Engineering strain for directional synthesis of bacitracin L, and recombinant vector and application thereof

Abstract

The invention discloses an engineering strain for directional synthesis of bacitracin L as well as a recombinant vector and application of the engineering strain, and relates to the technical field of microbiology and biology. The engineering strain is bacillus amyloliquefaciens M86 into which the recombinant vector pCN33-PbmyD-sfGFP-LacI-YhiH is introduced; the recombinant vector pCN33-PbmyD-sfGFP-LacI-YhiH comprises a bmyD promoter, an enhanced green fluorescent protein sfGFP gene, a lactose operon regulation and control element LacI-MCS and an encoding gene of a transcription factor YhiH, and the encoding gene of the recombinant vector pCN33-PbmyD-sfGFP-LacI-YhiH comprises a bmyD promoter, an enhanced green fluorescent protein sfGFP gene, a lactose operon regulation and control element Expression of the transcription factor YhiH is regulated and controlled by a lactose operon, and the transcription factor YhiH can be specifically combined with a bmyD promoter, so that transcription of a bmyDABC gene cluster is activated, synthesis of bacitracin L is promoted, and directional synthesis of bacitracin L can be realized.

Description

Technical Field

[0001] This invention relates to the fields of microbiology and biotechnology, specifically to an engineered strain for the directed synthesis of bacillus spp. L, its recombinant vector, and its applications. Background Technology

[0002] Fungal diseases are a widespread problem in agricultural production. These diseases not only infect crops, leading to reduced yields or even crop failure and affecting product quality, but also pose a risk of infecting post-harvest produce, resulting in post-harvest losses. In addition to causing severe economic losses, the fungal infections produced by some fungi (such as aflatoxin B1) can remain in food, posing a serious threat to human health. Statistics show that 25-50% of stored agricultural products worldwide are infected by fungi annually, resulting in storage losses of up to 30%.

[0003] Currently, chemical fungicides are the most common means of addressing this problem, effectively inhibiting or killing pathogens and achieving good control effects. However, chemical fungicides also have many drawbacks: most chemical fungicides enter the environment, pollute water bodies, threaten the survival of other organisms, and affect the ecological balance; at the same time, the mechanism of action of chemical pesticides is fixed, making it easy for pathogens to develop resistance; in addition, most chemical fungicides are harmful to humans and animals, and when used on agricultural products, they accumulate during the vegetative growth process of plants, eventually leading to chemical pesticide residues in agricultural products, which may cause serious consequences such as cancer, birth defects, or even death.

[0004] Currently, a variety of new methods have been developed to inhibit the growth of plant pathogenic fungi. Biopesticides are a promising type of green pesticide, and spore-forming Bacillus bacteria may be one of the most effective biological fungicides for plant protection.

[0005] Bacillus amyloliquefaciens ( Bacillus amyloliquefaciens As a typical rhizosphere growth-promoting bacterium, *Bacillus amyloliquefaciens* possesses broad application potential in the field of plant disease biological control due to its strong ability to synthesize antimicrobial secondary metabolites. Lipopeptides are key metabolites for its biocontrol effect. In biocontrol applications, *Bacillus amyloliquefaciens* must first colonize the host plant before initiating the synthesis and secretion of lipopeptides. Its biocontrol efficacy mainly depends on the spatiotemporal synthesis dynamics of secondary metabolites and is limited by complex transport regulatory networks and environmental factors. This leads to significant fluctuations in the synthesis efficiency of antimicrobial lipopeptides and a lack of timeliness, thus restricting its practical application in agricultural production. Summary of the Invention

[0006] The inventors of this invention discovered that directly using lipopeptides as biopesticides can rapidly inhibit pathogenic microorganisms. However, the synthesis of lipopeptides requires simultaneous conditions such as directional transcription of the synthetic gene cluster, efficient coding, and sufficient substrate to achieve directional and efficient synthesis. However, the complex structure of the lipopeptide synthetic gene cluster and insufficient substrate supply severely restrict lipopeptide synthesis; furthermore, the diverse types and functions of synthesized lipopeptides result in unstable efficacy.

[0007] The purpose of this invention is to solve the above-mentioned technical problems and to propose an engineered strain for the directed synthesis of bacillus styracifolium L, its recombinant vector, and its application.

[0008] This invention is achieved through the following technical solution: An engineered strain capable of targeted synthesis of bacillus tincture L, wherein the engineered strain is introduced into the recombinant vector pCN33-P bmyD -sfGFP-LacI-YhiH Bacillus amyloliquefaciens M86; The recombinant vector pCN33-P bmyD -sfGFP-LacI-YhiH contains: bmyD The gene encoding the promoter, the enhanced green fluorescent protein sfGFP gene, the lactose operon regulatory element LacI-MCS, and the transcription factor YhiH; The expression of the transcription factor YhiH is regulated by the lactose operon and can specifically bind to... bmyD Promoter, thereby activating bmyDABC Transcription of gene clusters promotes the synthesis of bacitracin L.

[0009] Furthermore, the pCN33-P bmyD The sequence of -sfGFP-LacI-YhiH is SEQ ID No:10.

[0010] Furthermore, the recombinant vector pCN33-P bmyD -sfGFP-LacI-YhiH is a shuttle plasmid that can replicate in Escherichia coli and Bacillus, and carries ampicillin resistance genes and erythromycin resistance genes.

[0011] A recombinant vector, wherein the vector is pCN33P bmyD -sfGFP-LacI-YhiH is constructed through the following steps: S1. Using pcN33-sfGFP as a template, a linearized pcN33-sfGFP vector was obtained by reverse PCR, and then... bmyD pCN33-P was constructed by homologous recombination of promoter fragments. bmyD -sfGFP; the bmyDThe promoter fragment is derived from the genome of Bacillus amyloliquefaciens M86 and is 300 bp in length; S2, with pCN33-P bmyD Using sfGFP as a template, a linearized vector was obtained by reverse PCR. This vector was then homologously recombinated with the lactose operon regulatory element LacI-MCS from the pET-19b vector to construct pCN33-P. bmyD -sfGFP-LacI-MCS; S3, with pCN33-P bmyD Using -sfGFP-LacI-MCS as a template, a linearized vector was obtained by reverse PCR. This vector was then homologously recombinated with the YhiH gene CDS fragment from the Bacillus amyloliquefaciens M86 genome to construct the pCN33-P vector. bmyD -sfGFP-LacI-YhiH.

[0012] Furthermore, in step S1, the primers used for reverse PCR are PCN33-sgGFP linearized-F / R. bmyD The primers for amplifying the promoter fragment are: bmyD -P(300bp)-F / R, and the 5' end of the primer contains a 15bp homologous sequence complementary to the end of the linearized vector; In step S2, the primers used for reverse PCR are pCN33- bmyD -PF / R, the amplification primers for the lactose operon regulatory element LacI-MCS are pET-19b-F / R; In step S3, the primers used for reverse PCR are pET-19b-linearization-F / R, and the primers used for amplifying the CDS fragment of the YhiH gene are yhiH-CDS-F / R.

[0013] Application of transcription factor YhiH in increasing the yield of bacitracin L, wherein YhiH specifically binds to Bacillus amyloliquefaciens. bmyDABC Gene clusters bmyD Promoter, activate bmyDABC The transcription of bacitracin L promotes its synthesis.

[0014] Furthermore, YhiH was expressed in a targeted manner using the aforementioned engineered strain, and its expression was induced by IPTG to increase the yield of bacitracin L.

[0015] Furthermore, YhiH was expressed in a targeted manner using the aforementioned engineered strain, and the strain was cultured in BDSM medium containing 1.25–20 g / L lactose, 0–1.5 g / L K2HPO4, and 0–3.0 g / L glycine to increase the yield of bacitracin L.

[0016] Furthermore, the BDSM medium formula is as follows: lactose 5 g / L, L-glutamate sodium 5 g / L, tryptone 5 g / L, yeast extract 1 g / L, MgSO4•7H2O 0.5 g / L, KCl 0.5 g / L, K2HPO4 0.75 g / L, and glycine 3 g / L.

[0017] Furthermore, YhiH was expressed in a targeted manner using the aforementioned engineered strain, and the strain was cultured in a medium containing fatty acid chain synthesis precursors and / or amino acid chain synthesis substrates.

[0018] The application of transcription factors YqeW, YqgH, YtrA, ComK, SpoOJ, LexA, GEO3564, or CcpB in regulating the production of bacitracin L, wherein YqeW, YqgH, YtrA, ComK, or SpoOJ specifically bind to Bacillus amyloliquefaciens. bmyDABC Gene clusters bmyD Promoter and suppressor bmyDABC Transcription of LexA, GEO3564, and CcpB can specifically bind to Bacillus amyloliquefaciens. bmyDABC Gene clusters bmyD Promoter and activation bmyDABC The transcription of the gene precisely regulates the synthesis of bacitracin L, and the encoding genes of YqeW, YqgH, YtrA, ComK, SpoOJ, LexA, GEO3564 or CcpB are derived from the genome of Bacillus amyloliquefaciens M86.

[0019] A strain of Bacillus amyloliquefaciens M86, producing high levels of bacillus-1,000 L, with accession number CGMCC No. 34810, was deposited on June 9, 2025, at the China General Microbiological Culture Collection Center and classified as *Bacillus amyloliquefaciens*.

[0020] Furthermore, the genome of Bacillus amyloliquefaciens M86 has the following mutations compared to the genome of the original strain 1841, whose genome is accessed via NCBI Submission Accession (SUB14919781): I. SNP mutation: Compared to the wild-type YplP protein of the original strain 1841 shown in SEQ ID NO:1, the 111th amino acid of the YplP protein changes from R to C. Compared to the wild-type XkdE protein of the original strain 1841 shown in SEQ ID NO:2, the 270th amino acid of the XkdE protein changes from P to S; Compared to the wild-type GutR protein of the original strain 1841 shown in SEQ ID NO:3, the 402nd amino acid of the GutR protein changes from N to D; Compared to the wild-type YdzE protein of the original strain 1841 shown in SEQ ID NO:4, the 109th amino acid of the YdzE protein changes from V to F; Compared to the wild-type GEO1829 protein of the original strain 1841 shown in SEQ ID NO:5, the 35th amino acid of the GEO1829 protein changes from L to Q; Compared to the wild-type GEO2348 protein of the original strain 1841 shown in SEQ ID NO:6, the 93rd amino acid of the GEO2348 protein changes from Q to E; Compared to the wild-type GEO3080 protein of the original strain 1841 shown in SEQ ID NO:7, the 303rd amino acid of the GEO3080 protein changes from P to T; Compared to the wild-type YqeW protein of the original strain 1841 shown in SEQ ID NO:8, the 91st amino acid of the YqeW protein changes from D to G; Compared to the wild-type YqgH protein of the original strain 1841 shown in SEQ ID NO:9, the 122nd amino acid of the YqgH protein changes from G to E; Synonymous mutations of GEO0859, GEO1366, and GEO3080 proteins; II. INDEL mutation: A T-TC insertion exists at locus 312941 in the genome, which is located upstream of the gene clusters GEO00285, yoaW, yndM, and GEO00288. A T-TC insertion exists at position 814535 of the genome, which is located upstream of the ykvZ, GEO00683, and ykvY gene clusters. An A-AC insertion exists at locus 1862323 in the genome, which is located downstream of the ycgO and ycgN gene clusters; A C-CT insertion exists at locus 2434315 in the genome, and this insertion is located downstream of the yxjB and ycgN gene clusters. An A-AC insertion exists at position 824120 in the genome, which leads to a change in the structure of the protein encoded by the ykvP gene; A T-TC insertion exists at position 1434230 in the genome, which leads to a change in the protein structure encoded by the cotJA gene; A T-TG insertion exists at position 2334423 in the genome, which leads to a change in the protein structure encoded by the xdhA gene; A C-CG insertion exists at position 2544952 in the genome, which leads to a change in the structure of the protein encoded by the ywfA gene; An A-AG insertion exists at position 3226061 in the genome, which leads to a change in the structure of the protein encoded by the GEO3075 gene; III. Chromosomal structural variations: There is a 1218 bp deletion between positions 29527 and 30745 in the genome; There is a 41815 bp intrachromosomal translocation in the genomic locus range of 1528892-1570707.

