Genetically engineered bacterium of high-yield cyclic lipopeptide as well as construction method and application of genetically engineered bacterium

Abstract

The invention discloses a genetically engineered bacterium for high-yield cyclic lipopeptide as well as a construction method and application of the genetically engineered bacterium, the cyclic lipopeptide yields of three mutant strains Bv-srf-loc, Bv-bl-fen and BvLSBF constructed by the invention are all remarkably improved, antifungal and bacterial activity determination is performed on lipopeptide antibiotic primary extracts of the three strains, and the results show that the yield of the cyclic lipopeptide is remarkably improved. The results show that the Bv-srf-loc is an antibacterial active strain, the Bv-bl-fen is an antifungal active strain, and the BvLSBF is an antibacterial and fungal active strain. The three mutant strains constructed by the invention are used for biological control tests on rice sheath blight disease and tomato fusarium wilt, and results show that the three strains have efficient control effects on plant fungal diseases and bacterial diseases, and are good biological control agents. The invention is beneficial for improving the antibacterial and antifungal activity of plants and improving the biological control capability of strains.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a genetic engineering bacterium with high yield of cyclolipopeptide and a construction method and application thereof. Background Art

[0002] Due to the demand for sustainable agricultural systems and the desire for chemical-free food, alternatives to chemical control of plant diseases have been intensively investigated. Microorganisms, especially Bacillus velez (Bv), are the best candidates for biocontrol of these pathogenic microorganisms, including bacteria, fungi, and nematodes, due to its ability to form endospores, produce a variety of bioactive compounds, and promote plant growth. Based on the current whole genome rearrangement and the unique phylogenetic characteristics of Bv, some well-known biocontrol strains Bacillus amyloliquefaciens FZB42T, Bacillus amyloliquefaciens FZB24, Bacillus subtilis QST713, Bacillus subtilis GB03, and Bacillus subtilis 916 were reclassified as Bv. More specifically, these Bv strains exhibit excellent genetic ability to synthesize secondary metabolites, including cyclic lipopeptides (CLPs) (i.e., surfactin, iturin, fentomycin (or plipastatin) and locillomycin) and polyketides (i.e., macrocyclic active proteins, bacillus and difficile), which are produced by non-ribosomal peptide synthetases (NRPSs) or polyketide synthetases (PKSs) at the end of the exponential phase. In particular, CLPs produced by Bv can induce systemic resistance in plants and inhibit plant pathogens. Because of the eco-friendly and probiotic properties of Bv, it has been increasingly studied and used in the agricultural industry to control plant diseases recently, but the inhibitory effect in the existing technology needs to be improved, and a more efficient inhibitory effect is required, while maintaining eco-friendliness.

[0003] Since the biocontrol agents developed from Bv and its active products are similar to other biological pesticides, they have inherent disadvantages such as narrow antibacterial spectrum, slow effect, susceptibility to environmental influences, and poor stability compared with chemical pesticides. With the widespread application of the clustered regularly interspaced short palindromic repeats and their associated genes (CRISPR-Cas9) genome editing system in bacterial genome DNA editing, it is possible to use this revolutionary technology to systematically transform the genomes of existing excellent biocontrol Bacillus strains in an all-round way to overcome their inherent disadvantages and develop a new generation of biofungicides with eco-friendly, high antibacterial activity, broad antibacterial spectrum and long shelf life. Since the construction of a continuous genome editing strategy based on the CRISPR-Cas9 system in negative bacteria Escherichia coli and Leucococcus citrinum, many CRISPR-Cas9 platforms belonging to positive bacteria Bacillus have been discovered. Genome iterations have been developed and used for large genome deletions, gene insertions and point mutations. However, compared with the development of CRISPR-Cas9 gene editing kits for Bacillus subtilis, Bacillus anthracis, Bacillus cereus, Bacillus licheniformis, and Bacillus mycoides, there are fewer reports on B. velezensis. Therefore, the development of efficient and high-fidelity CRISPR-Cas 9 platforms for B. velezensis is urgent and essential for further utilization in sustainable agriculture. In the widely used class II CRISPR-Cas9 system, CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA) for bacterial genome editing usually contain three key elements, namely sgRNA, trans-activating CRISPR RNA (tracrRNA), and Cas9. Since B. velezensis is a typical positive bacterium and an excellent industrial host strain, three typical CRISPR-Cas9 systems have been developed, including all-in-one, double plasmid, and chromosome integration, and work effectively in different B. velezensis strains. However, all three CRISPR-Cas9 systems must solidify the plasmid containing the previous sgRNA and introduce the plasmid containing the new sgRNA to complete multiple rounds of genome editing in B. velezensis. Currently, the removal of B. subtilis plasmids mainly uses three methods: temperature-sensitive replication, traditional negative selection, and programmed sgRNA removal system, but all of them require a long time and high-intensity labor. Summary of the invention

[0004] Purpose of the invention: In view of the problems existing in the prior art, the present invention provides a genetically engineered bacterium with high yield of cyclolipopeptides. The engineered bacterium constructed by the present invention not only improves the efficiency of gene editing but also significantly increases the yield of cyclolipopeptides. The strain can be used to prevent and control animal and plant pathogens, as a biological control agent, to reduce diseases, effectively improve the antibacterial and antifungal activity of animals and plants, and increase the yield of the strain.

[0005] The invention also provides a construction method and application of the genetically engineered bacteria.

