Engineered bacteria for increasing lipopeptide production, their construction methods, and applications.

Abstract

This invention discloses an engineered bacterium that increases lipopeptide production, its construction method, and its uses. The construction method involves eliminating the endogenous plasmid of Bacillus amyloliquefaciens to obtain engineered bacterium 1 that increases lipopeptide production. Compared with Bacillus amyloliquefaciens that has not undergone the elimination of the endogenous plasmid, engineered bacterium 1 of this invention can produce more lipopeptide substances such as surfactantin, fengycin, and iturin A during the culture process.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, specifically relating to engineered bacteria for increasing lipopeptide production, their construction methods, and applications. Background Technology

[0002] Of the losses caused by pests, 80% are due to plant pathogenic fungi. Pesticides are indispensable production materials for humans to obtain food and ensure stable and abundant agricultural yields. However, while pesticides have made significant contributions to human civilization, limitations in understanding have led to the negative impacts of highly toxic and persistent pesticides on the environment upon which humans depend. With societal progress and the advancement of civilization, the demand for harmonious coexistence between humans and nature has increased, leading to higher requirements for pesticides. Therefore, the research and development of highly efficient, low-toxicity, easily degradable, safe, and environmentally compatible green pesticides has become the mainstream and direction of pesticide research today. The use of biopesticides is considered the most promising, more rational, and safer method of crop management. Compared with traditional chemical pesticides, biopesticides have advantages such as safety for humans, animals, and non-target organisms, good environmental compatibility, low likelihood of developing resistance, ease of biodiversity protection, and wide availability. Therefore, the development and application of highly efficient biopesticides are of paramount importance to human health, environmental protection, and the sustainable development of agriculture. Bacillus is a commonly used microorganism, with 4%–5% of its genome related to antibiotic synthesis, possessing the potential to produce more than twenty structurally diverse antimicrobial compounds. Among these antimicrobial compounds, surfactants, iturobrine, and the fenestrant family of lipopeptides have recognized application potential in biotechnology and biopharmaceuticals due to their superior surface activity properties. Currently, research on lipopeptides mainly focuses on the following aspects: 1) screening and modification of high-yield strains; 2) improvement of fermentation culture methods; 3) optimization of culture conditions; and 4) selection and application of inexpensive fermentation raw materials. For a long time, the problems of high by-product content, high cost, and low yield have severely restricted the microbial production and application of lipopeptides, making the construction of genetically engineered strains that efficiently synthesize lipopeptide products an urgent need. Summary of the Invention

[0003] The purpose of this invention is to overcome the shortcomings of the prior art and provide an engineered bacterium that can increase lipopeptide production.

[0004] A second objective of this invention is to provide a method for constructing engineered bacteria that increase lipopeptide production.

[0005] A third objective of this invention is to provide an engineered bacterial fermentation method for obtaining lipopeptides to increase lipopeptide production.

[0006] A fourth objective of this invention is to provide another engineered bacterium that increases lipopeptide production.

[0007] The fifth objective of this invention is to provide another method for constructing engineered bacteria that increase lipopeptide production.

[0008] The sixth object of the present invention is to provide the use of another engineered strain for fermenting lipopeptides to increase lipopeptide production.

[0009] The technical solution of this invention is summarized as follows:

[0010] A method for constructing an engineered bacterium that increases lipopeptide production includes the following steps: eliminating the endogenous plasmid of Bacillus amyloliquefaciens to obtain an engineered bacterium 1 that increases lipopeptide production.

[0011] The preferred Bacillus amyloliquefaciens is Bacillus amyloliquefaciens HM-618, with accession number CGMCC No.7097.

[0012] The engineered bacteria 1 that improves lipopeptide production was constructed using the above method.

[0013] The above-mentioned engineered strain 1, which increases lipopeptide production, is used to obtain lipopeptides through fermentation.

[0014] Another method for constructing engineered bacteria that increase lipopeptide production includes the following steps: knocking out the non-ribosomal peptide synthase genes of surfactantin and fengycin in Bacillus amyloliquefaciens; then eliminating endogenous plasmids to obtain engineered bacteria 2 that increase lipopeptide production.

