Engineering bacterium capable of improving yield of abelegen as well as construction method and application of engineering bacterium

Abstract

The invention discloses an engineering bacterium capable of increasing the yield of pelegen as well as a construction method and application of the engineering bacterium, and the construction method comprises the following steps: (1) constructing a plasmid pDSGa suitable for knockout of a long fragment of a bacillus amyloliquefaciens genome; (2) knocking out a non-ribosome peptide synthetase encoding gene itu of iturin A of bacillus amyloliquefaciens HM-618, and obtaining an engineering bacterium 1 after the plasmid is lost at 42 DEG C; (3) knocking out the surfactin non-ribosome peptide synthetase encoding gene srfAA of the engineering bacterium 1 to obtain an engineering bacterium 2; and (4) knocking out the membrane protein coding gene yvbJ of the engineering bacterium 2 to obtain an engineering bacterium 3, namely the engineering bacterium for improving the yield of the abinogen. According to the method disclosed by the invention, the genome editing efficiency of the bacillus amyloliquefaciens is improved, the synthesis capabilities of iturin A and surfactin of the bacillus amyloliquefaciens are deficient, the yield of progen is improved, and the separation and purification of products are facilitated.

Description

Technical Field

[0001] The invention belongs to the technical field of genetic engineering, and specifically relates to an engineering bacterium for increasing the yield of fengycin, and a construction method and use thereof. Background Art

[0002] Fengycin is a class of lipopeptide substances with biological antibacterial activity synthesized by Bacillus. It has significant antifungal activity and can significantly inhibit the activity of filamentous fungi (Guo Q, Dong L, Wang P, Li S, Zhao W, Lu X, Zhang X, Ma P. The PhoR / PhoP two-component system regulates fengycin production in Bacillus subtilis NCD-2 under low-phosphate conditions [J]. Journal of Integrative Agriculture, 2018, 17 (1): 149–157). Compared with chemical fertilizers or pesticides, fengycin is an eco-friendly and cost-effective alternative (Ali S, Hameed S, Imran A, Iqbal M, Lazarovits G. Genetic, physiological and biochemical characterization of Bacillus sp. strain RMB7 exhibiting plant growth promoting and broad spectrum antifungal activities [J]. Microbial Cell Factories, 2014, 13: 144). However, the low yield of fengyuan limits its application. At present, research on increasing the yield of fengyuan mainly focuses on exogenous regulation and endogenous regulation. Studies by Chen et al. have shown that exogenous addition of proline can significantly increase the production level of lipopeptides (Chen XY, Sun HZ, Qiao B, Miao CH, Hou ZJ, Xu SJ, Xu QM, Cheng JS. Improved the lipopeptide production of Bacillus amyloliquefaciens HM618 under co-culture with therecombinant Corynebacterium glutamicum producing high-level proline[J]. Bioresource Technology, 2022, 349: 126863).Ding et al. added exogenous alkanoic acids to the bacterial growth medium to promote the production of lipopeptides in Bacillus amyloliquefaciens Pc3 and enhance its antifungal activity (Ding L, Guo W, Chen X. Exogenous addition of alkanoic acids enhanced production of antifungallipopeptides in Bacillus amyloliquefaciens Pc3[J]. Applied Microbiology and Biotechnology, 2019, 103(13): 5367–5377). At the same time, enhancing the synthesis of fengycin through endogenous regulation has gradually become a research hotspot, targeting promoters (Yaseen Y, Gancel F, Drider D, Béchet M, Jacques P. Influence of promoters on the production of fengycin in Bacillus spp. [J]. Research in Microbiology, 2016, 167 (4): 272–281), regulatory factors (Sun J, Liu Y, Lin F, Lu Z, Lu Y. Cod Y, Com A, Deg U and Spo0 A controlling lipopeptides biosynthesis in Bacillus amyloliquefaciens fmb J [J]. Journal of Applied Microbiology, 2021, 131 (3): 1289–1304), metabolic pathways (Gao GR, Hou ZJ, Ding MZ, Bai S, Wei SY, Qiao B, Xu QM, Cheng JS, Yuan YJ. Improved production of fengycin The synthesis of fengycin was further enhanced by the modification of fengycin in Bacillus subtilis by integrated strain engineering strategy[J].ACS Synthetic Biology,2022,11(12):4065–4076).