[0006] Technical solution: In order to achieve the above-mentioned object, the present invention provides a genetically engineered bacterium that produces four cyclolipopeptides in high yield. The genetically engineered bacterium uses Bacillus Velezii as a starting strain, and replaces any one or more of the following promoters to obtain the genetically engineered bacterium: the promoter encoding the Locillomycin production gene is replaced with a strong promoter PB, the promoter encoding the Bacillomycin L production gene is replaced with a strong promoter P43, the promoter encoding the Surfactin production gene is replaced with a strong promoter PA, and the promoter encoding Fengycin is replaced with a strong promoter PrepU.

[0007] Wherein, the Bacillus Velez is Bacillus Velez 916, and the cyclolipopeptides include Locillomycin, Bacillomycin L, Surfactin and Fengycin.

[0008] The method for constructing the genetically engineered bacteria capable of producing four cyclolipopeptides in high yield according to the present invention comprises the following steps:

[0009] (1) P43 sgRNA expression cassettes were constructed using the P43 promoter and sgRNAs of Locillomycin, Bacillomycin L, Surfactin, and Fengycin, respectively, to obtain P43 sgRNA (loc), P43 sgRNA (bl), P43 sgRNA (srf), and P43 sgRNA (fen) expression cassettes;

[0010] (2) Inserting the expression cassette constructed in step (1) into the pTK or pTC-based vector, and then inserting gene fragments with strong promoters PB, P43, PA, PrepU and upstream and downstream homology arm fragments of Locillomycin, Bacillomycin L, Surfactin, and Fengycin, respectively, to obtain editing vectors pTK-PB, pTK-P43, pTC-PA, and pTC-PrepU;

[0011] (3) The editing vector pTN-Cas9 and any one or more editing vectors constructed in step (2) are transformed into Bacillus velezensis to construct a genetically engineered bacterium that can produce four cyclic lipopeptides in high yield.

[0012] Wherein, in step (1), according to the nucleotide sequences encoding the production genes of Locillomycin, Bacillomycin L, Surfactin, and Fengycin, the corresponding sgRNA sequences sgRNA (loc), sgRNA (srf), sgRNA (bl), and sgRNA (fen) are designed, and then the P43 promoter is fused with them to construct the P43sgRNA expression cassette, thereby obtaining the P43sgRNA (loc), P43sgRNA (bl), P43sgRNA (srf), and P43sgRNA (fen) expression cassettes.

[0013] Furthermore, in step (1), the synthesized P43 promoter, sgRNA (loc) gene fragment or sgRNA (srf) gene fragment, sgRNA (bl) gene fragment, sgRNA (fen) gene fragment are used as templates to construct P43sgRNA (loc), P43sgRNA (bl), P43sgRNA (srf), P43sgRNA (fen) expression cassettes by overlapping PCR technology, and their sequences are shown in SEQ ID NO.1-4, respectively.

[0014] In step (2), pTK is used as a vector to insert the P43sgRNA (loc) expression frame to obtain a pTK-sgRNA (loc) recombinant plasmid; a gene fragment with a strong promoter PB and upstream and downstream homology arm fragments encoding the Locillomycin production gene are designed and synthesized, as shown in SEQ ID NO.5, and inserted into the pTK-sgRNA (loc) recombinant plasmid to obtain a pTK-PB recombinant plasmid, the sequence of which is shown in SEQ ID NO.9.

[0015] In which, in step (2), pTC is used as a vector to insert the P43sgRNA (srf) expression frame to obtain a pTC-sgRNA (srf) recombinant plasmid; a gene fragment with a strong promoter PA and upstream and downstream homologous arm fragments of a production gene encoding surfactin is designed and synthesized, as shown in SEQ ID NO.6, and inserted into the pTC-sgRNA (srf) recombinant plasmid to obtain a pTK-PB recombinant plasmid, the sequence of which is shown in SEQ ID NO.10.

[0016] In step (2), pTK is used as a vector to insert the P43sgRNA (bl) expression frame to obtain a pTK-sgRNA (bl) recombinant plasmid; a gene fragment with a strong promoter P43 and upstream and downstream homology arm fragments encoding the Bacillomycin L production gene is designed and synthesized, as shown in SEQ ID NO.7, and inserted into the pTK-sgRNA (bl) recombinant plasmid to obtain a pTK-P43 recombinant plasmid, the sequence of which is shown in SEQ ID NO.11.

[0017] In step (2), pTC is used as a vector to insert the P43sgRNA (fen) expression frame to obtain a pTK-sgRNA (fen) recombinant plasmid; a gene fragment with a strong promoter PA and upstream and downstream homologous arm fragments of the production gene encoding Fengycin is designed and synthesized, as shown in SEQ ID NO.8, and inserted into the pTK-sgRNA (fen) recombinant plasmid to obtain a pTC-PrepU recombinant plasmid, the sequence of which is shown in SEQ ID NO.12.

[0018] In step (3), the editing vector pTN-Cas9 and the pTK-PB and pTC-PA9 constructed in step (2) are transferred into the mutant strain Bv-bl-fen to obtain Bv-srf-loc; the editing vector pTN-Cas9 and the pTK-P43 and pTC-PrepU constructed in step (2) are transferred into Bacillus velez 916 to obtain Bv-bl-fen; the editing vector pTN-Cas9 and the pTK-PB and pTC-PA constructed in step (2) are transferred into Bacillus velez 916 to obtain BvLSBF.

[0019] Furthermore, the present invention first takes 2uL of each of the pTK-PB, pTC-PA, and pTN-Cas9 plasmids and transfers them into 400uL of the mutant strain Bv-bl-fen competent cells, and then cultures them at 37°C and 100rpm / min for 0.5h, cultures them at 180rpm for 1.5h, and collects the bacteria by centrifugation at 5000rpm for 4min. Finally, 100uL of the resuspended bacteria is evenly spread on an LB resistance plate containing neomycin (10μg / mL), kanamycin (10μg / mL) and chloramphenicol (10μg / mL), and placed in a 37°C incubator for overnight culture.