[0021] The aforementioned application of Bacillus amyloliquefaciens M86 in the synthesis of high-yield bacillus L, antagonism of fungi, and antagonism of bacteria.

[0022] A method for the tunable, directed synthesis of high-yield bacillus L based on the aforementioned Bacillus amyloliquefaciens M86 includes the following steps: a. The original strain 1841 was randomly mutated using ARTP mutagenesis, and the mutant strains that survived the mutagenesis were cultured in high-throughput culture. Then, Botrytis cinerea was used as the test strain to screen mutant strains with resistance to Botrytis cinerea, and the mutant strain Bacillus amyloliquefaciens M86 was obtained. b. Culture Bacillus amyloliquefaciens M86 in Landy medium for at least 12 hours to obtain a high-yield bacitracin L fermentation broth, wherein the bacitracin L content in the high-yield bacitracin L fermentation broth is not less than 423.33 mg / L.

[0023] Furthermore, in step b, activation is employed. bmyDABC Bacillus amyloliquefaciens M86 was cultured in BDSM medium containing synthetic gene clusters.

[0024] Furthermore, the BDSM culture medium contains 1.25–20 g / L lactose, 0–1.5 g / L K₂HPO₄, and 0–3.0 g / L glycine.

[0025] Furthermore, the BDSM medium contains 5 mg / L lactose, 0.75 g / L K2HPO4, and 3 g / L glycine.

[0026] Furthermore, the BDSM culture medium is formulated as follows: lactose 5 g / L, L-glutamate sodium 5 g / L, tryptone 5 g / L, yeast extract 1 g / L, MgSO4·7H2O 0.5 g / L, KCl 0.5 g / L, K2HPO4 0.75 g / L, and glycine 3 g / L.

[0027] Furthermore, in step b, fatty acid chain synthesis precursors and amino acid chain synthesis substrates are added to the culture medium for culturing Bacillus amyloliquefaciens M86.

[0028] Furthermore, the fatty acid chain synthesis precursor includes Val; the amino acid chain synthesis substrate includes L-Asn, L-Tyr, L-Ser, L-Glu, and L-Thr.

[0029] Furthermore, the concentration ratio of L-Asn, L-Tyr, L-Ser, L-Glu and L-Thr is 1~2:1~2:1~2:1~2:1~2.

[0030] Furthermore, the concentration ratio of L-Asn, L-Tyr, L-Ser, L-Glu and L-Thr is 2:1:2:1:1.

[0031] Furthermore, the concentration ratios of Val, L-Asn, L-Tyr, L-Ser, L-Glu, and L-Thr were 1 to 18:2:1:2:1:1.

[0032] Furthermore, the concentration ratio of Val, L-Asn, L-Tyr, L-Ser, L-Glu, and L-Thr was 7:2:1:2:1:1.

[0033] Application of high-yield bacitracin L fermentation broth in bacterial inhibitors / bactericides, wherein the bacteria include Shigella, Escherichia coli, Salmonella, Staphylococcus aureus, Micrococcus luteus, Enterococcus, Bacillus belye, Bacillus subtilis 1560, and Bacillus licheniformis. The high-yield bacitracin L fermentation broth is obtained by the aforementioned method. High-yield bacitracin L fermentation broth refers to fermentation broth in which bacitracin L reaches a concentration of 1000 mg / L or higher in a 300 ml fermentation system.

[0034] Application of high-yield bacitracin L fermentation broth in fungal inhibitors / fungicides, wherein the fungi include Aspergillus niger, Hansenula d'Barry yeast, Alternaria alternata, Alternaria tomato, Aspergillus flavus, Colletotrichum gloeosporioides, Fusarium moniliforme, Fusarium solani, Geotrichum candida, Penicillium fingernail, Penicillium citrinum, Trichoderma, Rhizopus nigricans, Rhizopus oryzae, Sclerotinia sclerotiorum, Streptomyces lavenderensis, and Trichoderma viride. The high-yield bacitracin L fermentation broth is obtained by the method described above. High-yield bacitracin L fermentation broth refers to a mixture in which bacitracin L reaches a concentration of 1500 mg / L or higher in a 300 ml fermentation system.

[0035] Application of high-yield bacitracin L fermentation broth in the preparation of agricultural product fungicides and pesticide formulations. The high-yield bacitracin L fermentation broth is obtained by the method described above. High-yield bacitracin L fermentation broth refers to a mixed solution in which bacitracin L reaches a concentration of 1900 mg / L or higher in a 300 ml fermentation system.

[0036] Furthermore, the high-yield bacillus styracifolium L fermentation broth is used in the preparation of drugs for treating skin infections, food poisoning, sepsis, neonatal sepsis, urinary tract infections in pregnant women, dysentery, and urinary tract infections, characterized in that the high-yield bacillus styracifolium L fermentation broth is obtained by the method described above.

[0037] Compared with the prior art, the present invention has the following advantages and beneficial effects: I. In this invention, the mutant strain M86 was obtained through ARTP mutagenesis. In ordinary Landy medium, its bacitracin L yield reached ≥423.33 mg / L, which is about 1.73 times higher than that of the original strain. Through medium optimization (using BDSM or BDSM+Val+(5+) medium), the yield was significantly improved: using BDSM medium, the yield reached 1349.01 mg / L; using BDSM+Val+(5+) medium, the yield reached 1992.51 mg / L.

[0038] II. In this invention, an engineered strain (M86-YhiH) capable of targeted synthesis of bacitracin L was successfully constructed. The gene cluster responsible only for bacitracin synthesis was identified. bmyDABC The key promoter of transcription, P bmyD Developed by P bmyD A promoter-regulated sfGFP fluorescence signaling system is used for real-time monitoring and precise assessment of promoter activity. This system utilizes the specific binding and activation of the yhiH transcription factor by P... bmyD Promoter. Establishment of a precise induction system: The constructed M86-YhiH engineered strain, its P... bmyD Promoter activity can be precisely regulated by a galactose-regulated system, enabling the quantitative production of bacitracin L.

[0039] III. In this invention, the developed BDSM+Val+(5+) medium not only significantly increases the yield of the original M86 strain but is also suitable for engineered strains, yielding higher concentrations of bacitracin L. A strategy of targeted addition of synthetic precursors is implemented to precisely compensate for insufficient precursors within the bacteria, effectively utilizing the excess synthase induced during synthesis. bmyDABC This further improves the synthesis efficiency.

[0040] IV. This invention proposes and implements a "dual-pathway targeted promotion" strategy: Pathway 1: By optimizing the culture medium formulation and targetedly adding key components (such as Val), targeted activation of metabolic flux and targeted synthesis of target products are achieved. Pathway 2 (molecular level): Through the aforementioned molecular tools (P... bmyD The -sfGFP, yhiH, galactose-induced system enables precise regulation of gene transcription and targeted activation of synthetic pathways.

[0041] V. The engineered strain for the directional synthesis of bacillus tincture L, its recombinant vector and its application proposed in this invention have the potential for industrial application. This scheme (high-yield strain, high-efficiency culture medium, and precise regulation) significantly improves production efficiency and lays a solid foundation for its promotion to industrial and large-scale production.

[0042] VI. The fermentation broth of bacitracin L obtained in this invention has activity in antagonizing fungi and bacteria, and can be used to prepare agricultural fungicides and pesticide formulations, which has practical application prospects. Attached Figure Description

[0043] Figure 1 This is a screening diagram of mutant strains with enhanced antagonistic activity against gray mold.

[0044] Figure 2 This is a graph showing the genetic stability of the mutant strain M86.

[0045] Figure 3 This is a growth curve diagram of mutant strain M86 and original strain 1841.

[0046] Figure 4 This is a graph showing the cellular energy metabolism of mutant strain M86 and original strain 1841 at different growth times.

[0047] Figure 5 This is a high-quality sequencing data image of the M86 genomic DNA.

[0048] Figure 6 This is a depth map of the M86 gene sequencing.

[0049] Figure 7 This is a curve showing the coverage of the M86 gene.

[0050] Figure 8 This is a statistical graph of the base skewness of mutations in M86.

[0051] Figure 9 This is a graph showing the spectral analysis results of SNP mutations in M86.

[0052] Figure 10 This is a graph showing the spectral analysis results of the INDEL mutation in M86.

[0053] Figure 11 It is the M86 mutation map.

[0054] Figure 12 The different extracts from the M86 fermentation broth inhibited the growth of Botrytis cinerea. Among them: methanol represents the blank group; supernatant represents the fermentation supernatant; pro-extract represents the protein extract; and lipopeptide extract represents the lipopeptide extract.

[0055] Figure 13It is the crude lipopeptide yield of 1841 and M86.

[0056] Figure 14 The graph shows the inhibitory effect of lipopeptide extracts from 1841 and M86 at different fermentation times on Botrytis cinerea.

[0057] Figure 15 This is a volcano plot showing the differential expression of lipopeptide synthesis gene clusters in M86 at 12h and 48h.

[0058] Figure 16 It is in 1841 and M86 bmyDABC Differences in the expression of gene clusters at different bacterial growth stages.

[0059] Figure 17 yes difficidin Differences in expression between 1841 and M86.

[0060] Figure 18 yes Locillibactin Differences in expression between 1841 and M86.

[0061] Figure 19 yes bacilysin Plot showing the expression differences between 1841 and M86.

[0062] Figure 20 yes surfactin Plot showing the expression differences between 1841 and M86.

[0063] Figure 21 yes Bacillibactin Plot showing the expression differences between 1841 and M86.

[0064] Figure 22 yes Fengycin Plot showing the expression differences between 1841 and M86.

[0065] Figure 23 yes macrolactin Differences in H expression between 1841 and M86.

[0066] Figure 24 yes Bacillaene Plot showing the expression differences between 1841 and M86.

[0067] Figure 25 This is a graph showing the test results of the anti-gray mold activity of the primary separation liquid.

[0068] Figure 26 This is a graph showing the test results of the anti-gray mold activity of the secondary separation liquid.

[0069] Figure 27 This is the mass spectrum of fraction a2.

[0070] Figure 28 It is C14 - Bacillomycin L and C 15 - Bacillomycin A three-dimensional structural diagram of L, where: A is C 14 - Bacillomycin A three-dimensional structural diagram of L, where B is C. 15 - Bacillomycin A three-dimensional structural diagram of L.

[0071] Figure 29 The difference lies in M86 and 1841. Bacillomycin L-homology first-order mass spectrometry.

[0072] Figure 30 This is a bar chart showing the intensity of sfGFP in different sugar sources.

[0073] Figure 31 These are sfGFP intensity curves for different sugar sources, where: A is the sfGFP intensity curve for different concentrations of glucose, and B is the sfGFP intensity curve for different concentrations of sucrose.

[0074] Figure 32 These are sfGFP intensity curves for different sugar sources, where: A represents the sfGFP intensity curve for different concentrations of maltose; and B represents the sfGFP intensity curve for different concentrations of lactose.

[0075] Figure 33 This is a comparison graph of sfGFP intensity after maltose and lactose are mixed with different glucose concentrations.

[0076] Figure 34 Different sugar sources affect bacillomycin The effect of L on production results is shown in the figure.

[0077] Figure 35 This is a bar chart showing the intensity of sfGFP in different metal ions.

[0078] Figure 36 This is a graph showing the sfGFP intensity at different K2HPO4 concentrations.

[0079] Figure 37 Different K2HPO4 concentrations have an effect on bacillomycin The impact curve of L production.

[0080] Figure 38 This is a bar chart showing the intensity of sfGFP in different amino acids.

[0081] Figure 39 This is a graph showing the sfGFP intensity at different Gly concentrations.

[0082] Figure 40 Different Gly concentrations have the effect on bacillomycin The impact curve of L production.

[0083] Figure 41 Is it BDSM medium and Landy medium that affect Bacillomycin The results of the influence of L are shown in Figure A, where A represents the difference in sfGFP fluorescence signal between BDSM and Landy media, and B represents the difference in sfGFP fluorescence signal between BDSM and Landy media. bacillomycin L-yield differences.