[0015] The preferred Bacillus amyloliquefaciens is Bacillus amyloliquefaciens HM-618, with accession number CGMCC No.7097.

[0016] The engineered bacteria 2 that improves lipopeptide production was constructed using the above method.

[0017] The above-mentioned engineered bacteria 2, which increases lipopeptide production, is used to obtain lipopeptides through fermentation.

[0018] Advantages of this invention:

[0019] Compared with Bacillus amyloliquefaciens that has not undergone elimination of endogenous plasmids, the engineered bacteria 1 of the present invention, which increases lipopeptide production, is able to produce more lipopeptide substances such as surfactantin, fengycin, and iturin A during the culture process.

[0020] The engineered bacteria 2 of the present invention, which improves lipopeptide production, can significantly increase the production of iturin A during the cultivation process. Attached Figure Description

[0021] Figure 1 This is a gel electrophoresis image of Bacillus amyloliquefaciens HM618 with endogenous plasmid DNA knocked out by colony PCR. M: marker; 1-10: negative control (Bacillus amyloliquefaciens HM618); 11-19: positive strain (HM618 strain with endogenous plasmid plas1 eliminated).

[0022] Figure 2 This was verified by shake-flask fermentation of Bacillus amyloliquefaciens HM618-N (engineered strain 1 that increases lipopeptide production).

[0023] Figure 3 This is a DNA gel electrophoresis image of the nonribosomal peptide synthase gene clusters of surfactantin and fengycin knocked out in Bacillus amyloliquefaciens HM618.

[0024] Figure 4 This is a shake-flask fermentation verification of Bacillus amyloliquefaciens HM618-NDSF (engineered strain 2 that increases lipopeptide production). Detailed Implementation

[0025] The technical solution of the present invention will be further described below with reference to specific embodiments. Unless otherwise specified, the technical means used in the present invention are all methods known to those skilled in the art. In addition, the embodiments should be understood as illustrative, not limiting the scope of the present invention, and the essence and scope of the present invention are defined 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 fall within the protection scope of the present invention.

[0026] Nonribosomal peptide synthase genes of surfactantin (Bacillus amyloliquefaciens HM618, DKG78_02285, DKG78_02290, DKG78_02295, DKG78_02300).

[0027] The nonribosomal peptide synthase gene of fengycin (Bacillus amyloliquefaciensHM618, gene number DKG78_10110, DKG78_10115, DKG78_10120, DKG78_10125, DKG78_10130)

[0028] Bacillus amyloliquefaciens HM-618, accession number CGMCC No. 7097, was deposited on January 8, 2013, at the China General Microbiological Culture Collection Center, located at No. 3, Courtyard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. Other Bacillus amyloliquefaciens species can also be used in this invention.

[0029] The present invention will be described in more detail below through specific embodiments.

[0030] Example 1

[0031] Constructing CRISPR / Cas9 gene editing plasmids suitable for Bacillus amyloliquefaciens: 1. Using pJOE8999 as a template, high-fidelity Takara Prime was used for gene editing. HS DNA Polymerase was used to amplify two linear fragments, frag 1 and frag 2, by PCR, respectively, after removing the BamH I site sequence. The primers were as follows:

[0032] frag 1-F(SEQ ID NO.1):TTAACGTGAGTTTTCGTTCCACTGAGCGTCAG;

[0033] frag 1-R(SEQ ID NO.2):

[0034] AAGACTGGGACCCAAAAAAATATGGTGGTTTTTGATAGTCCAACG;

[0035] frag 2-F (SEQ ID NO.3):

[0036] TTTTTTTGGGTCCCAGTCTTTTTTACGAGCAATAAGCTTGTCCG;

[0037] frag 2-R (SEQ ID NO. 4):GGAACGAAAACTCACGTTAAGGGATTTTGGTCAT.

[0038] The PCR reaction system is shown in Table 1.