[0003] Although the enhancement of fengyrin production by endogenous and exogenous regulation has been well studied, there has been no report on increasing fengyrin production by knocking out the itu gene encoding the iturin A non-ribosomal peptide synthetase of Bacillus amyloliquefaciens, the srfAA gene encoding the surfactant non-ribosomal peptide synthetase, and the yvbJ gene encoding the membrane protein by the CRISPR method. Summary of the invention

[0004] The first object of the present invention is to overcome the deficiencies of the prior art and provide an engineering bacterium for increasing the yield of Fengyuansu.

[0005] The second object of the present invention is to provide a method for constructing the above-mentioned engineering bacteria for increasing the yield of Fengyuansu.

[0006] The third object of the present invention is to provide the use of the above-mentioned engineering bacteria for increasing the yield of Fengyuansu to prepare Fengyuansu by fermentation.

[0007] The technical solution of the present invention is summarized as follows:

[0008] The method for constructing an engineering bacterium for increasing the yield of Fengyuansu comprises the following steps:

[0009] (1) Construction of plasmid pDSGa suitable for knocking out long fragments of the Bacillus amyloliquefaciens genome:

[0010] a. Using pJOE8999a as a template, the fragments pDSGa-F1 and pDSGa-F2 were amplified by PCR using primers pDSGa-1-HF, pDSGa-1-HR and pDSGa-2-HF, pDSGa-2-HR, respectively;

[0011] The nucleotide sequence of pJOE8999a is shown in SEQ ID NO.1;

[0012] The nucleotide sequence of the pDSGa-1-HF is shown in SEQ ID NO.2;

[0013] The nucleotide sequence of the pDSGa-1-HR is shown in SEQ ID NO.3;

[0014] The nucleotide sequence of the pDSGa-2-HF is shown in SEQ ID NO.4;

[0015] The nucleotide sequence of the pDSGa-2-HR is shown in SEQ ID NO.5;

[0016] b. Linearize pJOE8999a using Bsa I restriction endonuclease to obtain a linearized pJOE8999a vector;

[0017] c. The pDSGa-F1, pDSGa-F2 fragments and the linearized pJOE8999a vector were homologously recombined and connected into a circle to obtain a plasmid pDSGa suitable for knocking out a long fragment of the Bacillus amyloliquefaciens genome;

[0018] The nucleotide sequence of the plasmid pDSGa is shown in SEQ ID NO.6;

[0019] (2) Construction of engineered bacteria 1:

[0020] a. Design sgRNA sequences Δitu-sgRNA1 and Δitu-sgRNA2 according to the CRISPR RGEN Tools website, and connect them to the sgRNA cassette of the plasmid pDSGa; transform them into E. coli DH5α competent cells, screen positive clones and extract the recombinant plasmid named pDSGa-Δitu-sgRNA;

[0021] The nucleotide sequence of Δitu-sgRNA1 is shown in SEQ ID NO.8;

[0022] The nucleotide sequence of Δitu-sgRNA2 is shown in SEQ ID NO.9;

[0023] b. Using the genome of Bacillus amyloliquefaciens HM-618 as a template, primers Δitu-U-HF, Δitu-U-HR and primers Δitu-D-HF, Δitu-D-HR were used to amplify Δitu-U and Δitu-D respectively;

[0024] The nucleotide sequence of the Δitu-U-HF is shown in SEQ ID NO.10;

[0025] The nucleotide sequence of the Δitu-U-HR is shown in SEQ ID NO.11;

[0026] The nucleotide sequence of the Δitu-D-HF is shown in SEQ ID NO.12;

[0027] The nucleotide sequence of the Δitu-D-HR is shown in SEQ ID NO.13;

[0028] The pDSGa-Δitu-sgRNA was linearized by digestion with Sfi I restriction endonuclease to obtain a linearized pDSGa-Δitu-sgRNA vector; Δitu-U, Δitu-D and the linearized pDSGa-Δitu-sgRNA vector were homologously recombined and connected into a circle, and then transformed into the competent Escherichia coli DH5α, and positive clones were screened and extracted to name the recombinant plasmid pDSGa-Δitu;

[0029] c. The pDSGa-Δitu recombinant plasmid was transformed into Bacillus amyloliquefaciens HM-618, and the itu gene encoding iturin A non-ribosomal peptide synthetase of the Bacillus amyloliquefaciens HM-618 was knocked out by induction with 0.2% mannose at a mass concentration. After the plasmid was lost at 42°C, an engineered bacterium 1 was obtained;