[0020] The invention discloses an application of the genetically engineered bacteria with high yield of cyclolipopeptides in high yield of Locillomycin, Bacillomycin L, Surfactin and Fengycin as well as in antibacterial and biological control.

[0021] The invention discloses an application of the genetically engineered bacteria with high cyclolipopeptide production in the preparation of biological control agents.

[0022] Furthermore, the biologically controlled diseases include rice sheath blight and tomato wilt.

[0023] The present invention is constructed using a multi-plasmid gene editing system, including an editing vector pTN-Cas9, and two of the editing vectors pTK-PB, pTK-P43, pTC-PA, and pTC-PrepU, whose sequences are shown in SEQ ID NOs. 9-12, respectively.

[0024] Among them, the editing vector pTN-Cas9 is based on the pTN vector and has a Cas9 protein expression frame inserted into it; the Cas9 protein expression frame includes a P43 promoter, a gene encoding the Cas9 protein, and a T1T2 terminator.

[0025] Among them, the editing vector pTK-PB is based on pTK, double-enzyme digestion is performed, P43-sgRNA (loc) is inserted and replaced with the strong promoter PB.

[0026] Among them, the editing vector pTK-P43 uses pTK as the basic vector, performs double enzyme digestion, inserts P43-sgRNA (bl) and replaces the strong promoter P43.

[0027] Among them, the editing vector pTC-PA is based on pTC, double enzyme digestion is performed, P43-sgRNA (srf) is inserted and the strong promoter PA is replaced.

[0028] Among them, the editing vector pTC-PrepU uses pTC as the basic vector, performs double enzyme digestion, inserts P43-sgRNA (fen) and replaces the strong promoter P43.

[0029] The present invention integrates the optimized BvCas9 under the constitutive promoter P43, the sgRNAs under the specific promoter Psrf and the homologous recombination fragment into three thermosensitive shuttle vectors, and constructs a food-grade three-plasmid platform containing Bv916CRISPR-Cas9. The gene editing system is used to realize the insertion, deletion and replacement of genes in Bv916, and in particular, 4 NRPSs gene clusters are edited, the production of 4 cyclolipopeptides is increased, and the antibacterial and fungal activities are significantly enhanced. The present invention analyzes four cyclolipopeptides (Locillomycin, Bacillomycin L, Surfactin, Fengycin) by liquid chromatograph, obtains an efficient liquid chromatographic analysis chart, and measures its content, and draws a content bar chart. The present invention finds that the content of cyclolipopeptides in mutant strains Bv-srf-loc, Bv-bl-fen, and BvLSBF is increased, especially the mutant strain BvLSBF. Mass spectrometry analysis further showed that the strategy of replacing four endogenous cyclolipopeptide promoters with constitutively expressed strong promoters can significantly improve the expression levels of four cyclolipopeptides in B. velezensis 916. The present invention analyzes the antibacterial and fungal activity of lipopeptide antibiotics secreted by the original strain B. velezensis 916 (Bacillus velezensis 916) against plants, combined with the changes in the inhibition bandwidth in the culture dish, indicating that Bv-bl-fen has antifungal activity, Bv-srf-loc has antibacterial activity, and BvLSBF has antifungal and bacterial activity.

[0030] The construction method and multi-plasmid gene editing proposed in the present invention are crucial to the further utilization of sustainable agriculture, are beneficial to achieving high yield of Bacillus Velezii, and are beneficial to enhancing the antifungal and antibacterial activities of animals and plants.

[0031] The genetically engineered bacteria Bv-srf-loc, Bv-bl-fen and BvLSBF preferably constructed in the present invention can effectively enhance the production of four cyclic lipopeptides (Locillomycin, Bacillomycin L, Surfactin and Fengycin). The yield of the cyclolipopeptide Bacillomycin L of Bv-bl-fen and BvLSBF can reach about 600-700 mg / L, the yield of the cyclolipopeptide Fengycin of Bv-bl-fen and BvLSBF can reach about 130 mg / L, the yield of the cyclolipopeptide Locillomycin of BvLSBF can reach about 80 mg / L, and the yield of the cyclolipopeptide Surfactin of Bv-bl-fen and BvLSBF can reach about 800 mg / L. Among them, BvLSBF can significantly increase the yield of the four cyclolipopeptides, especially for the cyclolipopeptide Locillomycin. The degree of improvement of BvLSBF even significantly exceeds that of the mutant strains Bv-srf-loc and Bv-bl-fen, producing a significant synergistic effect.