[0039] Table 1 HS enzyme PCR reaction system

[0040]

[0041] The PCR reaction program settings are shown in Table 2.

[0042] Table 2. PCR reaction procedure for Hs enzyme

[0043]

[0044] The amplified frag 1 and frag 2 fragments were recovered using the Cycle Pure Kit D6492 kit.

[0045] 2. Using the ClonExpressⅡOne Step Cloning Kit, the fragments frag 1 and frag 2 were recombined. The homologous recombination ligation reaction system (Table 3) was prepared and placed in a 37°C water bath for 30 min, followed by an ice bath for 5 min. Then, the system was transformed into Escherichia coli DH5α competent cells by chemical transformation. Positive transformants were screened, plasmid strains were preserved, and plasmids were extracted.

[0046] Table 3 Homologous recombination linkage reaction system

[0047]

[0048] The constructed plasmid vector was named pJOE8999a (SEQ ID NO.13), also known as a CRISPR / Cas9 gene editing plasmid suitable for Bacillus amyloliquefaciens. This plasmid can be used for gene editing in Bacillus amyloliquefaciens.

[0049] Example 2: Eliminating the endogenous plasmid Plas1 of Bacillus amyloliquefaciens HM-618 (CGMCC No. 7097) through gene editing

[0050] 1. Constructing the gene-editing plasmid Plas1, an endogenous plasmid for knocking out Bacillus amyloliquefaciens HM618.

[0051] The Red replication protein gene site sequence information of the Bacillus amyloliquefaciens HM618 endogenous plasmid was determined in the National Genomics Institute (NCBI) database. The appropriate PAM sequence (5'-NGG-3') and its upstream 20 bp gene sequence were searched using CRISPR RGEN Tools (http: / / www.rgenome.net / cas-designer / ). The single-stranded annealed fragment sequence designed in this embodiment is Q-HMplas-20bp-S (SEQ ID NO.5):

[0052] TGAGTAAAATGTACCTACGCTTCAGCCCGAAGGCTTATGTTTTAGAGCTAGAAATA G; and Q-HMplas-20bp-A (SEQ ID NO. 6):

[0053] The single-stranded annealed fragment CTATTTCTAGCTCTAAAACATAAGCCTTCGGGCTGAAAGCGTAGGTACATTTTACTC A was synthesized by Qingke Company. The synthesized single strand was annealed by PCR to form a double-stranded fragment. The above double-stranded fragment was ligated with the pJOE8999a vector fragment linearized with Bsa I using homologous recombination. The ligation was then transformed into E. coli DH5α competent cells. Positive clones were screened and plasmids were extracted. The extracted recombinant plasmid was named pJOE8999a-ΔP and stored at -20℃ for later use.

[0054] 2. Transform the pJOE8999a-ΔP plasmid into Bacillus amyloliquefaciens HM618.

[0055] 2.1 Preparation of competent cells of Bacillus amyloliquefaciens HM618

[0056] Competent cells of *Bacillus amyloliquefaciens* HM618 were prepared using the Spizizen chemical transformation method. First, activated single colonies of *Bacillus amyloliquefaciens* HM618 were obtained by streaking in three zones. These single colonies were inoculated into first-generation cells in tubes containing 5 mL of GMI medium and incubated at 37°C and 200 rpm for 12 h. Then, 500 μL of the first-generation GMI culture was inoculated into second-generation cells in tubes containing 4.5 mL of GMI medium and incubated at 37°C and 200 rpm until OD (digesterone) was reached. 600 After 3, 750 μL of second-generation GM I culture medium was inoculated into a test tube containing 4.25 mL of GM II culture medium and cultured at 37°C and 200 rpm for 1.5 h to obtain Bacillus amyloliquefaciens HM618 chemocompetent cells.

