Pseudomonas for producing nitropyrrole, engineering bacterium and construction method and application of pseudomonas

Abstract

The invention discloses Pseudomonas protegens for producing nitropyrrole, which is preserved in the China Center for Type Culture Collection, and the preservation number is CCTCC (China Center for Type Culture Collection) M20241968. The invention further discloses a preparation method of the Pseudomonas protegens for producing nitropyrrole. The invention also discloses an engineering bacterium for producing nitropyrrole and a construction method of the engineering bacterium. The engineering bacterium is a mutant strain for blocking a bypass metabolic pathway of the pseudomonas. For example, the pseudomonas is taken as a target, a core gene in a BGC1 biosynthetic gene cluster in the pseudomonas is knocked out through a gene knockout method, or a core gene in a BGC16 biosynthetic gene cluster is knocked out, or a mutant strain of the core genes in two biosynthetic gene clusters of BGC1 and BGC16 is knocked out at the same time. The invention also discloses a biocontrol microbial inoculum containing the pseudomonas or the engineering bacteria. The invention further discloses application of the pseudomonas or the engineering bacteria or the biocontrol microbial agent in prevention and treatment of rhizoctonia solani, and the pseudomonas or the engineering bacteria or the biocontrol microbial agent has a remarkable antibacterial effect.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering bacteria, and in particular to a Pseudomonas for producing nitropyrrolidine, an engineering bacteria, and a construction method and application thereof. Background Art

[0002] Sheath blight is one of the three major fungal diseases of rice, which can cause a more than 40% reduction in rice production in severe cases. When sheath blight is prevalent, the use of chemical pesticides will cause soil pollution, pesticide residues and pathogen resistance, while biological control is a safe and effective means. Direct use of beneficial microorganisms or compounds derived from microorganisms is one of the main measures of biological control. Pyrro l nitrin (PRN) from Pseudomonas has significant inhibitory activity against rice sheath blight.

[0003] It is still of great significance to screen new biocontrol bacteria with anti-pathogenic fungal activity and to improve the production of active metabolites through genetic engineering. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a Pseudomonas producing nitropyrrolidone, an engineered bacterium capable of high-yielding nitropyrrolidone, and a construction method and application thereof.

[0005] In order to solve the above technical problems, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a Pseudomonas protegens for producing nitropyrrolidone, the strain is named JP2-4390, the strain is deposited in the China Center for Type Culture Collection (CCTCC), the deposit number is CCTCC M 20241968, the deposit date is September 13, 2024, and the deposit address is: Wuhan University, Wuhan, China, Postal Code: 430072. Its system classification is Pseudomonas, that is, the strain is Pseudomonas JP2-4390.

[0007] In a second aspect, the present invention provides an engineered bacterium for producing nitropyrrolidone, wherein the engineered bacterium is a mutant strain that blocks the above-mentioned bypass metabolic pathway of Pseudomonas. The bypass metabolic pathway is one or more other bypass metabolic pathways other than the production of nitropyrrolidone. The engineered bacterium is still Pseudomonas.

[0008] Furthermore, the engineered bacteria is a mutant strain that uses the Pseudomonas as a target, knocks out the core gene in the BGC1 biosynthetic gene cluster, or knocks out the core gene in the BGC16 biosynthetic gene cluster, or knocks out the core genes in both the BGC1 and BGC16 biosynthetic gene clusters by a gene knockout method; the Pseudomonas contains 16 biosynthetic gene clusters, which are named BGC1 to BGC16 in sequence according to their positions in the genome.

[0009] Furthermore, the core gene in the BGC1 biosynthetic gene cluster is located at 533,163bp-534,359bp; the core gene in the BGC16 biosynthetic gene cluster is located at 7,079,833bp-7,080,678bp.

[0010] In a third aspect, the present invention provides a method for constructing the above-mentioned engineered bacteria for producing nitropyrrolidone, taking the Pseudomonas as a target, and knocking out the core gene in the biosynthetic gene cluster therein by a gene knockout method, comprising:

[0011] Knockout of core genes in the BGC1 biosynthetic gene cluster;

[0012] or knocking out the core genes in the BGC16 biosynthetic gene cluster;

[0013] Or simultaneously knock out the core genes in the two biosynthetic gene clusters BGC1 and BGC16.

[0014] Furthermore, the core genes in the biosynthetic gene cluster of the Pseudomonas sp. are knocked out by homologous recombination gene knockout method, including:

[0015] Amplify the upstream and downstream homology arms of the core gene; connect the upstream and downstream homology arms by over l ap PCR fusion; insert the connected arms into the suicide plasmid pK18mobsacB; then introduce the suicide plasmid into the Pseudomonas; and screen the mutant strains.