[0032] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0033] The present invention recombines the P43sgRNA expression frame with the cyclolipopeptide protein encoding gene composition vector replaced with a strong promoter, including pTK-PB, pTK-P43, pTC-PA, and pTC-PrepU. Then, the above-mentioned editing vector and the editing vector pTN-Cas9 are used to perform gene editing on the four cyclolipopeptides in B. velezensis, and the promoter of Locillomycin is replaced with the strong promoter PB, the promoter of Bacillomycin L is replaced with the strong promoter P43, the promoter of Surfactin is replaced with the strong promoter PA, and the promoter of Fengycin is replaced with the strong promoter PrepU. Finally, three strains, Bv-srf-loc, Bv-bl-fen, and BvLSBF, can be obtained. Subsequent monitoring by liquid chromatograph shows that the cyclolipopeptide yields of these three strains are significantly improved. Compared with B.velezensis 916, the production of Bacillomycin L in Bv-bl-fen and BvLSBF increased by 9.2 times and 10.9 times, respectively; the production of Fengycin in Bv-bl-fen and BvLSBF increased by 5.9 times and 6.2 times, respectively; the production of Locillomycin in Bv-srf-loc and BvLSBF increased by 2.6 times and 6.8 times, respectively; the production of Surfactin in Bv-srf-loc and BvLSBF increased by 6.5 times and 5.9 times, respectively, compared with B.velezensis 916; the antifungal and bacterial activity of the initial extracts of lipopeptide antibiotics of the three strains were determined, and the results showed that Bv-srf-loc was an antibacterial strain, Bv-bl-fen was an antifungal strain, and BvLSBF was an antibacterial and antifungal strain. The invention is beneficial to improving the anti-bacterial and anti-fungal activities of plants and improving the biological control ability of strains. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 This is the pTK-PB plasmid map;

[0035] Figure 2 This is the pTC-PA plasmid map;

[0036] Figure 3 is the pTK-P43 plasmid map;

[0037] Figure 4 This is the pTC-PrepU plasmid map;

[0038] Figure 5Schematic diagram of the coordinated gene editing of four cyclolipopeptides in B. velezensis 916 using four plasmid gene editing systems;

[0039] Figure 6 The HPLC analysis of cyclolipopeptide Bacillomycin L produced by B. velezensis 916 and its mutants and the corresponding bar chart of the content of four strains;

[0040] Figure 7 The HPLC analysis of the cyclolipopeptide Fengycin produced by B. velezensis 916 and its mutants and the bar chart of the content of the corresponding four strains;

[0041] Figure 8 The HPLC analysis of cyclolipopeptide Locillomycin produced by B. velezensis 916 and its mutants and the bar chart of the content of the corresponding four strains;

[0042] Fig. 9 The HPLC analysis of the cyclolipopeptide Surfactin produced by B. velezensis 916 and its mutants and the bar chart of the content of the corresponding four strains;

[0043] Fig.10 Mass spectrometry analysis of cyclolipopeptides produced by B. velezensis 916 and its mutants Bv-srf-loc, Bv-fen-bl, and Bv-LSBF;

[0044] Fig.11 for the antifungal activity of B. velezensis 916 and its mutants;

[0045] Fig.12 for the antibacterial activity of B. velezensis 916 and its mutants;

[0046] Fig.13 The experiment was conducted on the biological control of rice sheath blight by B. velezensis 916 and its mutants;

[0047] Fig.14 This is a biological control experiment on tomato Fusarium wilt using B. velezensis 916 and its mutants. DETAILED DESCRIPTION

[0048] The present invention is further described below in conjunction with the accompanying drawings and embodiments.

[0049] Unless otherwise specified, the materials and reagents used in the examples of the present invention can be obtained from commercial sources. Experimental methods without specific conditions in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0050] The resistance gene and the artificially synthesized multiple cloning site in the present invention are both existing known genes.

[0051] The vectors and strains used in the examples were provided by Huaiyin Institute of Technology.

[0052] For details on the construction of the editing vector pTN-Cas9 and its sequence, please refer to Chinese Patent: 2022117424369.

[0053] The pTK vector and pTC vector were constructed by the applicant. For details, please refer to the construction of pTK and pTC shuttle plasmid vectors in the applicant's prior Chinese patent: 2021116808047.

[0054] The pTN-Cas9 vector was constructed by the applicant, for details, see SEQ ID NO.1 in the applicant's prior Chinese patent 2022117424369.

[0055] The strain B. velezensis 916 used in the embodiment is the strain Bacillus subtilis 916, which is a conventional wild-type Bacillus velezensis strain provided by Huaiyin Institute of Technology, Unusual Biosynthesis and Structure of Locillomycins from Bacillus subtilis 916, GENETICS AND MOLECULARBIOLOGY, Volume 81 Issue 19, https: / / doi.org / 10.1128 / AEM.01639-15.

[0056] The strains in the examples of the present invention were all prepared into competent cells by conventional methods.

[0057] Preparation method of competent cells: Pick the host bacteria (single colony) the night before and inoculate it into a 5ml LB liquid test tube medium, shake at 37℃ overnight, take 100μl of the overnight culture solution the next morning, inoculate it into 5ml SPI Medium prepared in a 50ml centrifuge tube, shake at 37℃, and start measuring OD600 after 3h. When the culture grows to the end of the logarithmic phase, quickly take 200μl and inoculate it into 2ml SPI Medium. Medium, 37℃100rpm1.5h, add 20μl100×EGTA solution, 37℃100rpm10min, use 1.5ml centrifuge tubes to divide into 500μl each tube, add appropriate plasmid 5-10μl to the tube, gently mix at 37℃100rpm30min, transfer the centrifuge tube to 250rpm, 37℃1.5h, centrifuge at 4000rpm to collect bacteria, discard part of the supernatant, keep 100μl of the resuspended strain, spread on the corresponding resistance plate, 37℃ overnight.