[0057] The preparation of GMI and GMII culture media includes:

[0058] 10×Spizizen basic salt solution: 12 g / L sodium citrate, 183 g / L K₂HPO₄, 60 g / L KH₂PO₄, 20 g / L (NH₄)₂SO₄, balance water. The (NH₄)₂SO₄ must be prepared as a stock solution and filtered through a 0.22 μm sterile syringe filter for sterilization. The remaining components are prepared as a stock solution and sterilized at 121°C for 20 min.

[0059] GMI medium (100 mL): 10 mL 10×Spizizen basic salt solution, 2 mL 2% casein hydrolysate stock solution, 2 mL 5% yeast extract stock solution, 2 mL 40% glucose stock solution, 1 mL 0.5% L-tryptophan stock solution, 100 μL 20% MgSO4·7H2O stock solution, and finally add 82.9 mL sterile water.

[0060] GMⅡ medium (100mL): 10mL 10×Spizizen basic salt solution, 1mL 2% casein hydrolysate stock solution, 2mL 40% glucose stock solution, 800μL 20% MgSO4·7H2O stock solution, and finally 86.2mL sterile water.

[0061] 2.2pJOE8999a-ΔP was transformed into Bacillus amyloliquefaciens HM618 competent cells.

[0062] Take 1 mL of GM II culture medium into a 1.5 mL sterile EP tube, add 1000 ng of pJOE8999a-ΔP plasmid, and incubate at 37℃ and 200 rpm for 3.5 h. After incubation, centrifuge at 6000 rpm for 2 min, resuspend 200 μL of culture medium, and spread it onto LB agar plates containing 25 μg / mL kanamycin. Incubate at 30℃ until single colonies grow.

[0063] 3. Inducing knockout of the endogenous plasmid Plas1 in Bacillus amyloliquefaciens HM618

[0064] The single colonies from step 2.2 were inoculated into test tubes containing 1% mannose and 25 μg / mL kanamycin in LB medium and incubated at 37°C and 200 rpm for 12 h. The colonies were then streaked into antibiotic-free LB agar plates. After single colonies grew, PCR identification was performed using identification primers. The primer sequences were: JD-Q-HMplas-F (SEQ ID NO.7): GTATGGTGAGCTGCTGAACTTTG and JD-Q-HMplas-R (SEQ ID NO.8): CACTCCGTGACCGCTCCTA. The reaction solution was prepared using the 2×Taq PCR Master Mix system from Beijing Solarbio Science & Technology Co., Ltd. The colony PCR reaction system and reaction procedure are shown in Tables 4 and 5. Strains that could not amplify bands using the identification primers were screened, i.e., Bacillus amyloliquefaciens strains with the endogenous plasmid Plas1 knocked out but still containing pJOE8999a-ΔP. The PCR reaction identification results are shown in [Table 4]. Figure 1 .

[0065] Table 4 Colony PCR Reaction System

[0066]

[0067] Table 5 Colony PCR reaction procedure

[0068]

[0069] 4. Loss of gene editing tool plasmid pJOE8999a-ΔP

[0070] The colonies obtained in step 3 were inoculated into 5 mL LB medium test tubes and cultured at 42°C and 200 rpm for 12 h. Single colonies were obtained by streaking in three zones. The single colonies were then spotted onto LB solid plates without antibiotics and LB solid plates containing 25 μg / mL kanamycin, respectively, and incubated at 30°C for 12 h. Colonies that grew on the antibiotic-free solid plate but did not grow on the kanamycin solid plate were selected and inoculated into 5 mL LB medium test tubes. After culturing for 12 h, the bacteria were preserved to obtain the final Bacillus amyloliquefaciens with the endogenous plasmid Plas1 eliminated, named Bacillus amyloliquefaciens HM618-N (i.e., engineered bacteria 1 that increases lipopeptide production).

[0071] Example 3: Shake-flask fermentation verification of Bacillus amyloliquefaciens HM618-N

[0072] The method for producing lipopeptides using the above-mentioned engineered strain Bacillus amyloliquefaciens HM618-N via shake-flask fermentation is as follows:

[0073] Seed activation culture: The 80℃ preserved strain was streaked into LB solid plates for activation and incubated at 37℃ for 12 hours.