[0016] Furthermore, when the core genes in the BGC1 biosynthetic gene cluster were knocked out:

[0017] Primers for amplifying the upstream homology arms of the core gene include Pk18-BGC1-upF and Pk18-BGC1-upR, the sequences of which are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively;

[0018] Primers for amplifying the downstream homology arms of the core gene include Pk18-BGC1-dwF and Pk18-BGC1-dwR, the sequences of which are shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively;

[0019] When knocking out the core genes in the BGC16 biosynthetic gene cluster:

[0020] Primers for amplifying the upstream homology arms of the core gene include Pk18-BGC16-upF and Pk18-BGC16-upR, the sequences of which are shown in SEQ ID NO: 10 and SEQ ID NO: 11, respectively;

[0021] Primers for amplifying the downstream homology arms of the core gene include Pk18-BGC16-dwF and Pk18-BGC16-dwR, and their sequences are shown in SEQ ID NO: 12 and SEQ ID NO: 13, respectively.

[0022] In a fourth aspect, the present invention provides a biocontrol agent, comprising the aforementioned Pseudomonas producing pyrrolidone, or the aforementioned engineered bacteria producing pyrrolidone, or the engineered bacteria constructed by the aforementioned method for constructing engineered bacteria producing pyrrolidone. It is understandable that the aforementioned biocontrol agent may also contain other necessary auxiliary materials for agricultural production; the aforementioned biocontrol agent may also be provided in different dosage forms as required. In addition, the biocontrol agent may also include other pest and disease control or other beneficial properties, such as a compound agent or further improvement on the basis of the aforementioned Pseudomonas or engineered bacteria.

[0023] In a fifth aspect, the present invention provides an application of the above-mentioned Pseudomonas producing pyrrolidone, or the above-mentioned engineered bacteria producing pyrrolidone, or the engineered bacteria constructed by the above-mentioned method of constructing an engineered bacteria producing pyrrolidone, or the above-mentioned biocontrol agent, for preparing a preparation for controlling rice sheath blight pathogen or for controlling rice sheath blight pathogen.

[0024] The present invention obtains a bacterium JP2-4390 having a significant inhibitory effect on rice sheath blight by isolating it from the rice rhizosphere, and the bacterium can inhibit rice sheath blight by producing nitropyrrolidone. 16S rDNA sequencing and phylogenetic analysis show that this is a strain of Pseudomonas. With the Pseudomonas as the target, a series of genetic engineering operations are performed to block the bypass metabolic pathway, and an engineered bacterium is constructed, which significantly increases the yield of nitropyrrolidone in the Pseudomonas, which can reach up to 324 mg / L, which is 91.7% higher than the wild-type JP2-4390, and can significantly inhibit rice sheath blight. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] The above is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention, the present invention is further described in detail below in conjunction with the accompanying drawings and specific implementation methods.

[0026] Figure 1This is a diagram showing the inhibitory effect of JP2-4390 on rice sheath blight using the confrontation test method, where A is the blank control and B is the antibacterial experiment of JP2-4390;

[0027] Figure 2 Phylogenetic tree constructed for 16S rDNA of JP2-4390;

[0028] Figure 3 This is a comparison chart of PRN production of wild-type JP2-4390 and its engineered strain; DETAILED DESCRIPTION

[0029] The specific embodiments of the present invention are further described below in conjunction with preferred embodiments. It should be noted that the specific embodiments described herein are only for the purpose of illustrating and explaining the present invention and are not limited to the present invention. Without departing from the spirit and essence of the present invention, modifications or replacements made to the method, steps or conditions of the present invention all belong to the scope of the present invention. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0030] The present invention collects rice rhizosphere samples from the rice experimental field of the Fuyang Base of the Chinese Academy of Rice Research in Fuyang District, Hangzhou, isolates bacteria, and determines the antagonistic ability of the isolated bacteria, in order to obtain biocontrol bacteria with significant inhibitory effects on rice sheath blight, and further aims to provide new research materials for the prevention and control of plant fungal diseases.

[0031] Pathogen: Rice sheath blight pathogen (Rhizoctonia solani) GD118.

[0032] PLA medium formula: 200g / L potato, 20g / L lactose, 20g / L agar powder, natural pH, high temperature sterilization at 115℃ for 15min.

[0033] LB medium formula: 10g / L tryptone, 5g / L yeast powder, 10g / L sodium chloride, natural pH, sterilized at 121℃ for 20min. If it is a solid medium, add 20g / L agar powder before sterilization. If it is LB sucrose medium, add 100g / L sucrose after sterilization.