[0058] Example 1

[0059] This experiment mainly involves the construction method of a vector recombinant plasmid that fuses the sgRNA expression frame of the P43 promoter and the cyclolipopeptide protein encoding gene replaced by a strong promoter

[0060] (1) Using the P43 promoter and sgRNA (loc) gene fragment synthesized by a biological company as templates, the P43sgRNA (loc) expression frame (sequence as SEQ ID NO.1) was fused by overlapping PCR technology, and then purified and recovered by double restriction digestion with BamH I / Xba I, and connected with the pTK vector that had been double-digested (BamH I / Xba I) at 16°C overnight, and transformed into Escherichia coli DH5a, and the plasmid was extracted to obtain the pTK-sgRNA (loc) recombinant plasmid. Design and synthesize a gene fragment with a strong promoter PB and upstream and downstream homology arm fragments of Locillomycin (sequence as SEQ ID NO.5), and then use Spe I / BgL II for double restriction digestion, and connect with the pTK-sgRNA (loc) recombinant plasmid that had been double-digested (Spe I / BgL II) overnight, transformed into Escherichia coli DH5a, and the plasmid was extracted to obtain the pTK-PB recombinant plasmid ( Figure 1 ), the sequence is as SEQ ID NO.9.

[0061] (2) Using the P43 promoter and sgRNA (srf) gene fragment synthesized by a biological company as templates, the P43sgRNA (srf) expression frame (sequence such as SEQ ID NO.2) was fused by overlapping PCR technology, and then purified and recovered by double restriction digestion with BamH I / Xba I, and connected with the pTC vector that had been double-digested (BamH I / Xba I) at a constant temperature overnight, and transformed into Escherichia coli DH5a, and the plasmid was extracted to obtain the pTC-sgRNA (srf) recombinant plasmid. Design and synthesize a gene fragment with a strong promoter PA and upstream and downstream homology arm fragments of surfactin (sequence such as SEQ ID NO.6), and then use Spe I / BgL II for double restriction digestion, and connect with the pTC-sgRNA (srf) recombinant plasmid that had been double-digested (Spe I / BgL II) overnight, transformed into Escherichia coli DH5a, and the plasmid was extracted to obtain the pTC-PA recombinant plasmid ( Figure 2 ), the sequence is as SEQ ID NO.10.

[0062] (3) Using the P43 promoter and sgRNA (bl) gene fragment synthesized by the biological company as templates, the P43sgRNA (bl) expression frame (sequence such as SEQ ID NO.3) was fused by overlapping PCR technology, and then purified and recovered by double restriction digestion with BamH I / Xba I, and connected with the pTK vector that had been double-digested (BamH I / Xba I) at a constant temperature overnight, and transformed into Escherichia coli DH5a, and the plasmid was extracted to obtain the pTK-sgRNA (bl) recombinant plasmid. Design and synthesize a gene fragment with a strong promoter P43 and upstream and downstream homologous arm fragments of Bacillomycin L (sequence such as SEQ ID NO.7), and then double-digested with Spe I / BgL II, and connected with the pTK-sgRNA (bl) recombinant plasmid that had been double-digested (Spe I / BgL II) overnight, transformed into Escherichia coli DH5a, and the plasmid was extracted to obtain the pTK-P43 recombinant plasmid ( Figure 3 ), the sequence is as SEQ ID NO.11.

[0063] (4) Using the P43 promoter and sgRNA (fen) gene fragment synthesized by a biological company as templates, the P43sgRNA (fen) expression frame (sequence such as SEQ ID NO.4) was fused by overlapping PCR technology, and then purified and recovered by double restriction digestion with BamH I / Xba I, and connected with the pTC vector that had been double-digested (BamH I / Xba I) at a constant temperature overnight, and transformed into Escherichia coli DH5a, and the plasmid was extracted to obtain the pTK-sgRNA (fen) recombinant plasmid. Design and synthesize a gene fragment with a strong promoter PA and a fragment with upstream and downstream homologous arms of Fengycin (sequence such as SEQ ID NO.8), and then use Spe I / BgL II for double restriction digestion, and connect with the pTK-sgRNA (fen) that had been double-digested (Spe I / BgL II) to obtain the pTC-PrepU recombinant plasmid ( Figure 4 ), the sequence is as SEQ ID NO.12.

[0064] Example 2

[0065] This experiment mainly uses the above-mentioned various recombinant plasmids to perform gene editing on cyclolipopeptides, among which the pTN-Cas9 plasmid was constructed by a biological company.

[0066] (1) pTK-PB, pTC-PA, and pTN-Cas9 in the plasmid pool were transferred into the B. velezensis 916 strain, and the Surfactin and Locillomycin genes were edited. Under the action of pTN-Cas9, the plasmids of pTC-PA and pTK-PB were eliminated, and the promoters of Surfactin and Locillomycin were converted into strong promoters PA and PB, respectively, to obtain the mutant strain Bv-srf-loc( Figure 5 A).

[0067] The specific process is: first take 2uL of pTN-Cas9, pTC-PA, and pTK-PB plasmids and transfer them into 100uL of B. velezensis In 916 competent cells, after shaking culture at 37°C and 100rpm / min for 0.5h, shake culture at 180rpm for 1.5h, add the bacterial solution to polyclonal antibody (neomycin / chloramphenicol / kanamycin, all 10ug / mL) nutrient broth medium at a volume ratio of 1:100, shake at 180rpm, and culture at 37°C overnight. Finally, take 100uL of bacterial solution and evenly spread it on LB resistance plates containing neomycin (10μg / mL), chloramphenicol (10μg / mL) and kanamycin (10μg / mL), and place it in a 37°C incubator for overnight culture; pick a single colony on the polyclonal antibody LB medium and perform PCR verification; inoculate the recombinant bacteria in non-antibody liquid LB, culture at 50°C, 200rpm, and culture for 48h. Dilute the bacterial solution 10 times and spread it on the polyclonal antibody / non-antibody LB plate, and culture it at 50°C overnight to screen out the recombinant bacteria that have eliminated the plasmid.