[0084] Figure 42 It is in M86 bmyDABC Statistical graph of differential expression of synthetic gene cluster transcription factors in BDSM medium, where A, B, C, and D represent... bmyD Transcriptional expression differences bmyA Transcriptional expression differences bmyB Transcriptional expression differences bmyC The transcriptional expression difference diagram.

[0085] Figure 43 This is a statistical graph showing the differential expression of glucose metabolism transcription factors in M86 cells in BDSM medium. A, B, C, and D represent... yhiH Transcriptional expression differences ccpB Transcriptional expression differences ccpA Transcriptional expression differences GEO3564 Differences in transcriptional expression.

[0086] Figure 44 This is a graph showing the fatty acid content of M86 at different fermentation stages.

[0087] Figure 45 They are fed with different fatty acids Bacillomycin L-mass spectrum.

[0088] Figure 46 They are fed with different fatty acids Bacillomycin L-yield differences.

[0089] Figure 47 Feeding of fatty acid synthesis precursors Bacillomycin The impact of L production.

[0090] Figure 48 This is a diagram showing the synergistic optimization results of the synthesis starters and substrates, where A represents different amino acid synthesis substrate pairs. Bacillomycin The effect of L-type production, B being the influence of D-type and L-type amino acids on L-type production. Bacillomcyin The effect of L yield, C being the effect of synergistic addition of synthetic starter and substrate on .... Bacillomycin The effect of L yield, D is the screening of the optimal synergistic ratio of the synthesis starter and substrate, E is the synergistic optimization of the culture medium. Bacillomycin The yield difference of L, and F is the yield difference in the co-optimized culture medium. Bacillomycin Mass spectrometry results for L.

[0091] Figure 49 yes Bacillomycin Antifungal spectrum of L-antagonistic fungi.

[0092] Figure 50 yes Bacillomycin Antibacterial spectrum of L-antagonistic bacteria.

[0093] Figure 51 yes Bacillomycin A photograph of the actual L antibacterial experiment.

[0094] Figure 52 It is a plasmid map.

[0095] Figure 53 This is a statistical chart showing the yield increase of bacitracin L in the M86-YhiH group and the blank group.

[0096] Figure 54 This is a graph showing the results of the EMSA experiment, where: A. Different candidate transcription factors and bmyD Promoter binding, B. SpoVG and bmyD Promoter binding, C.LexA and bmyD Promoter binding, D. CcpA and bmyD Promoter binding, E. GEO3564 and bmyD Promoter binding, F. FapR and bmyD Promoter binding, G. YhiH and bmyD Promoter binding, H.YfiS2 and bmyD Promoter binding, I. YtrA and bmyD Promoter binding, J. ComK and bmyD Promoter binding, K. CcpB and bmyD Promoter binding, L. SpoOJ and bmyD Promoter binding, M. YqeW-mut and bmyD Promoter binding, N. YqgH-mut and bmyD Promoter binding.

[0097] Figure 55 It is different transcription factors and different bmyD The image shows the detection results of sfGFP signal intensity bound to the promoter region, where: A. truncated bmyD Background sfGFP signal of the promoter, B.LexA and truncated bmyD The promoter's sfGFP signal, C.CcpA and truncated bmyD The promoter sfGFP signal, D. GEO3564 and truncated bmyD The promoter's sfGFP signal, E.CcpB and truncated bmyDThe promoter sfGFP signal, F.YhiH and truncated bmyD The promoter sfGFP signal, G. SpoOJ and truncated bmyD The promoter sfGFP signal, H.YqeW and truncated bmyD The promoter's sfGFP signal, I.ComK and truncated bmyD The promoter's sfGFP signal, J.YqgH and truncated bmyD The promoter's sfGFP signal, K.YtrA and truncated bmyD The sfGFP signal of the promoter. Detailed Implementation

[0098] The present invention will be further described in detail below with reference to embodiments, but the implementation of the present invention is not limited thereto.

[0099] This invention provides a Bacillus amyloliquefaciens M86, and the technical solution of this invention will be described in detail with reference to examples.

[0100] The following are the culture media and their corresponding formulations used in the embodiments of the present invention: 1. The formula for Potato Sucrose Agar (PDA) medium is: 200 g potato, 20 g sucrose, 20 g agar, natural pH. Boil the potatoes in an appropriate amount of tap water for half an hour, filter through eight layers of gauze, add 1000 ml of water, and autoclave at 121℃ for 20 minutes.

[0101] 2. The LB medium formula is as follows: 10g tryptone, 5g yeast extract, 10g sodium chloride, 20g agar powder, 1000ml distilled water, pH 7.2, autoclaved at 121℃ for 15 min.

[0102] 3. The liquid Landy medium formula is as follows: 5 g of sodium L-glutamate, 0.5 g of MgSO4·7H2O, 20 g of glucose, 0.15 mg of FeSO4, 0.5 g of potassium chloride, 1.0 g of K2HPO4, 5.0 mg of MnSO4•H2O, 0.16 mg of CuSO4•5H2O, 1000 ml of distilled water, pH 7.2, autoclaved at 121℃ for 15 min.

[0103] Example 1 In this embodiment, a mutant strain of Bacillus amyloliquefaciens M86, which produces high levels of bacillus L, was obtained through ARTP mutagenesis and breeding. The strain's accession number is CGMCC No. 34810. Furthermore, the controlled, directed synthesis of bacillus L was achieved using Bacillus amyloliquefaciens M86, resulting in high-yield production of bacillus L.

[0104] Meanwhile, the characteristics of Bacillus amyloliquefaciens M86 were studied in greater depth.

[0105] I. Obtain the mutant strain M86 that produces high levels of bacillus styrax L.

[0106] Existing research on Bacillus amyloliquefaciens 1841 has shown that it possesses a unique methylation modification system. This characteristic poses a significant challenge to enhancing the antibacterial activity of 1841 through gene editing. Therefore, this invention uses Bacillus amyloliquefaciens 1841 as the original starting strain for ARTP mutagenesis and uses Botrytis cinerea as an indicator strain to screen for mutant strains with enhanced resistance to Botrytis cinerea, aiming to obtain mutant strains with enhanced antibacterial activity.

[0107] 1. ARTP mutagenesis and mutant screening: Through ARTP mutagenesis and selection, this invention obtained a total of 60 strains that survived the mutagenesis treatment. Among them, 16 mutant strains (M58, M65, M68, M77, M78, M85, M86, M94, M96, M100, M111, M112, M113, M115, M116, M117) showed significantly enhanced antagonistic activity against Botrytis cinerea. Figure 1 .

[0108] Depend on Figure 1 It can be seen that the inhibition zone diameter of mutant strain M86 reached 38.22 mm, which was significantly higher than that of the other 15 mutant strains.

[0109] 2. Analysis of the genetic stability and growth characteristics of mutant strain M86: Subculture experiments were conducted on M86, and gray mold antagonism experiments were performed on M86 from the 5th, 10th, 15th, 20th, 25th, 30th, 35th, and 40th generations. The results showed that with increasing subculture number, the gray mold resistance of M86 gradually increased and tended to stabilize. Figure 2 It is evident that M86 is a strain with high antibacterial activity and genetic stability.

[0110] Furthermore, the growth of the mutant strain M86 was examined, see... Figure 3 The cellular energy metabolism capacity of strains M86 and 1841 at different growth stages was detected. The energy metabolism level of the strains was analyzed by detecting changes in heat during bacterial growth. (See attached document). Figure 4 .

[0111] Depend on Figure 3It can be seen that the growth rate of M86 is significantly higher than that of the original strain 1841. At 12 hours of fermentation, the absorbance of M86 cells is significantly higher than that of 1841, and there is a significant difference between the two (P<0.05). Between 12 and 36 hours of fermentation, M86 rapidly enters the stationary phase, while the growth of the original strain 1841 is relatively slow. This indicates that M86 is a rapidly growing and highly active strain. Figure 4 It can be seen that M86 exhibits higher energy metabolism activity than the original strain 1841 during growth. Under the same growth conditions, M86 reaches its peak energy metabolism (70 μM) after 11 hours of growth, while the peak energy metabolism of the original strain 1841 is only 45 μM, and it takes 12 hours to reach its peak. Compared with 1841, M86 has a significantly stronger energy metabolism capacity, indicating that it has higher metabolic activity during growth.

[0112] 3. Analysis of M86 mutation characteristics and their functional impact based on whole-genome resequencing To further analyze the mutational characteristics of M86, this invention resequencing its genome. Genomic DNA was extracted from M86, a sequencing library was constructed, and high-quality sequencing data was obtained using next-generation sequencing technology. Figure 5 Simultaneously, a genomic library with an insert length of 300 bp was constructed, referencing... Figure 6 , Figure 7 Using the complete genome sequence of the original strain 1841 as a reference (the genome accession number for the original strain 1841 is NCBI Submission accession (SUB14919781)), a comprehensive analysis of the genomic mutation sites of M86 was performed. Figure 8 .

[0113] Depend on Figure 8 It is known that ARTP-induced mutations are concentrated on adenine (A) and thymine (T) bases, and the main mutation type is point mutation (SNP). SNP mutation spectrum analysis revealed that the main SNP mutation types are T:A>C:G and C:G>T:A. Figure 9 Furthermore, insertion / deletion (INDEL) length analysis showed that single-base insertions were the predominant INDEL type. (See reference...) Figure 10 .

[0114] SNP mutation analysis: A total of 12 SNP mutation sites were detected in the M86 genome, all located in protein-coding regions. SNP mutations in 3 genes (GEO0859, GEO1366, and GEO3080) were silent mutations, while SNP mutations in 9 genes were nonsynonymous mutations, leading to changes in protein structure. (See [link to relevant documentation]). Figure 11 And Table 1. Arginine (R) at position 111 of YplP is mutated to cysteine ​​(C); proline (P) at position 270 of XkdE is mutated to serine (S); asparagine (N) at position 402 of GutR is mutated to aspartic acid (D); valine (V) at position 109 of YdzE is mutated to phenylalanine (F); leucine (L) at position 35 of GE01829 is mutated to glutamine (Q); glutamine (Q) at position 93 of GE02348 is mutated to glutamic acid (E); proline (P) at position 303 of GE03080 is mutated to threonine (T); aspartic acid (D) at position 91 of YqeW is mutated to glycine (G); glycine (G) at position 122 of YqgH is mutated to glutamic acid (E).

[0115] Table 1: SNP mutation site information for M86

[0116] INDEL mutation analysis: Nine single-base insertion sites were detected in the M86 genome, four of which were located upstream and downstream of gene clusters and may regulate gene transcription. (Reference) Figure 11 Table 2.

[0117] Specifically, the insertion sites are as follows: At site 312941 (T-TC), an insertion occurs upstream of the GEO00285, yoaW, yndM, and GEO00288 gene clusters. At site 814535 (T-TC), an insertion occurs upstream of the ykvZ, GE00683, and ykvY gene clusters. At site 1862323 (A-AC), an insertion occurs downstream of the ycgO and ycgN gene clusters. At site 2434315 (C-CT), an insertion occurs downstream of the yxjB and ycgN gene clusters. Furthermore, five insertion sites are located within the coding sequence (CDS) segment, causing alterations in the protein structure of the corresponding genes, potentially affecting protein function. Specifically, an insertion at site 824120 (A-AC) alters the protein structure encoded by the ykvP gene. An insertion at site 1434230 (T-TC) alters the protein structure encoded by the cotJA gene. An insertion at position 2334423 (T-TG) resulted in a structural change in the protein encoded by the xdhA gene. An insertion at position 2544952 (C-CG) resulted in a structural change in the protein encoded by the ywfA gene. An insertion at position 3226061 (A-AG) resulted in a structural change in the protein encoded by the GE03075 gene.

[0118] Table 2: INDEL mutation site information for M86.

[0119]

[0120] Chromosomal structural variation analysis: Chromosomal structural variations were also detected in the M86 genome. Specifically, a 1218 bp gene deletion (DEL) occurred between loci 29527 and 30745. An inter-chromosomal translocation (CTX) of 41815 bp occurred between loci 1528892 and 1570707. (See reference...) Figure 11 Table 3.