[0034] Relative inhibition rate (%) = [(control colony radius - treated colony radius) / control colony radius] × 100%

[0035] Example

[0036] 1. Isolation and purification of strains

[0037] Rice roots were randomly selected and dug from the rice planting area of ​​Fuyang Base of China National Rice Research Institute in Fuyang District, Hangzhou. The floating soil on the roots was gently washed with running water, and the soil closely attached to the roots was washed off by ultrasonic vibration. The washed soil suspension was gradiently diluted and applied to the LB culture dish, and the culture dish was placed in a 30℃ constant temperature incubator. Whenever the colonies grew, different forms of colonies were picked and streaked on fresh LB culture medium plates for purification, and then single colonies were picked and stored on LB solid slopes at 4℃ for short-term activity determination. At the same time, the single colony was inoculated with LB liquid, and after shaking and culturing at 30℃ overnight, the bacterial liquid was mixed with 50% sterilized glycerol in equal volumes and then stored at -80℃ for long-term use.

[0038] 2. Determination of the activity of strain JP2-4390 against rice sheath blight

[0039] The plate confrontation method was used: on the one hand, the test strain JP2-4390 was activated by shaking culture in LB liquid medium at 30℃ and 200rpm for 12 hours, and 15μL of culture solution was added to both sides of the center of the PLA medium, 2.5cm away from the center. On the other hand, the rice sheath blight pathogen GD118 was activated by culture at 30℃ for 2 days on PLA solid medium in advance, and a bacterial cake was punched along the edge of the hyphae with a 6mm puncher, and then the hyphae of the bacterial cake was inoculated with the center of the PLA medium inoculated with the test strain JP2-4390 facing down. Three plates were inoculated as replicates.

[0040] The plate inoculated with pathogenic bacteria GD118 but not with the test bacteria JP2-4390 was used as a control. Three plates were inoculated as replicates.

[0041] Figure 1 This is a graph showing the inhibitory effect of JP2-4390 on rice sheath blight pathogen using the confrontation test method, where A is the blank control and B is the antibacterial experiment of JP2-4390. The relative inhibition rate was calculated according to the above relative inhibition rate formula, and the relative inhibition rate of JP2-4390 on rice sheath blight pathogen GD118 was 60.5%.

[0042] 3. 16S rDNA sequencing and sequence analysis of strain JP2-4390

[0043] After the test strain JP2-4390 was activated on the LB plate, a single colony was picked and inoculated into LB liquid medium, and cultured at 30℃ and 200rpm for 12 hours. The bacteria were collected and the genome was extracted using DNAiso Reagent (TAKARA). The 16S rDNA of JP2-4390 was amplified using the bacterial 16S rDNA universal primers 27F and 1492R. The 50μL reaction system included: 2×PCR enzyme Mix 25μL, 27F 1μL, 1492R 1μL, DNA template (50ng / μL, measured by Nanodrop) 1μL, and ddH2O supplemented to 50μL. PCR amplification conditions: 98℃1min pre-denaturation, 98℃30sec, 57℃5sec, 72℃15sec, a total of 30 cycles, 72℃5min extension. It was sent to Shanghai Biotech Co., Ltd. for sequencing, and the resulting sequence is SEQ ID No: 1.