[0068] (2) According to the above method, pTK-P43, pTC-PrepU, and pTN-Cas9 in the plasmid pool were transferred into the B. velezensis 916 strain, and the genes of Bacillomycin L and Fengycin were edited. Under the action of pTN-Cas9, pTK-P43 and pTC-PrepU were eliminated, and the promoters of Bacillomycin L and Fengycin were converted into strong promoters P43 and PrepU, respectively, to obtain the mutant strain Bv-bl-fen( Figure 5 B).

[0069] (3) According to the above method, pTK-PB, pTC-PA, and pTN-Cas9 in the plasmid pool were again transferred into the mutant strain Bv-bl-fen, and the Surfactin and Locillomycin genes were edited. The promoters of Surfactin and Locillomycin were converted into strong promoters PA and PB, respectively, to achieve the elimination of the three plasmids and obtain the mutant strain BvLSBF ( Figure 5 C).

[0070] Example 3

[0071] This experiment mainly measured the absorbance of four cyclolipopeptides over time through high performance liquid chromatography analysis, and processed the data to obtain relevant bar charts, which directly reflected the changes in the yield of the strain. Mass spectrometry analysis further showed that the strategy of replacing the four endogenous cyclolipopeptide promoters with constitutively expressed strong promoters can significantly improve the expression levels of the four cyclolipopeptides in B. velezensis 916.

[0072] The original strain cultured to the logarithmic phase and the three mutant strains constructed in Example 2 were cultured and inoculated into LB medium at a volume ratio of 5%, and cultured at 37° C. and 100 rpm / min for 48 h. The supernatant was centrifuged and the contents of the four endogenous cyclolipopeptides were determined.

[0073] (1) Using a liquid chromatograph, a C18 column, acetonitrile-water-trifluoroacetic acid (Bacillomycin L is 40:60:0.5 [vol / vol / vol]) as the mobile phase, and a flow rate of 0.8 ml / min; the ultraviolet detection wavelength is 210 nm, and four absorbance curves of the cyclolipopeptide Bacillomycin L are measured, and the corresponding contents of the four strains Bv916, Bv-srf-loc, Bv-bl-fen, and BvLSBF are obtained ( Figure 6 ),Depend on Figure 6 As can be seen from A, compared with the peak height of Bv916, the peak height of Bv-srf-loc is only slightly increased, while the peak heights of Bv-bl-fen and BvLSBF are significantly increased. Figure 6 The production of cyclolipopeptide Bacillomycin L of mutant strains Bv-bl-fen and BvLSBF in B can reach about 600-700 mg / L, while the original strain is only about 50 mg / L, indicating that the production of cyclolipopeptide Bacillomycin L of these two mutant strains has increased significantly.

[0074] (2) Using a liquid chromatograph with a C18 column, the mobile phase was acetonitrile-water-trifluoroacetic acid (Fengycin was 50:50:0.5 [vol / vol / vol]), the flow rate was 0.8 ml / min, and the ultraviolet detection wavelength was 210 nm. Four absorbance curves of the cyclolipopeptide Fengycin were measured, and the corresponding contents of the four strains Bv916, Bv-srf-loc, Bv-bl-fen, and BvLSBF were obtained ( Figure 7 ),Depend on Figure 7 A shows that compared with the peak height of Bv916, the peak height of Bv-srf-loc is only slightly increased, while the peak heights of Bv-bl-fen and BvLSBF are significantly increased. Figure 7 The production of cyclolipopeptide Fengycin of mutant strains Bv-bl-fen and BvLSBF in B can reach about 130 mg / L, while that of the original strain is only about 40 mg / L, indicating that the production of cyclolipopeptide Fengycin of these two strains has increased significantly.

[0075] (3) Using a liquid chromatograph, a C18 column, acetonitrile-water-trifluoroacetic acid (Locillomycin is 50:50:0.5 [vol / vol / vol]) as the mobile phase, and a flow rate of 0.8 ml / min; the ultraviolet detection wavelength was 230 nm, and four absorbance curves of the cyclolipopeptide Locillomycin were measured, and the corresponding contents of the four strains Bv916, Bv-srf-loc, Bv-bl-fen, and BvLSBF were obtained ( Figure 8 ),Depend on Figure 8 As can be seen from A, compared with the peak height of Bv916, the peak height of Bv-srf-loc is only slightly increased, while the peak height of BvLSBF is significantly increased. Figure 8 The production of cyclolipopeptide Locillomycin of mutant strain BvLSBF in B can reach about 80 mg / L, while that of the original strain is only about 20 mg / L, indicating that the production of cyclolipopeptide Locillomycin of the mutant strain is significantly increased, and the mutant strain BvLSBF produces a significant synergistic effect.

[0076] (4) Using a liquid chromatograph, a C18 column, acetonitrile-water-trifluoroacetic acid (80:20:0.5 [vol / vol / vol] for Surfactin) as the mobile phase, and a flow rate of 0.8 ml / min; the UV detection wavelength was 210 nm, and four absorbance curves of the cyclolipopeptide Surfactin were measured, and the corresponding contents of the four strains Bv916, Bv-srf-loc, Bv-bl-fen, and BvLSBF were obtained ( Fig. 9 ),Depend on Fig. 9 A shows that compared with the peak height of Bv916, the peak height of Bv-bl-fen is only slightly increased, while the peak heights of Bv-srf-loc and BvLSBF are significantly increased. Fig. 9 The production of cyclolipopeptide Surfactin of mutant strains Bv-bl-fen and BvLSBF in B can reach about 800 mg / L, while that of the original strain is only about 100 mg / L, indicating that the production of cyclolipopeptide Surfactin of these two strains has increased significantly.