[0121] Table 3: DEL and ITX mutation sites in M86.

[0122]

[0123] II. Study on the composition of fermentation broth of Bacillus amyloliquefaciens M86 1. Identification of active substances against gray mold in M86 and study on the antibacterial effects of its lipopeptide components Previous studies have shown that the antibacterial active substances of *Bacillus amyloliquefaciens* mainly include antibacterial proteins, short peptides, and lipopeptides. In this invention, M86 was fermented, and proteins and lipopeptides were extracted from the fermentation broth. Their activity was detected using a botrytis cinerea antagonism assay. The activity detection results of the fermentation products are referenced... Figures 12-14 .

[0124] according to Figure 12 It was found that the fermentation broth of M86 exhibited significant anti-Botrytis cinerea activity, while the protein extract showed no anti-Botrytis cinerea activity. The lipopeptide extract, however, showed significant anti-Botrytis cinerea activity, with an inhibition zone diameter reaching 33.00 mm. Furthermore, lipopeptide yield determination showed that the crude lipopeptide yield of the original strain 1841 was 484.94 mg / L, while the crude lipopeptide yield of M86 was significantly increased to 985.17 mg / L. Figure 13 These results indicate that lipopeptides are the main components of the antibacterial activity of M86.

[0125] Time-dependent analysis of lipopeptide antibacterial activity: To further investigate the antibacterial activity of lipopeptides, crude lipopeptide extracts were extracted from 1841 and M86 at different fermentation times (24h, 48h, 72h, 96h), and their antibacterial activity was analyzed by a botrytis cinerea antagonism experiment. The results are referenced. Figure 14 .

[0126] Depend on Figure 14 It was found that the antibacterial activity of the lipopeptide extracts of both 1841 and M86 significantly increased with increasing fermentation time. After 48 hours of fermentation, the anti-Botrytis cinerea activity of M86 was significantly higher than that of 1841. In addition, the diameter of the inhibition zone of M86 was 28.00 mm at 48 hours of fermentation, 35.20 mm at 72 hours, and reached 40.00 mm at 96 hours.

[0127] 2. Differential expression of lipopeptide synthesis gene clusters in M86 and their bmyDABC Significant upward adjustment This invention validated 38 genes from 8 lipopeptide synthesis-related gene clusters predicted by anti-SMASH using RT-qPCR. The results are referenced. Figures 15-24 .

[0128] Depend on Figures 15-24 It can be seen that in the mutant strain M86, Bacillomycin Synthetic gene clusters ( bmyD , bmyA , bmyB , bmyC The expression level of ) was significantly higher than that of the other 7 gene clusters, according to reference Figure 15 By analyzing different fermentation times (12h, 36h, 48h, 72h, 96h) bmyD , bmyA , bmyB , bmyC Expression level detection revealed no significant difference in the expression levels of these genes between 1841 and M86 before 36 hours, but at 48 hours, 72 hours, and 96 hours, the expression levels of M86 showed a significant difference. bmyD , bmyA , bmyB , bmyC Expression levels were significantly upregulated, with the most significant difference observed at 48 hours, and the upregulation trends of the four genes were generally consistent. (See [link to relevant documentation]). Figure 16 Furthermore, in M86, Difficidin Synthetic gene clusters ( dfnD , dfnE , dfnF , dfnG , dfnH , dfnI , dfnJ The mRNA expression level of ) was also significantly upregulated, see [link to relevant documentation]. Figure 17 ; Locillibactin Synthetic gene clusters ( locD , locA , locB , mycB , locC mRNA expression levels were significantly upregulated, according to reference... Figure 18 ; Bacilysin Synthetic genes bacD mRNA expression levels were significantly upregulated, reference Figure 19 ; Surfactin Synthetic gene clusters ( srfAA , srfAB , srfAC The mRNA expression level of ) was significantly upregulated, according to reference Figure 20 ; Bacillibactin Synthesis gene mRNA expression level epsC Significantly upward, reference Figure 21 ; Fengycin Synthetic gene clusters ( fenA , fenB , fenC , fenD , fenE The mRNA expression level of ) was significantly downregulated, see [link to relevant documentation]. Figure 22 ;and Macrolactin H synthetic gene cluster ( mLnA , mLnB , mLnC , mLnD , mLnE , mLnF , mLnG The mRNA expression level of ) was significantly downregulated, according to reference Figure 23 ; Bacillaene Synthetic gene clusters ( baeJ , baeL , baeM , baeN , baeR The mRNA expression level of ) was significantly downregulated, according to reference Figure 24 .

[0129] In summary, the transcriptional expression analysis of eight lipopeptide synthesis gene clusters in the M86 mutant strain revealed significant differential regulatory patterns among these gene clusters during fermentation. Among them, Bacillomycin , Bacillibactin , Difficidin , Locillibactin , Surfactin and Bacilysin The synthetic gene clusters initiated transcription at 48 hours of fermentation, and the transcriptional expression level was significantly higher than that of the original strain 1841. Conversely, Macrolactin H, Fengycin and Bacillaene The transcription of the synthetic gene clusters was significantly suppressed at the same time point. Among the six gene clusters that were upregulated in transcription, Bacillomycin Synthetic gene cluster genes ( bmyD , bmyA , bmyB , bmyC The expression level of ) was particularly prominent, significantly higher than other lipopeptide synthesis gene clusters. This indicates that lipopeptide compounds in M86 are mainly expressed as... bmyD , bmyA , bmyB , bmyC Gene clusters composed of four genes Bacillomycin Mainly.

[0130] Bacillomycin belong Iturin Subtypes of the family, currently reported subtypes include Bacillomycin D、 Bacillomycin F, Bacillomycin L and Bacillomycin Lc. These subtypes have highly identical synthetic gene clusters in sequence, but their biological activities may vary due to structural differences.

[0131] The above research results indicate that the lipopeptide type after ARTP mutagenesis is determined by... bmyDABC Bacitracin synthesized by gene clusters.

[0132] 3. Lipopeptide compounds in M86 were separated and purified by HPLC.

[0133] In this invention, the extracted crude M86 lipopeptide was separated and prepared, and further purified by methanol reconstitution. Acetonitrile was used as the washing solution, and the mixture was separated using an HPLC system with a Symmetry Prep™ C18 column, initially yielding three fractions: a, b, and c. Further experiments using gray mold antagonism confirmed that fraction a exhibited significant anti-gray mold activity, while fractions b and c showed no antibacterial activity. (Refer to...) Figure .

[0134] During the separation process, the concentrations of fractions a and b exceeded the upper limit of HPLC. To obtain a pure product, fraction a, which exhibited antibacterial activity, was further separated and purified to obtain fractions a1, a2, and a3. An antagonistic experiment against *Botrytis cinerea* confirmed that a2 had significant anti-*Botrytis cinerea* activity, while a1 and a3 showed no activity. (Reference) ​ .

[0135] 4. Based on (ESI)-Q-TOF-MS parsing ​ The molecular structure of L and the types of its homologues.

[0136] In this invention, electrospray ionization-quadrupole-time-of-flight mass spectrometry ((ESI)-Q-TOF-MS) was used to analyze fraction a2, and the masses of the main compounds were found to be P4 (1021.5153 m / z), P5 (1035.5315 m / z), and P6 (1049.5473 m / z), respectively. (See [link to relevant documentation]). ​ Among them, the mass concentrations at 1021.5153 m / z and 1035.5315 m / z are relatively high. The MS / MS fragments of the three characteristic peaks P4, P5, and P6 mainly produce b-type and y-type ions. The b-type ions of the compound at 1021.5153 m / z include b1 (340.2593 m / z), b2 (503.3219 m / z), b3 (617.3638 m / z), and b4 (704.4381 m / z), while the corresponding main y-type ion is y5 (188.0710 m / z). In addition, other major fragments were detected, including Ser-Glu (216.0806 m / z), (D-Tyr) + (D-Asn) (278.1141 m / z), Thr + C14β-NH2FA + Asn (441.3095 m / z), and (CH2)14 (184.2058 m / z). For the compound at 1035.5315 m / z, b1 (354.2751 m / z) and b2 (517.3219 m / z) ions were observed, along with other major fragments such as Ser-Glu, (D-Tyr) + (D-Asn), Thr + C15β-NH2FA + Asn (455.3218 m / z), and (CH2)15 (198.2214 m / z). In the compound at 1049.5473 m / z, the b1 (368.2907 m / z) ion and other major fragments such as Ser-Glu, (D-Tyr) + (D-Asn), Thr + C16β-NH2FA + Asn (469.8375 m / z), and (CH2)16 (212.2370 m / z) were observed. Note that the b ions of P4, P5, and P6 all differ by one CH2 (14 m / z).

[0137] In summary, fraction a2 consists of three homologues (C14- ​ L, C15- ​ L,C16- ​ Composed of L, with molecular weights of 1021.5153 m / z, 1035.5315 m / z, and 1049.5473 m / z, respectively, its structure was determined by secondary mass spectrometry to be nβ-NH2FA+Asn+(D-Tyr)+(D-Asn)+Ser+Glu+(D-Ser)+Th. This is similar to bacitracin L ( ​ The structure of L) is consistent. This invention utilizes the chimeraX software to analyze C14- ​ L and C15- ​ L undergoes structural reconstruction, such as ​ As shown, C14- ​ L and C15- ​ The structure of the amino acid polypeptide segment of L is the same, and the amino group at the terminal Thr forms an amide bond with the hydroxyl group of the fatty acid under the action of ET enzyme, making... ​ The seven amino acids form a cyclic peptide structure, while C14- ​ L and C15- ​ L is separated from the fatty acid chain by a methyl group, C14- ​ L is formed from 14 carbon atoms to form a straight-chain fatty acid, while C15- ​ L is also a straight-chain fatty acid formed by 14 carbon atoms, but the hydrogen ion at its third carbon position is replaced by CH2, forming a C15 fatty acid with a trans fatty acid structure. From the above results, it can be seen that the fatty acid length of lipopeptides is the main factor in enhancing their function; long-chain and branched-chain fatty acids can improve the lipophilicity of lipopeptides and enhance their binding force to cell membranes.

[0138] During the two-stage separation and preparation of M86 lipopeptides, the remaining fractions showed no antibacterial activity. The a2 fraction, which exhibited antibacterial activity, was identified by mass spectrometry as bacitracin L (…). ​ Mass spectrometry analysis of the crude lipopeptide extracts of 1841 and M86 showed that the crude lipopeptides of 1841 and M86 had a larger proportion of 1021.5153 m / z and 1035.5315 m / z values, respectively. Furthermore, the detection signal of bacitracin L in M86 was significantly stronger than that in 1841. ​ Meanwhile, we captured and resolved other ion fragments of 1841 and M86, and found that, except for 1021.5153 m / z, 1035.5315 m / z, and 1049.5473 m / z, small amounts of C were present in 1841 and M86. 11 - ​ (979.4711 m / z), C 12- ​ L (993.4832 m / z), C13- ​ L (1007.5004 m / z). Secondary mass spectrometry resolved C. 11 - ​ L, C 12 - ​ L, C13- ​ L-ion fragments, with C 14 - ​ L, C 15 - ​ L, C 16 - ​ The b ions of L are all different by one -CH2- (14 m / z).

[0139] In summary, the antimicrobial lipopeptides in M86 are... ​ L can be synthesized into 6 types in M86. ​ L homologues, namely C 11 - ​ L, C 12 - ​ L, C 13 - ​ L, C 14 ​ L, C 15 - ​ L, C 16 - ​ L. Where C 14 - ​ L and C 15 - ​ L was synthesized in the largest quantity, C 16 - ​ L is next, and C is next. 11 - ​ L, C 12 - ​ L, C 13 - ​ The amount of L synthesized is relatively small. Additionally, C in M86... 14 - ​ L, C 15 - ​ L, C 16 - ​ The amount of L synthesized was significantly higher than that in 1841, and C 16 - ​ The amount of L synthesized was significantly increased compared to 1841. This was achieved by analyzing the synthesis of L in 1841 and M86. ​ L yield was measured, and it was found that M86 contained... ​ The yield of L was 423.33 mg / L, compared to 244.22 mg / L in 1841. ​The yield of L increased by approximately 1.73 times. In the fermentation broth of M86, ​ L accounts for approximately 57.78% of crude lipopeptides.