[0030] The nucleotide sequence of the gene itu is shown in SEQ ID NO.7;

[0031] (3) Construction of engineered bacteria 2:

[0032] The surfactin non-ribosomal peptide synthetase encoding gene srfAA of the engineered bacteria 1 was knocked out to obtain the engineered bacteria 2;

[0033] The nucleotide sequence of the gene srfAA is shown in SEQ ID NO.14;

[0034] (4) Construction of Engineering Bacteria 3:

[0035] The membrane protein encoding gene yvbJ of the engineered bacteria 2 was knocked out to obtain the engineered bacteria 3, i.e., the engineered bacteria with increased Fengyuansu production;

[0036] The nucleotide sequence of the gene yvbJ is shown in SEQ ID NO.15.

[0037] Preferably, the Bacillus amyloliquefaciens is Bacillus amyloliquefaciens HM-618, with the deposit number CGMCC No.7097.

[0038] The engineering bacteria for improving the production of Fengyuansu constructed by the above construction method.

[0039] The application of the above-mentioned engineered bacteria for increasing the yield of Fengyuansu to prepare Fengyuansu by fermentation.

[0040] Advantages of the present invention:

[0041] The method of the invention improves the genome editing efficiency of Bacillus amyloliquefaciens, makes the synthesis ability of iturin A and surfactin of Bacillus amyloliquefaciens lost, increases the yield of fengyrin and is beneficial to the separation and purification of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 The yield of fengyrin of Bacillus amyloliquefaciens HM-ΔituΔsrfAA (engineered bacteria 2) and Bacillus amyloliquefaciens HM-ΔituΔsrfAAΔyvbJ (engineered bacteria 3) after fermentation. DETAILED DESCRIPTION

[0043] The technical scheme of the present invention is further described below in conjunction with specific examples. Unless otherwise specified, the technical means used in the present invention are methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative rather than limiting the scope of the present invention, and the essence and scope of the present invention are limited only by the claims. For those skilled in the art, various changes or modifications to the material composition and dosage in these embodiments, without departing from the essence and scope of the present invention, also belong to the protection scope of the present invention.

[0044] The present invention constructs a plasmid pDSGa for knocking out a long fragment of the Bacillus amyloliquefaciens genome, and successfully knocks out the Bacillus amyloliquefaciens iturin A non-ribosomal peptide synthetase encoding gene itu to obtain an engineered bacterium 1; further knocks out the surfactin non-ribosomal peptide synthetase encoding gene srfAA to obtain an engineered bacterium 2; and further knocks out the membrane protein encoding gene yvbJ to obtain an engineered bacterium 3, i.e., an engineered bacterium with increased fengyrin production.

[0045] According to the present invention, the culture of the Bacillus amyloliquefaciens can be carried out by conventional methods in the art. The culture medium can be a synthetic culture medium, such as a typical culture medium containing a carbon source, a nitrogen source, a sulfur source, inorganic ions and other required organic and inorganic components. The Bacillus amyloliquefaciens can be cultured under aerobic conditions for 36 to 96 hours, the culture temperature is controlled at 22 to 40° C., and the pH can be adjusted between 6.0 and 8.5.

[0046] Preferably, the seed culture medium of the present invention comprises: 60 g / L glucose, 10 g / L peptone, 10 g / L yeast extract, 10 g / L beef extract, and 5 g / L NaCl; the fermentation culture medium comprises: 60 g / L glucose, 15 g / L yeast extract, 14 g / L sodium glutamate, 14 g / L MgSO 4 7H 2 O 0.2g / L, KCl 0.5g / L, KH 2 PO 4 0.4g / L, FeSO 4 0.03g / L, MnSO 4 0.03g / L,VB 1 5×10 -4 g / L,VB 7 5×10 -4 g / L, pH 6.8-7.2;

[0047] Source of pJOE8999a: pJOE8999a plasmid (SEQ ID NO.1) was obtained by synonymous mutation of the BamH I restriction endonuclease recognition site in pJOE8999 plasmid.

