay to determine the effect of ΔsrfAAΔbaeBE on the pathogenicity of potato pathogens

[0196] The lipopeptide solution was evenly sprayed onto the surfaces of leaves and tubers. An equal amount of distilled water was applied as a control group, while amystrobin was sprayed as a positive chemical control. After 24 hours of moisturizing in a dark environment at 25°C, 20 μL of spore suspension or bacterial cake was inoculated onto the surfaces of leaves or tubers, respectively. Subsequently, leaves were moisturized and incubated at 25°C under alternating dark and light conditions (8 hours of light, 16 hours of darkness) for 5 days; tubers were moisturized and incubated at 25°C in the dark for 5 days. Finally, the area of ​​lesions on leaves and tubers was observed and counted.

[0197] 1) The results of the experiment on the effect of ΔsrfAAΔbaeBE on the pathogenicity of potato early blight showed that with the increase of fengyogenin content, the control effect of ΔsrfAAΔbaeBE double knockout mutant on potato early blight was significantly improved. Figure 13As shown in the figure, the lesion area of ​​the leaves of the control group (CK) reached 1.77 cm 2 No lesions were observed on the leaves of the azoxystrobin-treated group (chemical positive control). In the wild-type (WT) lipopeptide-treated group, the lesion area was 0.41 cm 2 Compared with CK, the disease area was reduced by 76.70%, and the control effect was 76.84%. After the ΔsrfAAΔbaeBE double knockout mutant was treated with lipopeptide, the lesion area was further reduced to 0.24 cm 2 Compared with the wild-type WT group, the lesion area of ​​the ΔsrfAAΔbaeBE double knockout mutant group decreased by 41.46%, and the control effect increased by 12.49% compared with the WT group.

[0198] 2) The results of the experiment on the effect of ΔsrfAAΔbaeBE on the pathogenicity of potato black mole showed that the ΔsrfAAΔbaeBE double knockout mutant had significantly improved efficacy against potato black mole compared with the wild-type strain. Figure 14 The lesion area of ​​the control group was 8.92 cm 2 No lesions were observed in the positive control azoxystrobin treatment. After wild-type (WT) lipopeptide treatment, the lesion area was 7.10 cm 2 , which was reduced by 20.40% compared with the control group, with a prevention efficiency of 20.40%; after treatment with lipopeptide from the ΔsrfAAΔbaeBE double knockout mutant, the lesion area was 5.11cm 2 , which was reduced by 42.71% compared with the control group, and the prevention effect was 42.71%; the lesion area of ​​the ΔsrfAAΔbaeBE double knockout mutant was reduced by 28.17% compared with the wild WT, and the prevention effect was increased by 109.36% compared with the WT.

[0199] This indicates that increasing the fengycin content significantly enhances the antibacterial effect of the lipopeptide solution, significantly improving the control of the ΔsrfAAΔbaeBE double-knockout mutant against potato early blight and black spot. Furthermore, in vitro experiments further demonstrate that enhancing fengycin biosynthesis through genetic modification can further enhance the biocontrol potential of the HN-Q-8 strain, thereby expanding its application.

[0200] Example 4

[0201] Effects of srfAA and baeBE genes on other secondary metabolites

[0202] 1. Effect on surfactin production

[0203] To investigate the effect of the srfAA gene on surfactin synthesis in the strain, the extracted lipopeptide solution was used for quantitative detection of surfactin by HPLC. The results showed that within the retention time range of 12 to 24 minutes, the peak area of ​​the ΔsrfAA mutant and the ΔsrfAAΔbaeBE double knockout mutant was significantly reduced within this retention time.

[0204] The changes in the production of surfactant in the mutant strains are as follows Figure 15 As shown. The surfactin peak area of ​​the wild type (WT) is 411.11 (mAU*s). In contrast, the surfactin peak area of ​​the ΔsrfAA mutant strain decreased significantly to 116.4 (mAU*s), which is about 0.28 times that of the WT. The surfactin peak area of ​​the ΔbaeBE mutant strain is 493.9 (mAU*s), which increased by 0.20 times compared with the WT, but the difference was not significant. The surfactin peak area of ​​the ΔsrfAAΔbaeBE double knockout mutant strain is 153.11 (mAU*s), which is 0.63 times that of the WT. The results showed that the deletion of the srfAA gene significantly reduced the surfactin production of the strain, while the deletion of the baeBE gene had no direct effect on the synthesis of surfactin.