[0140] III. Adjustable directional synthesis of bacillus L 1. Bacitracin L Synthesis Gene Cluster ​ Targeted activation In this invention, sfGFP is used as the detection probe to... ​ The promoter is loaded into the sfGFP front promoter region to obtain P bmyD The -sfGFP plasmid was successfully transformed into M86, resulting in the M86-sfGFP strain. The fluorescence signal intensity was used to monitor... ​ Transcriptional levels of the promoter. Based on the M86-sfGFP strain, glucose in Landy medium was replaced with different sugar sources (including glucose, sucrose, maltose, and lactose), and the sfGFP fluorescence signal intensity was detected after 48 h of fermentation. Further, gradient media with different sugar sources were set up, and the sfGFP fluorescence signal was detected at 24 h and 48 h, respectively, to explore the effects of different sugar sources and concentration gradients on promoter transcription. ​ The effect on promoter transcriptional activity.

[0141] The effects of different glycogen sources on the transcriptional activity of the bmyD promoter, see reference. Figures 30 to 33 .

[0142] The experimental results show that glucose has no significant effect on the sfGFP fluorescence signal; the sfGFP signal increased by 1.51 times in sucrose medium; the sfGFP signal increased by 4.13 times in maltose medium; and the sfGFP signal increased by 4.96 times in lactose medium. (Reference) Figure 30 .

[0143] Concentration gradient experiments showed that glucose and sucrose activated the sfGFP signal at low concentrations, but the signal gradually weakened with increasing concentration, exhibiting an inhibitory effect at high concentrations. (Reference) Figure 31 (A represents the sfGFP intensity in different glucose groups, and B represents the sfGFP intensity in different sucrose groups).

[0144] In contrast, maltose and lactose activated sfGFP signaling at low concentrations at 24 h, but the activation effect was not significant at high concentrations, and the signal intensity difference between high and low concentrations was small. As the cells grew to 48 h, both high concentrations of maltose and lactose significantly activated sfGFP signaling (see...). Figure 32(A represents the sfGFP intensity in different maltose concentrations, and B represents the sfGFP intensity in different lactose concentrations). Specifically, the sfGFP signal gradually increases in the 1.25-10 g / L concentration range of maltose, and gradually decreases in the 10-30 g / L concentration range; the sfGFP signal gradually increases in the 1.25-20 g / L concentration range of lactose, and gradually decreases in the 25-30 g / L concentration range.

[0145] Furthermore, when maltose and lactose are added together with glucose, although maltose and lactose can activate sfGFP signaling, the presence of glucose significantly inhibits sfGFP signaling. The inhibitory effect becomes more pronounced with increasing glucose concentration. (See [link to relevant documentation]). Figure 33 (A represents the sfGFP intensity after mixing maltose with different glucose concentrations, and B represents the sfGFP intensity after mixing lactose with different glucose concentrations).

[0146] Based on the above findings, further investigation was conducted on the effects of different sugar source culture media on... Bacillomycin The impact of L production, and the results of the investigation are referenced. Figure 34 .

[0147] Depend on Figure 34 It can be seen that in a 10 g / L glucose medium, Bacillomycin The yield was 366.86 mg / L in Lactose medium; 786.56 mg / L in 10 g / L maltose medium; and 1177.44 mg / L in 10 g / L lactose medium. When lactose was used as the sugar source, Bacillomycin L production increased significantly, reaching 2.84 times that of glucose medium.

[0148] Based on the above findings, in this invention, using 1.25~20 g / L lactose as a carbon source can significantly promote... Bacillomycin The transcription and translation of L-synthetic gene clusters are significantly improved. Bacillomycin The production of L.

[0149] Different ion pairs bmyD The influence of promoters.

[0150] Fermentation experiments were conducted in Landy medium with the addition of 0.5 g / L of K₂HPO₄, KH₂PO₄, K₃PO₄, CaSO₄, MgSO₄, MgCl₂, KCl, Fe₂SO₄, CuSO₄, and MnSO₄, respectively. The results showed that K₂HPO₄, KH₂PO₄, K₃PO₄, CaSO₄, and MgSO₄ significantly enhanced the sfGFP signal, with K₂HPO₄ showing the most significant enhancement, reaching 2.05-fold. (Reference) Figure 35Further investigation was conducted into the effect of different concentration gradients of K2HPO4 on the sfGFP signal. The results showed that the sfGFP signal was highest at a concentration of 0.5 g / L, and gradually weakened with increasing K2HPO4 concentration. (Reference) Figure 36 Meanwhile, different concentrations of K2HPO4 have different effects on... Bacillomycin Experiments on the effect of L on yield showed that within a concentration gradient of 0–1.5 g / L, Bacillomycin L production gradually increased, with a 1.5-fold increase at a concentration of 0.5 g / L and a 2.5-fold increase at a concentration of 1.5 g / L. However, as the K₂HPO₄ concentration continued to increase, Bacillomycin L production declined significantly, reference Figure 37 .

[0151] Based on the above findings, in this invention, 0-1.5 g / L K₂HPO₄ is considered as an additive to the culture medium to promote... Bacillomycin L production.

[0152] Different amino acids bmyD The influence of promoters.

[0153] In this invention, the effects of different amino acids on the sfGFP fluorescence signal were also screened, including aspartic acid (Asp), glycine (Gly), threonine (Thr), asparagine (Asn), phenylalanine (Phe), tyrosine (Tyr), serine (Ser), valine (Val), tryptophan (Trp), glutamic acid (Glu), leucine (Leu), and proline (Pro). The results are shown in […]. Figure 38 .

[0154] Depend on Figure 38 It can be seen that 0.5 g / L of Gly inhibited the sfGFP fluorescence signal, while other amino acids had no significant effect on the sfGFP signal.

[0155] Further investigation was conducted into the effects of different concentration gradients of Gly on the sfGFP signal. The results are shown in [the table below]. Figure 39 .

[0156] Depend on Figure 39 It can be seen that when the Gly concentration is between 0 and 2.5 g / L, the sfGFP fluorescence signal increases with increasing concentration, and reaches the highest signal intensity at 2.5 g / L; when the Gly concentration increases further, the sfGFP signal weakens.

[0157] Meanwhile, different concentrations of Gly Bacillomycin Experiments on the effect of Gly on L production showed that, within the concentration range of 0–3.0 g / L, as the Gly concentration increased, Bacillomycin L production gradually increased at a concentration of 3.0 g / L Lly. BacillomycinL-yield reached 650 mg / L, representing a 1.78-fold increase compared to the unadded Gly, according to reference... Figure 40 .

[0158] Based on the above findings, this invention uses a concentration of 0~3.0 g / L. Gly As an additive to the culture medium to promote Bacillomycin L production Orthogonal experimental design and optimization.

[0159] Based on the previously screened exogenous regulatory elements (lactose, K2HPO4, and glycine) and their different concentration gradients, bmyD To investigate the regulatory role of the promoter, this invention uses Landy medium as the base and designs a three-factor, four-level orthogonal experimental scheme with lactose (5 g / L, 10 g / L, 15 g / L, 20 g / L), K₂HPO₄ (0.25 g / L, 0.75 g / L, 1.25 g / L, 1.75 g / L), and Gly (2.0 g / L, 3 g / L, 4 g / L, 5 g / L) as variables, as shown in Table 4. Sixteen combinations were set up, with three parallel experiments for each combination to ensure data reliability.

[0160] Sixteen culture media were prepared using an orthogonal experimental design. After fermentation on a shaker for 48 hours, the sfGFP probe signal intensity of each group was detected and statistically analyzed. The experimental results showed that the optimal ratio of exogenous regulatory elements for activating sfGFP was 5 g / L lactose, 3 g / L glycine, and 0.75 g / L K2HPO4 (see Table 4).

[0161] Table 4

[0162] Construction and validation of the optimal fermentation medium: Combining Landy's basal medium, this invention constructs a highly efficient fermentation medium for activation. Bacillomycin The optimal fermentation medium for L-synthetic gene clusters is BDSM medium, with the following formula: lactose 5g / L, L-glutamate sodium 5g / L, tryptone 5g / L, yeast extract 1g / L, MgSO4·7H2O 0.5g / L, KCl 0.5g / L, K2HPO4 0.75g / L.

[0163] Further fermentation experiments confirmed that the M86-sfGFP strain cultured in BDSM medium exhibited a 12-fold increase in sfGFP probe signal intensity compared to the M86-sfGFP strain cultured in Landy medium. Figure 41 (A). It is evident that the BDSM culture medium of the present invention can significantly improve... bmyD Transcriptional activity of the promoter.

[0164] Bacillomycin Increased L yield: In fermentation experiments conducted on BDSM medium, Bacillomycin The yield of L was significantly increased. Experimental results show that after fermentation in BDSM medium, Bacillomycin L The yield reached 1399.01 mg / L, which was 3.19 times higher than that of Landy medium. Figure 41 (B).

[0165] Optimization of external control elements bmyDABC And the effects on the expression of transcription factors related to glucose metabolism.

[0166] To further investigate the effects of BDSM culture medium... Bacillomycin To investigate the regulatory mechanisms of transcription and translation of L-synthetic gene clusters and their association with related transcription factors, this invention uses Landy and BDSM media as fermentation substrates to ferment and culture the M86 strain. Cells were collected at 24h, 36h, and 48h to extract mRNA, and the expression of related genes was analyzed by qRT-PCR.

[0167] Dynamic analysis of gene expression in M86: qRT-PCR results showed that... bmyD, bmyA, bmyB and bmyC Transcriptional expression was significantly advanced in BDSM medium. In Landy medium, bmyD The expression level of [a substance] gradually increases with bacterial growth, while in BDSM medium, bmyD, bmyA, bmyB and bmyC The expression level of [the substance] significantly increased at 24 h, gradually decreased at 36 h, and was essentially non-expressed at 48 h. (See [reference]). Figure 42 (A, B, C, and D represent respectively) bmyD Transcriptional expression differences bmyA Transcriptional expression differences bmyB Transcriptional expression differences bmyC (Differences in transcriptional expression). This result indicates that BDSM medium can accelerate... Bacillomycin The transcription and translation of the L-synthetic gene cluster provide an important basis for subsequent synthesis of complex zymocytes.

[0168] Transcription factor expression trend analysis: capable of activating bmyD promoter transcription factors yhiH and ccpB Expression trends in cells after fermentation in BDSM medium and Bacillomycin The expression for L is basically the same, see reference. Figure 43 (A, B). This indicates that... yhiH and ccpB It may be possible to regulate in BDSM medium. bmyD The activity of the promoter, and thus affects BacillomycinExpression of L-synthetic gene clusters. In contrast, transcription factors... ccpA The expression level was low at 24 h and increased at 36 h. (Reference) Figure 43 (C) indicates that ccpA exist Bacillomycin L-synthesis plays a phased role in regulation, and its function may be closely related to the growth stage or metabolic state.

[0169] Bacillomycin L-synthesis starting material analysis and directional optimization.

[0170] The aforementioned experiments, using sfGFP as a detection probe, confirmed that BDSM medium can significantly activate... Bacillomycin The transcription and translation of the L-synthetic gene cluster, thereby providing... Bacillomycin The synthesis of L provides a sufficient complex of polyketide synthase and non-ribosomal peptide synthase. However, in Bacillus amyloliquefaciens... ​ Whether L-type synthetic substrates can meet the synthesis requirements, and the specific types of synthetic substrates, are still unclear, although it is known that... ​ The structure of L consists of fatty acids and amino acids, and contains C. 12 - 16 Six homologues were identified, but the types of their starting fatty acids remain unclear. Through analysis of... ​ The analysis and prediction of the L-synthetic gene cluster suggest that fatty acids are the initiating substrates for the AT domain. Therefore, this invention uses fatty acid metabolomics analysis to elucidate the components involved in... ​ The types of fatty acids synthesized by L.