[0077] (5) Using a mass spectrometer, the four cyclolipopeptides produced by Bv916 ​​and its mutants Bv-srf-loc, Bv-fen-bl and BvLSBF were analyzed by mass spectrometry, and the corresponding mass spectrometry analysis graphs were obtained ( Fig.10 ), as can be seen from the figure, Bv916 ​​and its mutants Bv-srf-loc, Bv-fen-bl and BvLSBF can all produce four cyclolipopeptides, among which the production of each cyclolipopeptide of the BvLSBF strain is significantly improved.

[0078] Example 4

[0079] The antifungal and antibacterial activities of lipopeptide antibiotics secreted by B. velezensis 916 and its mutants against plants were studied.

[0080] (1) Determination of the inhibitory activity of B. velezensis 916 and its mutants on fungal growth.

[0081] An agar block with a diameter of 5 mm and containing mycelium of wild-type rice sheath blight was inoculated in the center of a 9 cm LB plate, and 1 μl of the culture solution of wild-type Bv916 ​​and its mutant strains cultured to the logarithmic growth phase was added 2.5 cm away from the edge of the block. The plates were blown dry on a clean bench and then inverted in a constant temperature incubator at 28°C for culture. The inhibition area was determined within 3 to 7 days. Fig.11 The inhibition bandwidth of mutant a (BvLSBF) was the most significant, the inhibition bandwidth of mutant b (Bv-bl-fen) was significant, the inhibition bandwidth of mutant c (Bv-srf-loc) was slightly blurred, and the wild-type strain (Bv916) had no obvious inhibition bandwidth. The experimental results showed that Bv-bl-fen and BvLSBF strains had strong antifungal activity.

[0082] (2) Determination of the inhibitory activity of B. velezensis 916 and its mutants on bacterial growth.

[0083] Sterilize the Oxford cup at 121℃ and set aside. Under sterile operation, use an applicator to evenly apply the bacterial solution of wild-type logarithmic-phase tomato Fusarium wilt on the surface of the culture medium. Then put the Oxford cup into the prepared bacteria-containing plate and gently pressurize it to make it contact with the culture medium without gaps. Add 50μL of the culture solution of wild-type Bv916 ​​and its mutant strains cultured to the logarithmic phase into different cups, make 3 parallels, use sterile water as the blank control group, and culture at a constant temperature of 37℃ for 24h after pre-diffusion to observe the growth of the colonies. Fig.12 The wild-type strain a (Bv916) had an insignificant inhibition bandwidth, the mutant strain b (Bv-bl-fen) had a smaller inhibition bandwidth, the mutant strain c (Bv-srf-loc) had an obvious inhibition bandwidth, and the mutant strain d (BvLSBF) had an obvious inhibition bandwidth ( Fig.12 ). The experimental results showed that Bv-srf-loc and BvLSBF strains had strong antibacterial activity.

[0084] Example 5

[0085] Biological control experiments of wild-type Bacillus velezensis 916 and its mutants against rice sheath blight and tomato wilt.

[0086] (1) A 250 mL conical flask was filled with 100 matchsticks and 50 mL sterile PDA medium, and a 5 mm mycelium block was taken from the PDA medium containing rice sheath blight pathogen for inoculation. The inoculated conical flask was placed at 30°C without shaking for 1 week. After soaking rice seeds in water for 48 h, they were sown in a nursery containing organic sterile soil, and then the 28-day-old seedlings were transplanted into 15 cm × 30 cm pots containing the same sterile organic soil. In order to simulate its natural environment, the rice seedlings were grown outdoors. At the jointing and booting stage, 10 matchsticks containing rice sheath blight pathogens were inoculated between the stem and leaves of each rice plant (i.e., the rice sheath). Immediately after inoculation, 20 mL of fermentation liquid of wild-type Bv916 ​​and its mutants Bv-bl-fen, Bv-srf-loc, and BvLSBF cultured to the logarithmic phase were sprayed on the rice, and the control group was replaced with an equal amount of tap water. The severity of rice sheath blight was scored from the 3rd to the 20th day after inoculation. Each treatment group contained eight individual pots. It was observed that the wild type Bv916 ​​and the mutant strain Bv-srf-loc had no significant change in resistance to rice sheath blight compared with the control group, while the mutant strains Bv-bl-fen and BvLSBF had significantly enhanced resistance to rice sheath blight ( Fig.13 ).

[0087] (2) Plant tomato seeds with the same germination in pots, and start the experiment when the tomato seedlings grow 4 to 6 true leaves. Select tomato seedlings with basically the same growth and plant height for root irrigation treatment. Water the pots thoroughly one day before inoculation. Take 10mL of tomato wilt spore suspension and inoculate it at the root zone of the tomato the next day. Incubate at 25℃ for 24h, and then irrigate the pots with 20mL of fermentation liquid of wild type Bv916 ​​and its mutants Bv-bl-fen, Bv-srf-loc, and BvLSBF that have been cultured to the logarithmic phase. 10 plants are treated for each treatment, repeated 3 times, and irrigated twice a month. The control group is replaced by an equal amount of tap water. No additional tomato wilt control is performed for each treatment. Other agricultural operations and management measures and conditions are the same, and irrigation is continued for 3 months. It was observed that compared with the control group, the wild type Bv916 ​​and the mutant strain Bv-bl-fen had no significant change in resistance to tomato wilt, while the mutant strains Bv-srf-loc and BvLSBF had significantly enhanced resistance to tomato wilt ( Fig.14 ).