[0048] The present invention preferably uses Bacillus amyloliquefaciens HM-618, with a deposit number of CGMCC No.7097, which was deposited in the General Microbiological Center of China National Microbiological Culture Collection Administration Committee on January 8, 2013, at the General Microbiological Center of China National Microbiological Culture Collection Administration Committee, address: No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing. Other Bacillus amyloliquefaciens can also be used in the present invention.

[0049] The present invention will be described in more detail below through specific examples.

[0050] Example 1

[0051] 1. Construction of plasmid pDSGa for long-fragment knockout in Bacillus amyloliquefaciens genome

[0052] Using pJOE8999a as a template, the primers pDSGa-1-HF, pDSGa-1-HR and pDSGa-2-HF, pDSGa-2-HR were used to amplify the fragments pDSGa-F1 and pDSGa-F2 by PCR, respectively. The amplified pDSGa-F1 and pDSGa-F2 fragments were recovered by Cycle Pure Kit D6492.

[0053] The nucleotide sequence of pJOE8999a is shown in SEQ ID NO.1;

[0054] The nucleotide sequence of the pDSGa-1-HF is shown in SEQ ID NO.2;

[0055] The nucleotide sequence of the pDSGa-1-HR is shown in SEQ ID NO.3;

[0056] The nucleotide sequence of the pDSGa-2-HF is shown in SEQ ID NO.4;

[0057] The nucleotide sequence of the pDSGa-2-HR is shown in SEQ ID NO.5;

[0058] The Max Super-Fidelity DNA Polymerase PCR reaction system is shown in Table 1.

[0059] Table 1 Max Super-Fidelity DNA Polymerase PCR System

[0060]

[0061] The Max Super-Fidelity DNA Polymerase PCR reaction program settings are shown in Table 2.

[0062] Table 2 Max Super-Fidelity DNA Polymerase PCR Procedure

[0063]

[0064] 2. The pJOE8999a plasmid was linearized by digestion with Bsa I restriction endonuclease, and the linearized pJOE8999a vector was recovered by Cycle Pure Kit D6492.

[0065] Table 3 Bsa I restriction endonuclease digestion reaction system

[0066]

[0067] 3. The pDSGa-F1, pDSGa-F2 fragments and the linearized pJOE8999a vector were connected into a circular plasmid by homologous recombination reaction (as shown in Table 4) using the seamless cloning kit ClonExpressⅡOne Step Cloning Kit, and the obtained circular plasmid was transformed into Escherichia coli DH5α competent cells by chemical transformation, and coated on a 25μg / mL kanamycin resistance plate to screen positive transformants. Recombination conditions: 37°C, 30min. The plasmid pDSGa suitable for long-fragment knockout of the Bacillus amyloliquefaciens genome was obtained; the nucleotide sequence of the plasmid pDSGa is shown in SEQ ID NO.6.

[0068] Table 4 ⅡOne Step Cloning Kit Homologous Recombination Ligation Reaction System

[0069]

[0070] Example 2

[0071] The pDSGa-Δitu plasmid was used to knock out the iturin A non-ribosomal peptide synthetase encoding gene itu of Bacillus amyloliquefaciens HM-618, with the deposit number CGMCC No.7097:

[0072] 1. Construction of plasmid pDSGa-Δitu knocking out the itu gene encoding iturin A non-ribosomal peptide synthetase of Bacillus amyloliquefaciens HM-618

[0073] According to the sgRNA sequence results designed by the CRISPR RGEN Tools website, Δitu-sgRNA1 and Δitu-sgRNA2 were synthesized respectively.

[0074] pDSGa was digested with Bsa I restriction endonuclease to obtain a linearized vector, which was connected to Δitu-sgRNA1 by homologous recombination, transformed into E. coli DH5α competent cells, and coated on a 25 μg / mL kanamycin resistance plate to screen positive transformants, and linearized with Bsa HI restriction endonuclease, connected to the sgRNA cassette of plasmid pDSGa by homologous recombination with Δitu-sgRNA2, transformed into E. coli DH5α competent cells, and coated on a 25 μg / mL kanamycin resistance plate to screen positive transformants. The plasmid is plasmid pDSGa-Δitu-sgRNA. The nucleotide sequence of the Δitu-sgRNA1 is shown in SEQ ID NO.8; the nucleotide sequence of the Δitu-sgRNA2 is shown in SEQ ID NO.9;