[0171] Fatty acid metabolomics analysis: In this invention, fatty acids of strain M86 at different growth stages (12h, 24h, 48h, 72h) were extracted and measured. PCA analysis confirmed that there were differences in fatty acids in M86 cells at different fermentation times. A total of 48 fatty acids, including medium-chain and long-chain fatty acids, were detected in M86.

[0172] refer to ​ The contents of the five fatty acids (C10-O, C12-O, C14-O, C15-O, and C18-1n9c) varied significantly among different samples, and were related to... ​L yield showed a negative correlation. Myristic acid content was the highest, at 20.94 mg / g at 12h, 8.20 mg / g at 24h, 12.59 mg / g at 48h, and 23.67 mg / g at 72h. Myristic acid content gradually decreased from 12 to 24h, while gradually increasing from 24 to 72h. Pentadecanoic acid and oleic acid showed similar trends. Pentadecanoic acid content was 8.35 mg / g at 12h, 3.89 mg / g at 24h, 2.83 mg / g at 48h, and 3.13 mg / g at 72h. Oleic acid content was 7.51 mg / g at 12h, 2.76 mg / g at 24h, 2.52 mg / g at 48h, and 2.33 mg / g at 72h. Both pentadecanoic acid and oleic acid content gradually decreased with cell growth. While capric acid and lauric acid showed the same variation trend as pentadecanoic acid and oleic acid, their content in M86 was extremely low. Specifically, capric acid levels were 0.69 mg / g at 12h, 0.23 mg / g at 24h, 0.21 mg / g at 48h, and 0.23 mg / g at 72h; lauric acid levels were 1.96 mg / g at 12h, 0.56 mg / g at 24h, 0.59 mg / g at 48h, and 0.64 mg / g at 72h. This indicates that the changes in the content of these fatty acids have a significant impact on… ​ The synthesis of L has a significant impact.

[0173] Validation of fatty acids as synthesis starting materials: Based on the above metabolomics results, the inventors of this invention hypothesize that C10-O, C12-O, C14-O, C15-O, and C18-1n9c may be... ​ The starting fatty acid for L-synthesis.

[0174] To further confirm this prediction, this invention added 0.5 g / L of C10-0, C12-0, C14-0, C15-0, and C18-1n9c to Landy medium for fermentation experiments. The results were investigated as follows: ​ , ​ .

[0175] Depend on ​ It can be seen that after adding C14-0 ​ L production increased by 1.83 times after adding C15-0. ​ L production increased by 1.64 times; ​ It can be seen that C14- ​ L and C15- ​ The L content was significantly higher in Landy medium than in Landy medium. However, after adding lauric acid, lauric acid, and oleic acid... ​ L production decreased significantly, reference ​ This confirms that C14-0 and C15-0 are... ​ Starting materials for the synthesis of L.

[0176] Optimization of fatty acid synthesis precursors.

[0177] As demonstrated in the aforementioned experiments, C14-0 and C15-0 have poor solubility in the culture medium and tend to precipitate after sterilization, which limits their application in fermentation processes. Valine, isoleucine, and leucine can regulate fatty acid synthesis and oxidation through their metabolites and signaling pathways. The roles of these branched-chain amino acids in lipid metabolism depend not only on their direct metabolites but also on their influence on fatty acid synthesis and breakdown through complex signaling pathways.

[0178] Therefore, this invention further investigates how adding fatty acid synthesis precursors (Val, Ile, Leu) can promote fatty acid synthesis in strain M86, thereby indirectly improving... ​ The yield of L, the results of which can be found in [the table]. ​ .

[0179] Depend on ​ It can be seen that after adding 0.5 g / L of Val, ​ L-carboxylic acid (L-carboxylic acid) production increased by 1.22 times, while leucine and isoleucine had no significant effect on increasing production. Therefore, valine was identified as the main additive to promote fatty acid synthesis.

[0180] according to ​ -SMASH ​ A detailed analysis of the L-synthetic gene cluster revealed that its synthesis process uses fatty acids as starting materials and amino acids as substrates. ​ The PKS-NRPS complex synthesizes lipopeptides using starters and substrates. Preliminary investigations indicate that C... 14 and C 15 Fatty acids are ​ The synthesis of L can be promoted by adding the branched-chain fatty acid precursor Val, but the yield increase is limited, which may be related to the lack of amino acid substrate.

[0181] Identification and optimization of amino acid substrates: Through domain prediction of the PKS-NRPS complex, it was found that... ​ During the synthesis of L ​ Responsible for binding asparagine (Asn). ​ BmyC is responsible for assembling tyrosine (Tyr), asparagine (Asn), serine (Ser), and glutamate (Glu), with Tyr and Asn forming D-Tyr and D-Asn during the transformation process; BmyC is responsible for assembling Ser and threonine (Thr), with Ser forming D-Ser under the influence of the E domain. Therefore, it is speculated that Thr, Asn, Ser, Glu, and Tyr are... ​ Key substrates for L-amino acid chain synthesis.

[0182] Substrate feeding experiments showed that adding Thr, Asn, Ser, Glu, or Tyr alone had an effect on... ​ L yield had no significant impact, but when added in a 2:1:2:1:1 ratio (Asn:Tyr:Ser:Glu:Thr), ​ L production increased by 1.3 times, reference ​ (A). Furthermore, adding D-Ser and D-Asn in the D-(5+) form increased yield by 0.84 times; while adding them in the L-(5+) form increased yield by 1.34 times. (Ref.) ​ (B). This indicates that the simultaneous presence of Asn, Tyr, Ser, Glu, and Thr can significantly promote [the effect]. ​ Synthesis of L.

[0183] Co-optimization of starter and substrate: Based on the aforementioned findings, this invention involves mixing and adding the synthetic starting material (valine, Val) with the amino acid substrate (Asn:Tyr:Ser:Glu:Thr, abbreviated as (5+)).

[0184] The results showed that after co-addition of Val and (5+), ​ L production increased by 1.49 times, a 22.13% increase compared to adding Val alone, and a 14.62% increase compared to adding (5+) alone. (Reference) ​ (C).

[0185] Further optimization of the addition ratio of Val and (5+) revealed that when added at a ratio of 7:1, ​ L production reached optimal reference ​ (D).

[0186] Based on this, a "BDSM+Val+(5+)" medium was developed. Compared with BDSM medium, the BDSM+Val+(5+) medium has... ​ L-yield reached 1992.51 mg / L, an increase of 1.48 times compared to 1349.01 mg / L, according to reference. ​ (E). In the optimized culture medium, C 15 - ​ L and C 16 - ​ The content of L increased significantly, especially with the achievement of high yields of C. 16 - ​ L, reference ​ (F).

[0187] IV. Broad-spectrum antibacterial activity of bacitracin L and its application in agriculture and biomedicine. Lipopeptide compounds have attracted widespread attention due to their broad-spectrum antibacterial activity, but their specific antagonistic spectrum has not been systematically studied. This invention, using *Botrytis cinerea* as an indicator fungus, screened for a lipopeptide compound with strong antibacterial activity— ​ L. Existing research indicates that ​ L can effectively destroy Rhizoctonia solani ( ​ ​ The mycelium of ) and the pathogen of tomato wilt ( ​ It exhibits a strong inhibitory effect. However, it rarely shows... ​ Reports on the inhibitory effects of L on other pathogenic microorganisms also limit the scope of research. ​ Applications of L.

[0188] To further develop ​ The application of L in this invention has been extensively studied. Bacillomycin Experiments on the inhibitory effect of L on various pathogenic microorganisms.

[0189] The results of the experiment showed that Bacillomycin Fermentation broth of Bacillus amyloliquefaciens M86 with a concentration of 423.33~1992.51 mg / L showed excellent antibacterial effects against the following 16 fungi and 9 bacteria.

[0190] The 17 fungi are: Aspergillus niger Aspergillus niger Hansenula d'Bari yeast Debaryomyces Hansen's Alternaria Alternaria alternata Alternaria tomato Alternaria solani Aspergillus flavus Aspergillus flavus Colloidal anthrax bacteria Colletotrichum gloeosporioides Fusarium moniliforme Fusarium wilt Fusarium solani Fusarium solani White mold Geotrichum candidum Penicillium finger Penicillium digitatum Lemon green penicillium Penicillium lemon-green Trichoderma, Pestalotipsis funerea, and Rhizopus nigricans Rhizopus nigricans , Rhizopus oryzae Rhizopus rice Sclerotium sclerotiorum Sclerotinia sclerotiorum Streptomyces lavender Streptomyces lavender green Trichoderma Trichoderma green The nine bacteria included three Gram-negative bacteria and six Gram-positive bacteria; the three Gram-negative bacteria were Shigella. Shigella castellani, E. coli Escherichia coli ,salmonella Salmonella enteritis The six Gram-positive bacteria were Staphylococcus aureus. Staphylococcus aureus Micrococcus luteus Micrococcus luteus Enterococcus Streptococcus agalactiae Bacillus belesi Bacillus beautiful Bacillus subtilis 1560 Bacillus subtilis 1560 Bacillus licheniformis Bacillus lichen-like .

[0191] Different concentrations were determined using the filter paper disc method. Bacillomycin The antibacterial activity spectrum of the L fermentation broth is shown in Tables 5 and 6. The inhibition zone diameter was measured, and a plot was drawn. Bacillomycin L has an antifungal and antibacterial spectrum. Bacillomycin The antifungal spectrum of Bacillus amyloliquefaciens M86 fermentation broth at a concentration of 423.33 mg / L is shown in the figure. Figure 49 The antibacterial spectrum of antagonistic bacteria is shown in [reference needed]. Figure 50 See the actual pictures of the experimental results. Figure 51 .

[0192] Table 5: Different Concentrations Bacillomycin Statistical table of inhibition zone diameters of L fermentation broth against 16 fungi.

[0193]

[0194] Table 6: Different Concentrations Bacillomycin Statistical table of inhibition zone diameters of L fermentation broth against 9 types of bacteria.

[0195]

[0196] The results of the above investigation show that, Bacillomycin Fermentation broth of Bacillus amyloliquefaciens M86 at concentrations ranging from 423.33 to 1995.21 mg / L showed excellent antibacterial effects against the aforementioned 16 fungi and 9 bacteria, while lower concentrations... Bacillomycin The L mixture showed poor antibacterial effects against the aforementioned 16 fungi and 9 bacteria. Furthermore, the high concentration (1995.21 mg / L) obtained by this invention... Bacillomycin L fermentation broth exhibited significant antifungal activity, including against Aspergillus niger. Aspergillus niger (20.22mm), Hansenula d'Bary yeast Debaryomyces hansenii (18.82 mm), Alternaria Alternaria alternata (26.13 mm), Alternaria tomato Alternaria solani (21.98mm), Aspergillus flavus Aspergillus flavus (21.81 mm), Colloidal anthrax Colletotrichum gloeosporioides (27.61 mm), Fusarium moniliforme Fusarium wilt (23.11 mm), Fusarium solani Fusarium nightshade (20.86mm), Geotrichum candida Geotrichum candidum (20.18mm), Penicillium finger-shaped Penicillium fingered (19.85mm), *Penicillium citreovirde* (21.14mm), *Trichoderma* Pestalopsis funeraria (26.61mm), Rhizopus nigricans Rhizopus nigricans (20.85mm), Rhizopus oryzae Rhizopus rice (21.38mm), Sclerotinia sclerotiorum Sclerotinia sclerotiorum (25.85mm), Streptomyces lavenderus Streptomyces lavender (23.26mm), green Trichoderma Trichoderma green (24.83 mm). Bacillomycin L also showed significant antibacterial activity against the following 9 bacteria, including Staphylococcus aureus. Staphylococcus aureus (29.11 mm), Micrococcus luteus Micrococcus luteus (30.04mm), Enterococcus Streptococcus agalactiae (25.59mm), Bacillus belye Bacillus beleister (16.76 mm), Bacillus subtilis 1560 Bacillus subtilis 1560 (26.37 mm), Bacillus licheniformis Bacillus licheniformis (26.79 mm), Shigella Shigella castellani (18.30mm), Escherichia coli Escherichia coli (19.93mm), Salmonella Salmonella enteritidis (25.31mm).