[0088] Table 1 Biological control of rice sheath blight and tomato wilt by wild-type Bv916 ​​and its mutants

[0089]

Claims

1. A genetically engineered bacterium for high production of cyclolipopeptides, characterized in that: The genetically engineered bacteria is prepared by using Bacillus velez as a starting strain, and any one or more of the following promoters are replaced to obtain the genetically engineered bacteria: the promoter encoding the Locillomycin production gene is replaced with the strong promoter PB, the promoter encoding the Bacillomycin L production is replaced with the strong promoter P43, the promoter encoding the Surfactin production is replaced with the strong promoter PA, and the promoter encoding the Fengycin is replaced with the strong promoter PrepU.

2. The genetically engineered bacteria for high production of four cyclolipopeptides according to claim 1, characterized in that: The Bacillus Velez is preferably Bacillus Velez 916, and the cyclolipopeptide includes Locillomycin, Bacillomycin L, Surfactin and Fengycin.

3. A method for constructing a genetically engineered bacterium capable of producing four cyclolipopeptides in high yield according to claim 1, characterized in that: The steps include: (1) constructing P43 sgRNA expression cassettes using P43 promoter and sgRNAs designed according to the production genes encoding Locillomycin, Bacillomycin L, Surfactin, and Fengycin, respectively, to obtain P43 sgRNA (loc), P43 sgRNA (bl), P43 sgRNA (srf), and P43 sgRNA (fen) expression cassettes; (2) Inserting the expression cassette constructed in step (1) into the pTK or pTC-based vector, and then inserting gene fragments with strong promoters PB, P43, PA, PrepU and upstream and downstream homology arm fragments encoding Locillomycin, Bacillomycin L, Surfactin, and Fengycin production genes, respectively, to obtain editing vectors pTK-PB, pTK-P43, pTC-PA, and pTC-PrepU; (3) The editing vector pTN-Cas9 and any one or more editing vectors constructed in step (2) are transformed into Bacillus velezensis to construct a genetically engineered bacterium that can produce four cyclic lipopeptides in high yield.

4. The construction method according to claim 3, characterized in that: In step (1), the synthesized P43 promoter, sgRNA (loc) gene fragment or sgRNA (srf) gene fragment, sgRNA (bl) gene fragment, sgRNA (fen) gene fragment are used as templates to construct P43sgRNA (loc), P43sgRNA (bl), P43sgRNA (srf), P43sgRNA (fen) expression cassettes by overlapping PCR technology, and their sequences are shown in SEQ ID NO.1-4, respectively.

5. The construction method according to claim 3, characterized in that: In step (2), pTK is used as a vector to insert the P43sgRNA (loc) expression frame to obtain a pTK-sgRNA (loc) recombinant plasmid; a gene fragment with a strong promoter PB and upstream and downstream homology arm fragments encoding the Locillomycin production gene is designed and synthesized, as shown in SEQ ID NO.5, and inserted into the pTK-sgRNA (loc) recombinant plasmid to obtain a pTK-PB recombinant plasmid, the sequence of which is shown in SEQ ID NO.

9.

6. The construction method according to claim 3, characterized in that: In step (2), pTC is used as a vector to insert the P43sgRNA (srf) expression frame to obtain a pTC-sgRNA (srf) recombinant plasmid; a gene fragment with a strong promoter PA and upstream and downstream homology arm fragments encoding a surfactin production gene is designed and synthesized, as shown in SEQ ID NO.6, and inserted into the pTC-sgRNA (srf) recombinant plasmid to obtain a pTK-PB recombinant plasmid, the sequence of which is shown in SEQ ID NO.

10.

7. The construction method according to claim 3, characterized in that: In step (2), pTK is used as a vector to insert the P43sgRNA (bl) expression frame to obtain a pTK-sgRNA (bl) recombinant plasmid; a gene fragment with a strong promoter P43 and upstream and downstream homology arm fragments encoding the Bacillomycin L production gene is designed and synthesized, as shown in SEQ ID NO.7, and inserted into the pTK-sgRNA (bl) recombinant plasmid to obtain a pTK-P43 recombinant plasmid, the sequence of which is shown in SEQ ID NO.

11.

8. The construction method according to claim 3, characterized in that: In step (2), pTC is used as a vector to insert the P43sgRNA (fen) expression frame to obtain a pTK-sgRNA (fen) recombinant plasmid; a gene fragment with a strong promoter PA and upstream and downstream homologous arm fragments encoding the Fengycin production gene is designed and synthesized, as shown in SEQ ID NO.8, and inserted into the pTK-sgRNA (fen) recombinant plasmid to obtain a pTC-PrepU recombinant plasmid, the sequence of which is shown in SEQ ID NO.

12.

9. The construction method according to claim 3, characterized in that: In step (3), the editing vector pTN-Cas9 and the pTK-PB and pTC-PA constructed in step (2) are transferred into Bacillus velez 916 to obtain Bv-srf-loc; the editing vector pTN-Cas9 and the pTK-P43 and pTC-PrepU constructed in step (2) are transferred into Bacillus velez 916 to obtain Bv-bl-fen; the editing vector pTN-Cas9 and the pTK-PB and pTC-PA constructed in step (2) are transferred into the mutant strain Bv-bl-fen to obtain BvLSBF.

10. Use of the genetically engineered bacteria with high cyclolipopeptide production according to claim 1 in high production of Locillomycin, Bacillomycin L, Surfactin, Fengycin, and in antibacterial and biological control.

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