[0075] Using the genome of Bacillus amyloliquefaciens HM-618 as a template, primers Δitu-U-HF, Δitu-U-HR and Δitu-D-HF, Δitu-D-HR were used to amplify Δitu-U and Δitu-D respectively; the nucleotide sequence of the Δitu-U-HF is shown in SEQ ID NO.10; the nucleotide sequence of the Δitu-U-HR is shown in SEQ ID NO.11; the nucleotide sequence of the Δitu-D-HF is shown in SEQ ID NO.12; the nucleotide sequence of the Δitu-D-HR is shown in SEQ ID NO.13;

[0076] The obtained plasmid pDSGa-Δitu-sgRNA was linearized by Sfi I restriction endonuclease to obtain a linearized pDSGa-Δitu-sgRNA vector; Δitu-U, Δitu-D and the linearized pDSGa-Δitu-sgRNA vector were connected into a circle by homologous recombination, transformed into Escherichia coli DH5α competent cells, and spread on 25 μg / mL kanamycin resistance plates to screen positive transformants. The recombinant plasmid was named pDSGa-Δitu.

[0077] 2. Transform the pDSGa-Δitu recombinant plasmid into Bacillus amyloliquefaciens HM-618

[0078] 2.1 Preparation of competent cells of Bacillus amyloliquefaciens HM-618

[0079] The competent cells of Bacillus amyloliquefaciens HM-618 were prepared by the Bacillus Spizizen chemical transformation method.

[0080] First, an activated single colony of Bacillus amyloliquefaciens HM-618 was obtained by three-zone streaking, and the single colony was inoculated into a test tube containing 5 mL of GM I medium at 37°C and 200 rpm for 12 h.

[0081] Then, 500 μL of the first generation GM I culture medium was inoculated into a fresh test tube containing 4.5 mL of GM I medium and cultured at 37°C and 200 rpm until the OD 600 After the concentration was 3, 750 μL of the second-generation GM I culture solution was inoculated into a test tube containing 4.25 mL of GM II medium and cultured at 37°C, 200 rpm for 1.5 h to obtain chemically competent cells of Bacillus amyloliquefaciens HM-618.

[0082] Among them, the preparation of GM I and GM II culture medium:

[0083] 10×Spizizen basic salt solution: 12g / L sodium citrate, 183g / L K 2 HPO 4 , 60g / L KH 2 PO 4 , 20g / L(NH 4 ) 2 SO 4 , the balance is water. 4 ) 2 SO 4 The mother solution must be prepared and sterilized using a 0.22μm sterile syringe filter, and the remaining ingredients are prepared into mother solutions and sterilized at 121℃ for 20min.

[0084] GM I medium (100 mL): 10 mL 10× Spizizen basic salt solution, 2 mL 2% casein hydrolyzate solution, 2 mL 5% yeast extract solution, 2 mL 40% glucose solution, 1 mL 0.5% L-tryptophan solution, 100 μL 20% MgSO 4 7H 2 O stock solution, and finally add 82.9mL of sterile water.

[0085] GMⅡ medium (100mL): 10mL 10×Spizizen basic salt solution, 1mL 2% casein hydrolyzate mother solution, 2mL 40% glucose mother solution, 800μL 20% MgSO 4 7H 2 O stock solution, and finally add 86.2mL of sterile water.

[0086] 2.2 Transformation of pDSGa-Δitu recombinant plasmid into Bacillus amyloliquefaciens HM-618 competent cells

[0087] Take 1mL GM II culture medium into a 1.5mL sterile EP tube, add 3000ng of pDSGa-Δitu recombinant plasmid, and culture at 37℃, 200rpm for 3.5h. After the culture is completed, centrifuge at 6000rpm for 2min, keep 200μL culture medium, resuspend and apply to LB solid plate containing 25μg / mL kanamycin, and culture at 30℃ until a single colony grows.

[0088] 3. Induced knockout of the itu gene encoding iturin A non-ribosomal peptide synthetase in Bacillus amyloliquefaciens HM-618

[0089] The single colony in step 2.2 was inoculated into an LB test tube containing 0.2% mannose and 25 μg / mL kanamycin, and cultured at 37°C, 200 rpm for 12 hours. The correct transformant was streaked on a non-resistant plate and incubated at 42°C to lose the plasmid, and finally a Bacillus amyloliquefaciens HM-Δitu (engineered bacteria 1) with the gene itu encoding iturin A non-ribosomal peptide synthetase of Bacillus amyloliquefaciens HM-618 knocked out was obtained. The nucleotide sequence of the gene itu is shown in SEQ ID NO.7.