[0205] 2. Effects of srfAA and baeBE gene knockout on volatile substances in HN-Q-8 strain

[0206] Pick a single colony into liquid LB, shake at 37℃ and 200rpm for 24h, inoculate into LB medium (250mL shake flask, liquid volume 100mL) at a ratio of 2%, and culture at 37℃ and 200rpm until OD 600 =0.8.

[0207] Sample preparation: Before the experiment, the chromatographic column was conditioned to eliminate impurity peaks. The DVB / CAR / PDMS extraction tip was then conditioned at the gas chromatograph inlet until the sample was free of impurity peaks. 5.0 mL of bacterial culture was accurately measured and placed in a 20 mL headspace vial. The vial was equilibrated at 50°C in a water bath with magnetic stirring for 30 min. The conditioned extraction tip was then inserted and extracted for 40 min. The tip was then removed and immediately inserted into the inlet for 5 min of desorption. Gas chromatography conditions included: a first-dimensional column, HP-innowax (30 m × 0.25 mm × 0.25 μm); a second-dimensional column, DB17-MS (1.2 m × 0.18 mm × 0.18 μm); and helium as the carrier gas at a flow rate of 1.0 mL / min. The temperature program was as follows: an initial temperature of 50°C, then a rate of 4°C / min to 240°C, where it was held for 10 min. The inlet temperature was 250°C, and the sample was operated in splitless mode with a split ratio of 40.0 mL / min. Mass spectrometry conditions: EI source, electron energy 70 eV, transfer line temperature 250°C; ion source temperature 230°C; quadrupole temperature 150°C; solvent delay 3 min; proton scan range (m / z 40-550). GC-MS mass spectra were searched and analyzed using the NIST mass spectral database, and components with relative peak areas greater than 1% and retention indices greater than 800 were selected for dynamic compositional analysis.

[0208] GC-MS was used to detect the changes in volatile gases of the strains. The results were as follows Figure 16 As shown, both the ΔsrfAA and ΔbaeBE mutants significantly increased the synthesis of 2,4-di-tert-butylphenol, acetoin, methoxyphenyloxime, pentadecanoic acid, and 2,5-dimethylpyrazine, while inhibiting the synthesis of 2-dodecanone and dodecanal. The ΔsrfAA mutant alone promoted the synthesis of benzaldehyde and phenylethanol, while the ΔbaeBE mutant inhibited the synthesis of these two volatile compounds. Among them, acetoin, 2,4-di-tert-butylphenol, 2-dodecanone, benzaldehyde, and 2,5-dimethylpyrazine exhibited antibacterial activity. In summary, the ΔbaeBEΔsrfAA double knockout mutant promoted the synthesis of the antibacterial volatile compounds 2,4-di-tert-butylphenol, acetoin, and 2,5-dimethylpyrazine.

[0209] Example 5

[0210] Effects of srfAA and baeBE gene knockout on the growth and conidia production of HN-Q-8 strain

[0211] 1. Effects of srfAA and baeBE on bacterial colony morphology

[0212] Among the strains cultured on solid LB plates, the colony edges of the ΔbaeBE mutant strain showed more obvious irregularities compared to the WT. In addition, the ΔbaeBEΔsrfAA double knockout mutant and the ΔsrfAA mutant strain were morphologically similar, with a smooth surface and no wrinkles. However, there were differences in color and transparency. The colonies of the ΔsrfAA mutant strain were milky white and opaque, while the colonies of the ΔbaeBEΔsrfAA double knockout mutant strain were light yellow and translucent ( Figure 17 ).

[0213] 2. Biofilm Observation and Quantitative Analysis

[0214] Effects of gene knockout on biofilm formation of HN-Q-8 strain

[0215] Add 1 mL of LBGM medium to each well of a 48-well plate and inoculate the 24-hour-old seed solution at a 2% inoculum size. Incubate at 37°C for 48 hours for quantitative biofilm determination. After incubation, slowly aspirate the plate and rinse 2-3 times with sterile PBS to remove excess bacteria. After drying, stain with 1 mL of 0.1% crystal violet dye for 20 minutes. Rinse slowly with PBS until the effluent is colorless. Allow to dry at room temperature to remove excess water. Decolorize with 2 mL of 33% acetic acid for 15 minutes. Mix thoroughly and measure the absorbance at OD570 to determine biofilm formation.