[0197] Based on the above findings, it can be concluded that Bacillomycin L has great potential for application in agriculture.

[0198] Bacillomycin The potential of L in biomedical applications.

[0199] As can be seen from the aforementioned research, Bacillomycin L can antagonize Staphylococcus aureus. Staphylococcus aureus can cause skin infections, food poisoning, and sepsis, therefore... Bacillomycin L can be used in the preparation of drugs for treating skin infections, food poisoning, and sepsis.

[0200] As can be seen from the aforementioned research, Bacillomycin L can antagonize enterococci. Enterococci can cause neonatal sepsis and urinary tract infections in pregnant women. Bacillomycin L can be used in the preparation of drugs for treating neonatal sepsis and urinary tract infections in pregnant women.

[0201] As can be seen from the aforementioned research, Bacillomycin L can antagonize Shigella. Shigella can cause dysentery, which shows... Bacillomycin L can be used in the preparation of drugs for treating dysentery.

[0202] As can be seen from the aforementioned research, Bacillomycin L can antagonize Escherichia coli. Escherichia coli can cause food poisoning and urinary tract infections, indicating that... Bacillomycin L can be used in the preparation of drugs for treating food poisoning and urinary tract infections caused by Escherichia coli.

[0203] V. Carrier Construction.

[0204] a)pcN33- P bmyD -sfGFP vector.

[0205] Using the pcN33-sfGFP vector as a template, the linearized vector was obtained by reverse PCR using self-designed PCN33-sgGFP linearization-F / R primers.

[0206] The 15bp sequence at the end of the linearized vector was used as a homologous sequence and added to the 5' end of the forward / reverse amplification primer sequences of the promoter, respectively, and primers were synthesized. bmyD -P(300bp)-F / bmyD -PR primers.

[0207] Using the Bacillus amyloliquefaciens M86 genome fragment as a template, bmyD -P(300bp)-F / bmyD -PR primers were used for PCR amplification to obtain P bmyD Promoter segment.

[0208] The linearized pcN33-sfGFP vector amplified above and P bmyD The promoter fragment was subjected to DNA electrophoresis to remove interference from the genomic DNA and primer dimers present in the PCR reaction system. Following the instructions of a universal DNA purification and recovery kit (DP214, TIANGEN, China), DNA was recovered via gel extraction to obtain purified linearized pcN33-sfGFP vector and P... bmyD Promoter segment.

[0209] The linearized vector and the target fragment were constructed using homologous recombinase. Homologous recombination was performed according to the instructions of the ClonExpress II One Step Cloning Kit (C112-02, Vazyme, China).

[0210] The reaction solution from the complete homologous recombination reaction was mixed with DH5α competent cells and transformed using the same method as the DH5α competent cells (TSC-C14, Qingke, China).

[0211] Use primers bmyD-P-YAN-F / R, verified by colony PCR and sequencing, yielded pcN33-P bmyD The -sfGFP plasmid is a shuttle plasmid that can replicate in both *E. coli* and *Bacillus*. This plasmid contains both ampicillin and erythromycin resistance; ampicillin is suitable for replication in *E. coli*, while erythromycin is suitable for replication in *Bacillus* (*Bacillus* is sensitive to erythromycin, which increases the probability of positive clones). This plasmid contains enhanced green fluorescent protein (sfGFP), the expression of which is influenced by P. bmyD Strict control of the promoter.

[0212] Primer sequences are shown in Table 7.

[0213] Table 7

[0214] b) Construct pCN33-P bmyD -sfGFP-LacI-MCS.

[0215] The pcN33-P constructed as described above bmyD Using the -sfGFP vector as a template, and employing the self-designed pCN33- bmyD -PF / R reverse PCR was used to obtain pcN33-P bmyD -sfGFP linearized vector.

[0216] Using the pET-19b vector as a template, pET-19b-F / R primers were designed, and the galactose manipulation regulatory element LacI-MCS in the pET-19b vector was obtained by reverse PCR.

[0217] pcN33-P was obtained by referring to the above homologous recombination method. bmyD -sfGFP-LacI-MCS vector.

[0218] Primer sequences are shown in Table 8.

[0219] Table 8

[0220] c) pCN33-P bmyD -sfGFP-LacI-YhiH construction With pcN33-P bmyD Using -sfGFP-LacI-MSN as a vector, pET-19b-linearized-F / R primers were designed for reverse PCR amplification to obtain pcN33-P bmyD The -sfGFP-LacI-MCS linearized fragment aims to insert the transcription factor YhiH into the MCS region.

[0221] Using the genome of Bacillus amyloliquefaciens M86 as a template, primers yhiH-CDS-F / R were designed for PCR amplification to obtain the CDS fragment of the YhiH gene.

[0222] Following the methods described above, the glue was recovered and homologous recombination was performed to obtain pCN33-P. bmyD The 19b-YAN-F / R colony PCR and sequencing verification were performed using the sfGFP-LacI-YhiH vector.

[0223] Primer sequences are shown in Table 9.

[0224] Table 9

[0225] refer to Figure 1 , Figure 1 This is a plasmid map. The recombinant vector pCN33-P was constructed. bmyD The sequence of -sfGFP-LacI-YhiH is shown in SEQ ID No:10.

[0226] verify.

[0227] a) Electroconversion of Bacillus amyloliquefaciens.

[0228] Plasmid demethylation modification: pCN33-P bmyD -sfGFP-LacI-YhiH and empty pcN33-P bmyD The -sfGFP-LacI-MCS vector was transformed into E. coli EC135 / Pm. Bam competent cells and cultured on Amp and Spec antibiotic plates, with colony PCR verification. Subsequently, correct single clones were inoculated into Amp and Spec antibiotic medium and cultured at 37°C until the OD600nm reached 0.2-0.4. Then, 2% arabinose was added, and the strain was transferred to a shaker at 30°C for 12 h to induce the expression of the methyltransferase gene in the Pm. Bam plasmid, thus modifying the recombinant vector with methylation.

[0229] Electroporation of Bacillus amyloliquefaciens: Competent cells of Bacillus amyloliquefaciens M86 were prepared, and sterile, dried electroporation cuvettes (2 mm) were pre-chilled on ice. After the competent cells thawed on ice, 10–15 μL of recombinant vector was added to the competent cells in a clean bench and gently mixed. 90 μL of the mixture of competent cells and recombinant vector (10 μL) was transferred to the pre-chilled electroporation cuvette and incubated on ice for 30 min. Then, electroporation was performed at 2500 V. After electroporation, 750 μL of electroporation recovery medium was quickly added, and the mixture was gently mixed. The bacterial culture was then transferred to sterile EP tubes and incubated at 37 °C and 220 rpm for 3.5 h. After recovery, the culture was centrifuged at 4200 rpm for 4 min to collect the bacterial cells, which were then plated on plates containing erythromycin resistance and incubated overnight at 37 °C. Positive engineered strains with sfGFP fluorescence signals were selected.

[0230] b) Functional verification of the YhiH promoter.

[0231] Following the above procedures, the engineered strain M86-YhiH and the control strain M86-MCS were successfully obtained. After activation, M86-YhiH and M86-MCS bacterial suspensions (OD600nm = 1.0) were prepared.

[0232] Inoculate the cells at a rate of 1% into BDSM+Val+5+ medium and culture until the OD600 reaches 0.5. Then, add IPTG (working concentration 25mM) to induce the expression of YhiH.

[0233] After 48 hours of fermentation, the sfGFP signal was detected, and the yield of bacitracin L was also measured. The results are as follows: Figure 53 As shown, its bacitracin L production increased by 3.75 times.

[0234] In this invention, other transcription factors and bmyD The specific binding ability of the promoter.

[0235] Transcription factors play a central role in gene expression regulation; they activate or inhibit the transcription of target genes by binding to specific DNA sequences in promoter regions. Studies have identified 13 factors that can regulate gene expression in vivo. bmyD Transcription factors of the promoter. To further verify the relationship between these transcription factors and... bmyD The binding ability of the promoter was qualitatively analyzed using EMSA experiments in this scheme.

[0236] Overexpression and purification of transcription factors: His tags were inserted at the front end of 13 candidate transcription factors, which were then transformed into *E. coli* BL21. Overexpression of the transcription factors was induced by IPTG, and the His-tagged transcription factors were purified using a Co column. The purified proteins were SpoVG, LexA, CcpA, GEO3564, FapR, YhiH, YfiS2, YtrA, ComK, CcpB, SpoOJ, YqeW, and YqgH.

[0237] Transcription factors and bmyD Promoter binding: The purified 13 transcription factors were combined with... bmyD A 300bp DNA fragment of the promoter was added to the buffer at a 1:1 ratio to initiate the specific binding reaction between the transcription factor and DNA. bmyD Promoter fragments and the con group without transcription factors served as controls. The migration rate of the complex was analyzed by EMSA electrophoresis. The results showed that the three transcription factors SpoVG, FapR, and YfiS2 were involved in the complex migration. bmyD No significant promoter binding was observed, and the DNA migration position remained largely consistent with the control group (con). Complexes formed by LexA, CcpA, GEO3564, YhiH, YtrA, ComK, CcpB, SpoOJ, YqeW, and YqgH bound to the promoter migrated more slowly, with the DNA appearing at the upper part of the map. Figure 54 (A).

[0238] Binding ability verification at different concentrations: To further verify the binding ability, binding experiments were conducted with four different transcription factor concentrations (0.75, 1.5, 3.0, and 6.0 µM) and the same DNA concentration (10 ng). The results confirmed... bmyD No complex was formed between the promoter and different concentrations of SpoVG, and the DNA migration rate remained consistent with the control. Figure 54 B) bmyD The promoter forms a complex with LexA, and the migration rate of the complex decreases with increasing protein concentration. bmyD The promoter appears at the upper part of the map, showing a clear gradient. Figure 54 C). bmyD The promoter and CcpA can form a complex, and the migration rate of the complex decreases with increasing protein concentration. bmyD The promoter appears at the upper part of the map, showing a clear gradient. Figure 54 D). bmyD The promoter can form a complex with GEO3564; the migration rate of the complex decreases with increasing protein concentration. bmyD The promoter appears at the upper part of the map, showing a clear gradient. Figure 54E). bmyD No complex was formed between the promoter and different concentrations of FapR, and the DNA migration rate remained consistent with the control. Figure 54 F); bmyD The promoter can form a complex with YhiH; the migration rate of the complex decreases with increasing protein concentration. bmyD The promoter appears at the upper part of the map, showing a clear gradient. Figure 54 G); bmyD No complex was formed between the promoter and different concentrations of YfiS2, and the DNA migration rate remained consistent with the control. Figure 54 H); bmyD The promoter can form a complex with YtrA; the migration rate of the complex decreases with increasing protein concentration. bmyD The promoter appears at the upper part of the map, showing a clear gradient. Figure 54 I); bmyD The promoter and ComK can form a complex, and the migration rate of the complex decreases with increasing protein concentration. bmyD The promoter appears at the upper part of the map, showing a clear gradient. Figure 54 J); bmyD The promoter and CcpB can form a complex, and the migration rate of the complex decreases with increasing protein concentration, but the change in DNA translocation is relatively small. This may be related to the interaction between CcpB and... bmyD promoter binding strength related ( Figure 54 K); bmyD The promoter can form a complex with SpoOJ, and the migration rate of the complex decreases with increasing protein concentration. bmyD The promoter appears at the upper part of the map, showing a clear gradient. Figure 54 L); bmyD The promoter can form a complex with YqeW, and the migration rate of the complex decreases with increasing protein concentration. bmyD The promoter appears at the upper part of the map, showing a clear gradient. Figure 54 M); bmyD The promoter can form a complex with YqgH, and the migration rate of the complex decreases with increasing protein concentration. bmyD The promoter appears at the upper part of the map, showing a clear gradient. Figure 54 N).