[0090] Example 3

[0091] Knockout of the surfactin non-ribosomal peptide synthetase encoding gene srfAA in Bacillus amyloliquefaciens HM-Δitu (engineering bacteria 1):

[0092] 1. Construction of pJOE8999a-ΔsrfAA plasmid.

[0093] 1.1 Knock out the surfactin non-ribosomal peptide synthetase encoding gene srfAA in engineered bacteria 1 by CRISPR / Cas9 method. According to the sgRNA sequence results designed on the CRISPR RGEN Tools website, design ΔsrfAA-sgRNA-F primer (SEQ ID NO.16) and ΔsrfAA-sgRNA-R primer (SEQ ID NO.17), anneal and connect ΔsrfAA-sgRNA-F and ΔsrfAA-sgRNA-F to obtain the connection product ΔsrfAA-sgRNA; connect ΔsrfAA-sgRNA to the pJOE8999a plasmid linearized with Bsa I to obtain the pJOE-ΔsrfAA-sgRNA plasmid;

[0094] Using the genome of Bacillus amyloliquefaciens HM-618 as a template, primers ΔsrfAA-U-HF (SEQ ID NO.18), ΔsrfAA-U-HR (SEQ ID NO.19) and ΔsrfAA-D-HF (SEQ ID NO.20), ΔsrfAA-D-HR (SEQ ID NO.21) were used to amplify the upstream and downstream homologous arms of srfAA ΔsrfAA-U and ΔsrfAA-D, respectively.

[0095] The pJOE-ΔsrfAA-sgRNA plasmid was digested with Sfi I to obtain a linearized pJOE-ΔsrfAA-sgRNA vector. ΔsrfAA-U, ΔsrfAA-D and the linearized pJOE-ΔsrfAA-sgRNA vector were connected by homologous recombination and transformed into Escherichia coli DH5α, and the positive transformants were selected on a 25 μg / mL kanamycin resistance plate. The recombinant plasmid was named pJOE8999a-ΔsrfAA.

[0096] 1.2 PCR reaction system and procedure are the same as Table 1 and Table 2;

[0097] 1.3 Homologous recombination ligation reaction system is the same as Table 4;

[0098] 2. Construction of Bacillus amyloliquefaciens HM-ΔituΔsrfAA (engineered strain 2)

[0099] 1.1 Preparation of Bacillus amyloliquefaciens Competent Cells The competent cells of Bacillus amyloliquefaciens HM-Δitu (engineered bacteria 1) were prepared in the same manner as step 2.1 of Example 2;

[0100] 1.2 The pJOE8999a-ΔsrfAA plasmid in step 1 was transformed into competent cells of Bacillus amyloliquefaciens HM-Δitu (engineering bacteria 1) by chemical transformation method, and cultured at 37°C and 200rpm for 3.5h. After the culture was completed, centrifuged at 6000rpm for 2min, 200μL of culture solution was resuspended and applied to LB solid plates containing 25μg / mL kanamycin, and cultured at 30°C until a single colony grew; the single colony was inoculated into an LB test tube containing 0.2% mannose and 25μg / mL kanamycin, and cultured at 37°C and 200rpm for 12h, and the correct transformant was streaked on a non-resistant plate and placed in a 42°C incubator to lose the plasmid, and finally a Bacillus amyloliquefaciens HM-ΔituΔsrfAA (engineering bacteria 2) with the gene srfAA encoding the surfactant non-ribosomal peptide synthetase of engineering bacteria 1 knocked out was obtained. The nucleotide sequence of the gene srfAA is shown in SEQ ID NO.14;

[0101] Embodiment 4:

[0102] Knockout of the membrane protein encoding gene yvbJ in Bacillus amyloliquefaciens HM-ΔituΔsrfAA (engineered bacteria 2)

[0103] 1. Construction of pJOE8999a-ΔyvbJ plasmid.