[0216] The simultaneous knockout of srfAA and baeBE genes promoted biofilm formation. Figure 18 As shown. At 24 hours, the wild-type (WT) strain formed a relatively complete biofilm, but no wrinkles appeared. The ΔsrfAA mutant strain did not form a complete biofilm. The ΔbaeBE mutant strain began to show a small number of wrinkles in its biofilm, while the ΔbaeBEΔsrfAA double knockout mutant strain showed more wrinkles. At 48 hours, the biofilms were observed: the ΔsrfAA mutant strain formed a complete biofilm, but with almost no wrinkles. The ΔbaeBE mutant strain showed an increased number of wrinkles compared to the WT strain, while the ΔbaeBEΔsrfAA double knockout mutant strain showed biofilm wrinkles between those of the ΔsrfAA and WT strains.

[0217] Quantitative analysis of the biofilms at 48 hours using crystal violet staining revealed that the absorbance at OD570 of the ΔsrfAA mutant was 2.87, 1.26-fold higher than that of the WT. The absorbance of the ΔbaeBE mutant was 1.41, a 0.11-fold increase compared to the WT (1.27). Furthermore, the absorbance of the ΔbaeBEΔsrfAA double knockout mutant was 2.88, 1.27-fold higher than that of the WT. In summary, knockout of the srfAA gene reduced biofilm wrinkling but increased biofilm formation.

[0218] 3. Growth Curve Determination of HN-Q-8 Mutant Strain

[0219] Mutant growth curve determination

[0220] After the strain was activated in LB solid medium, a single colony was inoculated into LB liquid medium and cultured for 24 hours. The inoculation ratio was 2% and transferred into a 250 mL conical flask containing 100 mL of liquid LB medium. The mixture was mixed evenly and cultured at 37 ° C and 220 rpm. Samples were taken every 6 hours and the OD value was calculated. 600 The absorbance was measured and the growth curve was drawn.

[0221] In order to clarify the effects of srfAA and baeBE genes on the growth of the strain, a 72-h growth curve was drawn. Figure 19 As shown in Figure 3, compared with the wild-type (WT) strain, the growth rates of the ΔsrfAA mutant and the ΔbaeBE mutant did not change significantly, and both reached their maximum values ​​at 24 h. The maximum OD value of the ΔbaeBE mutant in the stationary phase 600 The value was higher than that of the WT strain, while the maximum OD 600 The value was lower than that of the WT strain and showed a downward trend at 42h. The ΔbaeBEΔsrfAA double knockout mutant reached the maximum OD at 18h. 600 value, followed by OD 600 The value began to decrease and stabilized at 54h. These results showed that gene knockout had a significant effect on the growth of the HN-Q-8 strain. Among them, the logarithmic growth phase of the ΔbaeBEΔsrfAA double knockout mutant was shortened, the growth rate was accelerated, but the stationary phase was shortened, the late growth was inhibited, and the OD value decreased.

[0222] 4. Effects of srfAA and baeBE on sporulation ability

[0223] Bacteria were grown in LB medium with shaking at 180 rpm and 37°C for 48 hours. Serial dilutions were performed. Half of each dilution was left unheated, while the other half was heated at 80°C for 20 minutes. The two dilutions were spread onto LB plates and incubated at 37°C for 12 hours. Sporulation rate was calculated as follows: Sporulation rate (%) = Number of colonies in heated culture medium / Number of colonies in unheated culture medium × 100%.

[0224] The lack of surfactin and bacillusene synthesis ability significantly affected the spore production ability of Bacillus. Figure 20As shown, the wild-type (WT) had a sporulation rate of 95.03%. In contrast, the sporulation rate of the ΔsrfAA mutant decreased to 48.22%, while the sporulation rate of the ΔbaeBE mutant decreased to 60.11%, both significantly lower than the WT strain. The sporulation rate of the ΔbaeBEΔsrfAA double knockout mutant further decreased to 3.74%, indicating that the srfAA and baeBE genes have a significant impact on the sporulation ability of Bacillus.

[0225] 5. Effects of srfAA and baeB on adsorption capacity

[0226] The adsorption capacity of Bacillus refers to its ability to adsorb heavy metal ions, organic matter, and other substances in the environment. This is of great significance in the fields of environmental remediation and biosorption. The changes in adsorption capacity were determined by the adsorption of Congo red by mutant strains, as shown below.