[0239] As can be seen from the above, among the 13 transcription factors, SpoVG, FapR, and YfiS2 cannot directly interact with... bmyD The startup sub-segments are combined, and LexA, CcpA, GEO3564, YhiH, YtrA, comK, CcpB, SpoOJ, YqeW, and YqgH can be combined with... bmyDPromoter segments directly bind to form complexes. Based on changes in mobility, LexA, CcpA, GEO3564, CcpB, YqeW, and YqgH interact with... bmyD The binding strength of the promoters is relatively low, while YhiH, YtrA, ComK, and SpoOJ bind to... bmyD The promoter binding strength is relatively strong.

[0240] The aforementioned studies have confirmed that multiple transcription factors can directly bind to the bmyD promoter, regulating its transcriptional activity. However, the specific binding sites of these transcription factors remain unclear. Existing research indicates that the binding of transcription factors to the promoter requires a specific 5-20 bp DNA sequence. Currently, [the study suggests that] [specific binding sites for these transcription factors are not specified]. [[ID= The specific binding sites of promoter-bound transcription factors are still unclear. To further determine the regulatory regions of these 10 transcription factors, this invention uses plasmids of the ten transcription factors lexA, ccpA, GEO3564, yhiH, ytrA, comK, spoOJ, yqeW, yqgH, and ccpB as templates to construct truncated plasmids. ​ Promoter regions (216 bp, 116 bp, 65 bp) were extracted and transformed into *E. coli* BL21. The interaction between transcription factors and different truncations was investigated using IPTG induction. ​ The binding sites of transcription factors to promoter regions were investigated, and the intensity of sfGFP signal was used as a detection indicator.

[0241] ​ Functional analysis of different sections of the promoter: First, detection ​ Fluorescence intensity of different segments of the promoter in the absence of exogenous transcription factors. The results confirmed that... ​ (300 bp) and ​ The fluorescence intensity of (216 bp) was basically the same, while ​ The fluorescence intensity (116 bp) increased by 2.13 times, while ​ The fluorescence intensity (65 bp) was reduced by 0.16 times. Combined with the above... ​ The analysis results of the starter sub-segment show that ​ The promoter sequence has three functional regions, two of which are transcription factor binding regions and one is a ribosome binding region. The RBS sequence of the ribosome binding region is located in... ​ Within the 0-65bp range before the start codon, transcription factors cannot bind, and the sfGFP fluorescent signal essentially disappears. ​ The region 65-116 bp before the start codon is the transcription factor activation region, while the region 116-300 bp contains the transcription factor repression region. ​The promoter may be regulated at multiple sites by multiple transcription factors during transcription, and is also significantly affected by the binding of transcriptional repressors in the 116-300 bp range. ​ Transcription ( ​ A).

[0242] Binding of Transcription Activators to Different Promoter Truncated Sections: Based on the above conclusions, we examined the binding of 10 candidate transcription factors to different promoter truncated sections. The results showed that the candidate transcription activators LexA, CcpA, GEO3564, YhiH, and CcpB, and the transcription repressors YtrA, ComK, SpoOJ, YqeW, and YqgH did not bind to different promoter truncated sections. ​ The 0-65 bp region binds to the sfGFP, preventing successful expression and resulting in a low signal. Five candidate transcription activators mainly bind to the sfGFP in the 65-300 bp region. ​ Promoter binding forms a complex ( ​ (BK). Based on the fluorescence signal intensity after different transcription factors bound to different promoter truncated segments, it was determined that LexA's binding site was located at... ​ The 65-116 bp range ( ​ B); CcpA binds to sfGFP in the 0-300 bp region, increases sfGFP binding by 2.07-fold, in the 0-216 bp region, increases sfGFP binding by 2.55-fold, and in the 0-116 bp region, increases sfGFP binding by 4.09-fold. The binding site of CcpA is located at... ​ The 65-116 bp range ( ​ C) GEO3564 increases sfGFP binding by 1.38-fold with the 0-300 bp region, 2.46-fold with the 0-216 bp region, and 3.34-fold with the 0-116 bp region. The binding site of GEO3564 is located at... ​ The 65-116bp range ( ​ D); ccpB binding to sfGFP increases sfGFP by 1.57-fold in the 0-300 bp region, by 1.54-fold in the 0-216 bp region, and by 3.81-fold in the 0-116 bp region. The binding site of ccpB is located at... ​ The 65-116 bp range ( ​ E); YhiH increases sfGFP binding by 1.44-fold in the 0-300 bp region, by 1.22-fold in the 0-216 bp region, and by 5.68-fold in the 0-116 bp region. The binding site of YhiH is located at...​ The 65-116 bp range ( ​ F).

[0243] Binding of transcriptional repressors to different promoter truncated regions: The binding sites of the five candidate repressors differed, as determined by the fluorescence signal intensity after binding to different promoter truncated regions. SpoOJ binding to the 0-300 bp region reduced sfGFP by 0.21-fold, but binding to the 0-216 bp region increased sfGFP by 218-fold, and binding to the 0-116 bp region increased it by 1.81-fold. The fluorescence intensity trend confirmed that the binding site of spoOJ is located at... ​ The 216-300 bp range ( ​ G); YqeW's binding to sfGFP decreased by 0.35-fold in the 0-300 bp region, but decreased by 0.18-fold in the 0-216 bp region, and increased by 0.57-fold in the 0-116 bp region. The fluorescence intensity trend confirmed that the binding site of YqeW is located at... ​ The 116-216 bp range ( ​ H); ComK binding to the 0-300 bp region reduced sfGFP by 0.25-fold, binding to the 0-216 bp region reduced sfGFP by 0.07-fold, and binding to the 0-116 bp region reduced sfGFP by 0.19-fold. The fluorescence intensity change trend confirmed that the binding site of ytrA is located at... ​ The 65-116 bp range ( ​ I); YqgH binding to sfGFP decreased by 0.64-fold in the 0-300 bp region, but by 0.20-fold in the 0-216 bp region, and by 1.17-fold in the 0-116 bp region. The fluorescence intensity trend confirmed that the binding site of YqeW is located at... ​ The 116-216 bp range ( ​ J); YtrA binding to sfGFP decreased by 0.08-fold in the 0-300 bp region, by 0.06-fold in the 0-216 bp region, and by 0.11-fold in the 0-116 bp region. The fluorescence intensity change trend confirmed that the binding site of ytrA is located at... ​ The 65-116 bp range ( ​ K).

[0244] Construction of transcriptional regulatory networks: based on the effects of transcription factors on different ​ The regulation of the promoter truncated segment is constructed in this invention. ​ The transcription factor regulatory network and regulatory sites were investigated, and it was found that transcription factors YhiH, CcpB, CcpA, GEO3564, LexA, YtrA, and ComK can interact with...​ The transcriptional activation and repression occur by binding to the 65-116 bp region preceding the start codon. YqgH and YqeW can bind to this region. ​ SpoOJ binds to the 116-216 bp region preceding the start codon for transcriptional repression; SpoOJ can bind to... ​ Transcriptional repression occurs by binding to the 216-300 bp region preceding the start codon.

[0245] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications or equivalent changes made to the above embodiments based on the technical essence of the present invention shall fall within the protection scope of the present invention.

Claims

1. An engineered strain capable of targeted synthesis of bacillus tincture L, characterized in that: The engineered strain was introduced using the recombinant vector pCN33-P. bmyD -sfGFP-LacI-YhiH Bacillus amyloliquefaciens M86; The recombinant vector pCN33-P bmyD -sfGFP-LacI-YhiH contains: bmyD The gene encoding the promoter, the enhanced green fluorescent protein sfGFP gene, the lactose operon regulatory element LacI-MCS, and the transcription factor YhiH; The expression of the transcription factor YhiH is regulated by the lactose operon and can specifically bind to... bmyD Promoter, thereby activating bmyDABC Transcription of gene clusters promotes the synthesis of bacitracin L.

2. The engineered strain according to claim 1, characterized in that: pCN33-P bmyD The sequence of -sfGFP-LacI-YhiH is SEQ ID No:

10.

3. The engineered strain according to claim 1, characterized in that: The recombinant vector pCN33-P bmyD -sfGFP-LacI-YhiH is a shuttle plasmid that can replicate in Escherichia coli and Bacillus, and carries ampicillin resistance genes and erythromycin resistance genes.

4. A recombinant vector, characterized in that, The carrier is pCN33-P bmyD -sfGFP-LacI-YhiH is constructed through the following steps: S1. Using pcN33-sfGFP as a template, a linearized pcN33-sfGFP vector was obtained by reverse PCR, and then... bmyD pCN33-P was constructed by homologous recombination of promoter fragments. bmyD -sfGFP; The bmyD The promoter fragment is derived from the genome of Bacillus amyloliquefaciens M86 and is 300 bp in length; S2, with pCN33-P bmyD Using sfGFP as a template, a linearized vector was obtained by reverse PCR. This vector was then homologously recombinated with the lactose operon regulatory element LacI-MCS from the pET-19b vector to construct pCN33-P. bmyD -sfGFP-LacI-MCS; S3, with pCN33-P bmyD Using -sfGFP-LacI-MCS as a template, a linearized vector was obtained by reverse PCR. This vector was then homologously recombinated with the YhiH gene CDS fragment from the Bacillus amyloliquefaciens M86 genome to construct the pCN33-P vector. bmyD -sfGFP-LacI-YhiH.

5. A recombinant vector according to claim 4, characterized in that: In step S1, the primers used for reverse PCR are PCN33-sgGFP linearized-F / R, P bmyD The primers for amplifying the promoter fragment are: bmyD -P(300bp)-F / R, and the 5' end of the primer contains a 15bp homologous sequence complementary to the end of the linearized vector; In step S2, the primers used for reverse PCR are pCN33- bmyD -PF / R, the amplification primers for the lactose operon regulatory element LacI-MCS are pET-19b-F / R; In step S3, the primers used for reverse PCR are pET-19b-linearization-F / R, and the primers for amplifying the CDS fragment of the YhiH gene are yhiH-CDS-F / R.

6. The application of transcription factor YhiH in increasing the yield of bacitracin L, characterized by: The YhiH can specifically bind to Bacillus amyloliquefaciens. bmyDABC Gene clusters bmyD Promoter, activate bmyDABC The transcription of bacitracin L promotes its synthesis.

7. The application according to claim 6, characterized in that: The engineered strains described in any of claims 1 to 3 are used to achieve targeted expression of YhiH, and the expression of YhiH is induced by IPTG to increase the yield of bacitracin L.

8. The application according to claim 6, characterized in that: The directed expression of YhiH was achieved using any of the engineered strains described in claims 1 to 3, and the strain was cultured in BDSM medium containing 1.25 to 20 g / L lactose, 0 to 1.5 g / L K2HPO4, and 0 to 3.0 g / L glycine to increase the yield of bacitracin L.

9. The application according to claim 7, characterized in that: The BDSM medium formula is as follows: lactose 5 g / L, L-glutamate sodium 5 g / L, tryptone 5 g / L, yeast extract 1 g / L, MgSO4·7H2O 0.5 g / L, KCl 0.5 g / L, K2HPO4 0.75 g / L, and glycine 3 g / L.

10. The application according to claim 6, characterized in that: The directed expression of YhiH was achieved by the engineered strain according to any one of claims 1 to 3, and the strain was cultured in a medium containing fatty acid chain synthesis precursors and / or amino acid chain synthesis substrates.

11. The application of transcription factors YqeW, YqgH, YtrA, ComK, SpoOJ, LexA, GEO3564, or CcpB in regulating the production of bacitracin L, characterized by: The YqeW, YqgH, YtrA, ComK, or SpoOJ can specifically bind to Bacillus amyloliquefaciens. bmyDABC Gene clusters bmyD Promoter and suppressor bmyDABC Transcription of LexA, GEO3564, and CcpB can specifically bind to Bacillus amyloliquefaciens. bmyDABC Gene clusters bmyD Promoter and activation bmyDABC The transcription of the gene precisely regulates the synthesis of bacitracin L, and the encoding genes of YqeW, YqgH, YtrA, ComK, SpoOJ, LexA, GEO3564 or CcpB are derived from the genome of Bacillus amyloliquefaciens M86.

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