[0074] Seed culture: Inoculate a single activated colony into a 250 mL Erlenmeyer flask containing 20 mL of seed culture medium and incubate at 37 °C and 200 rpm until OD500 reaches zero. 600 It is 10.

[0075] Fermentation culture: Take 1 mL of seed liquid and inoculate it into a 250 mL baffled Erlenmeyer flask containing fermentation medium. Add 5 mL of 45% glucose stock solution (final fermentation volume is 50 mL). Incubate at 30℃ and 180 rpm for 72 h.

[0076] Seed culture medium: 10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, with the remainder being water.

[0077] Fermentation medium: Sodium glutamate 5 g / L, MgSO4·7H2O 0.5 g / L, KCl 0.5 g / L, KH2PO4 0.5 g / L, FeSO4 0.15 × 10⁻⁶-3 g / L, MnSO4 0.15×10 -3 g / L, peptone 12g / L, yeast extract 4g / L, glucose 45g / L, pH 6.8, balance water.

[0078] Bacillus amyloliquefaciens HM618-N was cultured in shake flasks for 72 hours.

[0079] The production of surfactant was 863.88 mg / L, which was 43 times higher than that of the control strain Bacillus myloliquefaciens HM618.

[0080] The production of fengycin was 349.86 mg / L, which was 17 times higher than that of the control strain Bacillus amyloliquefaciens HM618;

[0081] The production of iturin A was 614.75 mg / L, which was 54 times higher than that of the control strain Bacillus myloliquefaciens HM618;

[0082] The yield of the three lipopeptides was 1828.49 mg / L, which was 35 times higher than that of the control strain Bacillus amyloliquefaciens HM618. The fermentation process curves are shown below. Figure 2 .

[0083] Example 4: Knock out the nonribosomal peptide synthase gene of surfactant and the nonribosomal peptide synthase gene of cytosine in Bacillus amyloliquefaciens HM618, and eliminate their endogenous plasmids.

[0084] First, using the CRISPR / Cas9 method, the non-ribosomal peptide synthase genes for surfactantin and fengycin in Bacillus amyloliquefaciens HM618 were knocked out. The designed genome identification primers were as follows:

[0085] JD-Q-srf-F (SEQ ID NO.9): TGAGTCGTCTGTCGCTCCTAAA;

[0086] JD-Q-srf-R (SEQ ID NO.10): GCTTGAAATCGTTCCCCTGA;

[0087] JD-Q-fen-F (SEQ ID NO. 11): GGGGCCAGACCAAGTAGCA;

[0088] JD-Q-fen-R (SEQ ID NO. 12): TGACGGTTGAACGGGAGC;

[0089] See genome knockout validation results Figure 3 A modified strain of Bacillus amyloliquefaciens HM618ΔsrfΔfen was constructed and named Bacillus amyloliquefaciens HM618-DSF.

[0090] Using the method described in Example 2, the endogenous plasmid of Bacillus amyloliquefaciens HM618-DSF was knocked out to obtain the Bacillus amyloliquefaciens HM618-NDSF strain (i.e., engineered strain 2 that increases lipopeptide production).

[0091] Example 5: Shake-flask fermentation verification of Bacillus amyloliquefaciens HM618-NDSF

[0092] The same shake-flask fermentation culture method as in Example 3 was used to simultaneously control Bacillus amyloliquefaciens HM618-DSF and Bacillus amyloliquefaciens HM618-NDSF, and the yields of surfactantin, fengycin, and iturin A were measured. The results showed that Bacillus amyloliquefaciens HM618-NDSF synthesized iturin A at a yield of 128.33 mg / L, while surfactantin and fengycin were undetectable. The control strain Bacillus amyloliquefaciens HM618-DSF produced iturin A at 3.29 mg / L, while surfactantin and fengycin were undetectable. This indicates that eliminating the endogenous plasmid in the Bacillus amyloliquefaciens HM618-DSF strain can increase the yield of the lipopeptide iturin A. (See results below.) Figure 4 .