[0227] Take 1mL of bacterial solution (OD 600nm =1.0) was added to 100 mL of LB broth and incubated at 37°C, 180 rpm for 12 h. Congo red adsorption was measured. 1 mL of cells was centrifuged at 8000 rpm for 2 min, washed once with 1 mL of liquid LB medium, resuspended in 1 mL of liquid LB medium, and diluted 10-fold. 5 μL of 4 mg / mL Congo red was added to 500 μL of the suspension, incubated at 220 rpm, 30°C for 2 h, centrifuged at 12000 rpm for 5 min, and the supernatant OD was measured. 490nm Liquid LB medium + Congo red was used as blank. The adsorption capacity (CR) was calculated according to formula I.

[0228] CR (μg / OD 600nm )=(OD 490nm-空白 -OD 490nm-样品 )×44.676 / OD 600nm Formula I.

[0229] turn out( Figure 21 ), the adsorption capacity of the ΔsrfAA mutant was 7.59 (μg / OD 600nm ), and the ΔbaeBE mutant was 6.25 (μg / OD 600nm ), while the adsorption capacity of the ΔbaeBEΔsrfAA double knockout mutant was 7.45 (μg / OD 600nm ), compared with 6.70 (μg / OD of the wild type (WT) strain 600nm ), the adsorption capacity of these mutants did not change significantly. This shows that the deletion of baeBE and srfAA genes does not significantly affect the adsorption capacity of the strain.

[0230] Although the above embodiment provides a detailed description of the present invention, it is only a part of the embodiments of the present invention, not all of the embodiments. Other embodiments can be obtained based on this embodiment without creativity, and these embodiments all fall within the scope of protection of the present invention.

Claims

1. Application of bacillusene or bacillusene combined with surfactin as targets for regulating the production of fengycin in Bacillus velezensis strain HN-Q-8; The deposit number of the Bacillus Velez strain HN-Q-8 is CGMCC No.19554.

2. The application according to claim 1, characterized in that The bacillusene or bacillusene combined with surfactin negatively regulates the fengycin production of the Bacillus velezensis strain HN-Q-8.

3. Use of bacillus cereus and / or surfactin as targets in regulating at least one of the antibacterial activity, spore production ability and biofilm formation of Bacillus velezensis strain HN-Q-8.

4. The application according to claim 3, characterized in that The bacillus cereus and / or surfactin negatively regulate the antibacterial activity and / or biofilm formation of Bacillus velezensis strain HN-Q-8; The bacillus cereus and / or surfactin positively regulate the spore production ability of the Bacillus velezensis strain HN-Q-8.

5. Use of knockout bacillusene genes and surfactin genes to increase the yield of fengycin and / or antibacterial activity of Bacillus velez strain HN-Q-8, reduce the spore production ability of Bacillus velez strain HN-Q-8, and promote biofilm formation of Bacillus velez strain HN-Q-8; The deposit number of the Bacillus Velez strain HN-Q-8 is CGMCC No.19554.

6. The application according to claim 5, characterized in that: The reagent for knocking out the expression of the bacillusene gene includes a bacillusene knockout vector; the bacillusene knockout vector includes a first upstream homology arm, a first resistance gene fragment and a first downstream homology arm; The nucleotide sequence of the upstream homology arm of the bacillusene vector is shown in SEQ ID NO: 1; the nucleotide sequence of the downstream homology arm of the bacillusene gene vector is shown in SEQ ID NO:

2.

7. The use according to claim 5, characterized in that The reagent for knocking out the expression of surfactin includes a surfactin knockout vector; the surfactin knockout vector includes a second upstream homology arm, a second resistance gene fragment and a second downstream homology arm; The nucleotide sequence of the upstream homology arm of the surfactin vector is shown in SEQ ID NO: 3; the nucleotide sequence of the downstream homology arm of the surfactin gene vector is shown in SEQ ID NO:

4.

8. A mutant strain of Bacillus velezensis strain HN-Q-8, characterized in that The Bacillus velez strain HN-Q-8 was knocked out for the bacillusene gene and surfactin gene; The deposit number of the Bacillus Velez strain HN-Q-8 is CGMCC No.19554.

9. Use of the mutant strain of the Bacillus velezensis strain HN-Q-8 according to claim 8 in producing Fengyuan and / or preventing and controlling potato diseases.

10. A method for producing Fengyuansu by fermentation, characterized in that: Cultivating the mutant strain of Bacillus velezensis according to claim 8, and isolating a lipopeptide extract from the culture solution; The nitrogen source in the culture medium includes 3-7 g / L sodium glutamate and 8-12 g / L peptone; The deposit number of the Bacillus Velez strain HN-Q-8 is CGMCC No.19554.

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