[0104] 1.1 Knock out the membrane protein encoding gene yvbJ in engineered bacteria 2 by CRISPR / Cas9. According to the sgRNA sequence designed on the CRISPRRGEN Tools website, design the ΔyvbJ-sgRNA-F primer (SEQ ID NO.22) and the ΔyvbJ-sgRNA-R primer (SEQ ID NO.23), anneal and connect ΔyvbJ-sgRNA-F and ΔyvbJ-sgRNA-F to obtain the connection product ΔyvbJ-sgRNA; connect ΔyvbJ-sgRNA to the pJOE8999a plasmid linearized with Bsa I to obtain the pJOE-ΔyvbJ-sgRNA plasmid;

[0105] Using the genome of Bacillus amyloliquefaciens HM-618 as a template, primers ΔyvbJ-U-HF (SEQ ID NO.24), ΔyvbJ-U-HR (SEQ ID NO.25) and ΔyvbJ-D-HF (SEQ ID NO.26), ΔyvbJ-D-HR (SEQID NO.27) were used to amplify the upstream and downstream homologous arms of yvbJ ΔyvbJ-U and ΔyvbJ-D, respectively.

[0106] The pJOE-ΔyvbJ-sgRNA plasmid was digested with Sfi I to obtain a linearized pJOE-ΔyvbJ-sgRNA vector. ΔyvbJ-U, ΔyvbJ-D and the linearized pJOE-ΔyvbJ-sgRNA vector were connected by homologous recombination and transformed into Escherichia coli DH5α, and the positive transformants were selected by coating on 25 μg / mL kanamycin resistance plates. The recombinant plasmid was named pJOE8999a-ΔyvbJ.

[0107] 1.2 PCR reaction system and procedure are the same as Table 1 and Table 2;

[0108] 1.3 Homologous recombination ligation reaction system is the same as Table 4;

[0109] 2. Construction of Bacillus amyloliquefaciens HM-ΔituΔsrfAAΔyvbJ (engineered strain 3)

[0110] 2.1 Preparation of Bacillus amyloliquefaciens Competent Cells The competent cells of Bacillus amyloliquefaciens HM-ΔituΔsrfAA (engineering bacteria 2) were prepared in the same manner as step 2.1 of Example 2;

[0111] 2.2 The pJOE8999a-ΔyvbJ plasmid in step 1 was transformed into competent cells of Bacillus amyloliquefaciens HM-ΔituΔsrfAA (engineering bacteria 2) by chemical transformation method, and cultured at 37°C, 200rpm for 3.5h. After the culture was completed, centrifuged at 6000rpm for 2min, 200μL of culture solution was resuspended and applied to LB solid plates containing 25μg / mL kanamycin, and cultured at 30°C until a single colony grew; the single colony was inoculated into an LB test tube containing 0.2% mannose and 25μg / mL kanamycin, and cultured at 37°C, 200rpm for 12h, and the correct transformant was streaked on a non-resistant plate and placed in a 42°C incubator to lose the plasmid, and finally a Bacillus amyloliquefaciens HM-ΔituΔsrfAAΔyvbJ (engineering bacteria 3) with the membrane protein encoding gene yvbJ of engineering bacteria 2 knocked out was obtained. The nucleotide sequence of the gene yvbJ is shown in SEQ ID NO.15.

[0112] Embodiment 5:

[0113] Verification of shake flask fermentation of Bacillus amyloliquefaciens HM-ΔituΔsrfAAΔyvbJ (engineered bacteria 3):

[0114] The method for producing Fengyuansu by using the above-mentioned Bacillus amyloliquefaciens HM-ΔituΔsrfAAΔyvbJ (engineered bacteria 3) in shake flask fermentation is as follows:

[0115] Seed activation culture: Take the 80℃ preserved strain and inoculate three zones on the activated solid plate and culture at 37℃ for 12h.

[0116] Seed culture: Take an activated single colony and inoculate it into a 250 mL round-bottomed Erlenmeyer flask containing 100 mL of seed culture medium, and culture it at 37°C and 180 rpm for 12 h.

[0117] Fermentation culture: 1 mL of seed solution was inoculated into a 250 mL baffled flask containing 100 mL of fermentation medium, and cultured at 30°C and 200 rpm for 72 h.

[0118] After 72 hours of shake flask fermentation, Bacillus amyloliquefaciens HM-ΔituΔsrfAAΔyvbJ (engineered bacteria 3) can produce 164.89 mg / L of fengycin, which is 54.28% higher than the control strain Bacillus amyloliquefaciens HM-ΔituΔsrfAA (engineered bacteria 2). Figure 1 .