[0093] Conclusions: Engineered strain 1, which enhances lipopeptide production by eliminating endogenous plasmids, showed a significant increase in the production of lipopeptides such as surfactant, fenestrator, and iturobrinein A after cultivation. Engineered strain 2, which enhances lipopeptide production by knocking out the non-ribosomal peptide synthase genes of surfactant and fenestrator, and simultaneously eliminating endogenous plasmids, produced a more homogeneous lipopeptide fermentation product and increased iturobrinein A production after cultivation.

Claims

1. A method for constructing engineered bacteria to increase lipopeptide production, characterized in that: The steps include: eliminating Bacillus amyloliquefaciens (BAM) Bacillus amyloliquefaciens The endogenous plasmid Plas1 of Bacillus amyloliquefaciens HM-618, with accession number CGMCC No. 7097, was used to obtain engineered bacteria 1 that increases lipopeptide production. The lipopeptides are surfactant, cytokinin, and ituronin. The process of eliminating the endogenous plasmid Plas1 of Bacillus amyloliquefaciens HM-618, with accession number CGMCC No. 7097, includes the following steps: synthesizing single-stranded molecules and annealing them via PCR to form double-stranded fragments; then, combining the double-stranded fragments with... Bsa The pJOE8999a vector fragment, linearized and digested with enzyme I, was ligated using homologous recombination and transformed into Escherichia coli DH5α competent cells. Positive clones were screened and plasmids were extracted. The extracted recombinant plasmid was named pJOE8999a-△P. The single-stranded annealed fragment sequences are SEQ ID NO.5 and SEQ ID NO.6, respectively. The pJOE8999a vector is shown in SEQ ID NO.

13. The pJOE8999a-△P plasmid was transformed into Bacillus amyloliquefaciens HM-618, and the endogenous plasmid Plas1 of Bacillus amyloliquefaciens HM-618 was knocked out, resulting in the loss of the gene editing tool plasmid pJOE8999a-△P, thus obtaining an engineered bacterium that increases lipopeptide production.

2. The engineered bacteria 1 for increasing lipopeptide production constructed by the construction method of claim 1.

3. Use of the engineered bacteria 1 for increasing lipopeptide production according to claim 2 to obtain lipopeptides through fermentation, wherein the lipopeptides are surfactants, cytosine, and itursin.

4. A method for constructing engineered bacteria to increase lipopeptide production, characterized in that: The process includes the following steps: knocking out the non-ribosomal peptide synthase genes of surfactant and fibrosin in *Bacillus amyloliquefaciens* HM-618 (CGMCC No. 7097); then eliminating the endogenous plasmid Plas1 to obtain engineered bacteria 2 with increased lipopeptide production, wherein the lipopeptide is iturobacterin. The elimination of the endogenous plasmid Plas1 from *Bacillus amyloliquefaciens* HM-618 (CGMCC No. 7097) includes the following steps: synthesizing single-stranded fragments and annealing them via PCR to form double-stranded fragments; then combining the double-stranded fragments with... Bsa The pJOE8999a vector fragment, linearized and digested with enzyme I, was ligated using homologous recombination and transformed into E. coli DH5α competent cells. Positive clones were screened and plasmids were extracted. The extracted recombinant plasmid was named pJOE8999a-△P. The single-stranded annealed fragment sequences are SEQ ID NO.5 and SEQ ID NO.6, respectively. The pJOE8999a vector is shown in SEQ ID NO.

13. The pJOE8999a-△P plasmid was transformed into Bacillus amyloliquefaciens HM-618, inducing the knockout of the endogenous plasmid Plas1 in Bacillus amyloliquefaciens HM-618, resulting in the loss of the gene editing tool plasmid pJOE8999a-△P.

5. The engineered bacteria 2 for increasing lipopeptide production constructed by the construction method of claim 4.

6. Use of the engineered bacteria 2 for increasing lipopeptide production according to claim 5 to obtain lipopeptides through fermentation, wherein the lipopeptide is itursin.

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