Claims

1. A method for constructing an engineered bacterium for increasing the yield of Fengyuansu, characterized in that The steps include: (1) Construction of plasmid pDSGa suitable for knocking out long fragments of the Bacillus amyloliquefaciens genome: a. Using pJOE8999a as a template, the fragments pDSGa-F1 and pDSGa-F2 were amplified by PCR using primers pDSGa-1-HF, pDSGa-1-HR and pDSGa-2-HF, pDSGa-2-HR, respectively; The nucleotide sequence of pJOE8999a is shown in SEQ ID NO.1; The nucleotide sequence of the pDSGa-1-HF is shown in SEQ ID NO.2; The nucleotide sequence of the pDSGa-1-HR is shown in SEQ ID NO.3; The nucleotide sequence of the pDSGa-2-HF is shown in SEQ ID NO.4; The nucleotide sequence of the pDSGa-2-HR is shown in SEQ ID NO.5; b. Linearize pJOE8999a using Bsa I restriction endonuclease to obtain a linearized pJOE8999a vector; c. The pDSGa-F1, pDSGa-F2 fragments and the linearized pJOE8999a vector were homologously recombined and connected into a circle to obtain a plasmid pDSGa suitable for knocking out a long fragment of the Bacillus amyloliquefaciens genome; The nucleotide sequence of the plasmid pDSGa is shown in SEQ ID NO.6; (2) Construction of engineered bacteria 1: a. Design sgRNA sequences Δitu-sgRNA1 and Δitu-sgRNA2 according to the CRISPR RGEN Tools website, and connect them to the sgRNA cassette of the plasmid pDSGa; transform them into E. coli DH5α competent cells, screen positive clones and extract the recombinant plasmid named pDSGa-Δitu-sgRNA; The nucleotide sequence of the Δitu-sgRNA1 is shown in SEQ ID NO.8; The nucleotide sequence of Δitu-sgRNA2 is shown in SEQ ID NO.9; b. Using the genome of Bacillus amyloliquefaciens HM-618 as a template, primers Δitu-U-HF, Δitu-U-HR and primers Δitu-D-HF, Δitu-D-HR were used to amplify Δitu-U and Δitu-D respectively; The nucleotide sequence of the Δitu-U-HF is shown in SEQ ID NO.10; The nucleotide sequence of the Δitu-U-HR is shown in SEQ ID NO.11; The nucleotide sequence of the Δitu-D-HF is shown in SEQ ID NO.12; The nucleotide sequence of the Δitu-D-HR is shown in SEQ ID NO.13; The pDSGa-Δitu-sgRNA was linearized by digestion with Sfi I restriction endonuclease to obtain a linearized pDSGa-Δitu-sgRNA vector; Δitu-U, Δitu-D and the linearized pDSGa-Δitu-sgRNA vector were homologously recombined and connected into a circle, and then transformed into the competent E. coli DH5α, and the positive clones were screened and extracted and named as pDSGa-Δitu recombinant plasmid; c. The pDSGa-Δitu recombinant plasmid was transformed into Bacillus amyloliquefaciens HM-618, and the itu gene encoding iturin A non-ribosomal peptide synthetase of the Bacillus amyloliquefaciens HM-618 was knocked out by induction with 0.2% mannose at a mass concentration. After the plasmid was lost at 42°C, an engineered bacterium 1 was obtained; The nucleotide sequence of the gene itu is shown in SEQ ID NO.7; (3) Construction of engineered bacteria 2: The surfactin non-ribosomal peptide synthetase encoding gene srfAA of the engineered bacteria 1 was knocked out to obtain the engineered bacteria 2; The nucleotide sequence of the gene srfAA is shown in SEQ ID NO.14; (4) Construction of engineered bacteria 3: The membrane protein encoding gene yvbJ of the engineered bacteria 2 was knocked out to obtain the engineered bacteria 3, i.e., the engineered bacteria with increased Fengyuansu production; The nucleotide sequence of the gene yvbJ is shown in SEQ ID NO.

15.

2. The construction method according to claim 1, characterized in that The Bacillus amyloliquefaciens is Bacillus amyloliquefaciens HM-618, with a preservation number of CGMCC No.7097.

3. An engineered bacterium for increasing the yield of Fengyuansu constructed by the construction method of claim 1 or 2.

4. Use of the engineered bacteria for increasing the yield of Fengyuansu according to claim 3 in fermenting Fengyuansu.

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