[0044] SEQ ID No:1

[0045] AGAGTTTGATCATGGCTCAGATTGAACGCTGGCGGCAGGCCTAACACATGCAAGTCGAGCGGCA

[0046] GCACGGGTACTTGTACCTGGTGGCGAGCGGCGGACGGGTGAGTAATGCCTAGGAATCTGCCTAG

[0047] TAGTGGGGGATAACGTCCGGAAACGGGCGCTAATACCGCATACGTCCTACGGGAGAAAGTGGGG

[0048] GATCTTCGGACCTCACGCTATTAGATGAGCCTAGGTCGGATTAGCTAGTTGGTGAGGTAATGGC

[0049] TCACCAAGGCGACGATCCGTAACTGGTCTGAGAGGATGATCAGTCACACTGGAACTGAGACACG

[0050] GTCCAGACTCCTACGGGAGGCAGCAGTGGGGAATATTGGACAATGGGCGAAAGCCTGATCCAGC

[0051] CATGCCGCGTGTGTGAAGAAGGTCTTCGGATTGTAAAGCACTTTAAGTTGGGAGGAAGGGCAGT

[0052] TACCTAATACGTGATTGTTTTGACGTTACCGACAGAATAAGCACCGGCTAACTCTGTGCCAGCA

[0053] GCCGCGGTAATACAGAGGGTGCAAGCGTTAATCGGAATTACTGGGCGTAAAGCGCGCGTAGGTG

[0054] GTTTGTTAAGTTGGATGTGAAAGCCCCGGGCTCAACCTGGGAACTGCATCCAAAACTGGCAAGC

[0055] TAGAGTATGGTAGAGGGTGGTGGAATTTCCTGTGTAGCGGTGAAATGCGTAGATATAGGAAGGA

[0056] ACACCAGTGGCGAAGGCGACCACCTGGACTGATACTGACACTGAGGTGCGAAAGCGTGGGGAGC

[0057] AAACAGGATTAGATACCCTGGTAGTCCACGCCGTAAACGATGTCAACTAGCCGTTGGGAGCCTT

[0058] GAGCTCTTAGTGGCGCAGCTAACGCATTAAGTTGACCGCCTGGGGAGTACGGCCGCAAGGTTAA

[0059] AACTCAAATGAATTGACGGGGGCCCGCACAAGCGGTGGAGCATGTGGTTTAATTCGAAGCAACG

[0060] CGAAGAACCTTACCAGGCCTTGACATCCAATGAACTTTCTAGAGATAGATTGGTGCCTTCGGGA

[0061] ACATTGAGACAGGTGCTGCATGGCTGTCGTCAGCTCGTGTCGTGAGATGTTGGGTTAAGTCCCG

[0062] TAACGAGCGCAACCCTTGTCCTTAGTTACCAGCACGTTATGGTGGGCACTCTAAGGAGACTGCC

[0063] GGTGACAAACCGGAGGAAGGTGGGGATGACGTCAAGTCATCATGGCCCTTACGGCCTGGGCTAC

[0064] ACACGTGCTACAATGGTCGGTACAAAGGGTTGCCAAGCCGCGAGGTGGAGCTAATCCCATAAAA

[0065] CCGATCGTAGTCCGGATCGCAGTCTGCAACTCGACTGCGTGAAGTCGGAATCGCTAGTAATCGC

[0066] GAATCAGAATGTCGCGGTGAATACGTTCCCGGGCCTTGTACACACCGCCCGTCACACCATGGGA

[0067] GTGGGTTGCACCAGAAGTAGCTAGTCTAACCTTCGGGAGGACGGTTACCACGGTGTGATTCATG

[0068] ACTGGGGTGAAGTCGTAACAAGGTAGCCGTA

[0069] The 16S rDNA sequence of SEQ ID No: 1 was compared with known Pseudomonas type species and a phylogenetic tree was constructed using MEGA 6.0 software (eg Figure 2 The results showed that the bacteria were clustered in the same branch as the Pseudomonas protegens model species CHA0. Based on this, it can be identified as Pseudomonas protegens.

[0070] 4. Construction of JP2-4390 engineered bacteria

[0071] The primers used for amplification are listed in Table 1 (the underlined parts are homology arms). All plasmids were constructed by homologous recombination.

[0072] According to the analysis of the biosynthetic gene clusters of JP2-4390 by antiSMASH software, the bacterium contains 16 biosynthetic gene clusters, which are named BGC1 to BGC16 in sequence according to their genomic positions, among which BGC9 is the biosynthetic gene cluster of nitropyrrolidone. Nitropyrrolidone is a compound that has a significant inhibitory effect on rice sheath blight. According to the prediction results, the first core genes of BGC1 and BGC16 were amplified 1000bp upstream and downstream as homologous arms, which were fused and connected by over l ap PCR, and connected to the suicide plasmid pK18mobsacB at the XbaI / EcoRI restriction site using the homologous recombination method. The suicide plasmids were introduced into JP2-4390 respectively by the 2.5kV electrotransformation program. Pseudomonas strains containing plasmids were screened in LB medium containing 50μg / ml kanamycin. Subsequently, the Pseudomonas strain containing the plasmid was plated on an LB plate containing 10% (w / v) sucrose, and finally the deletion mutants that could grow on LB plates containing 10% (w / v) sucrose but not on LB plates containing 50 μg / ml kanamycin were screened, and the gene knockout was confirmed by PCR and Sanger sequencing. Finally, the engineered bacteria JP2ΔBGC1 with the BGC1 core gene (533,163bp-534,359bp) knocked out, the engineered bacteria JP2ΔBGC16 with the BGC16 core gene (7,079,833bp-7,080,678bp) knocked out, and the engineered bacteria JP2ΔBGC1 / 16 with the core genes of both the BGC1 and BGC16 gene clusters knocked out were obtained.

[0073] Table 1 List of primers used for amplification

[0074]

[0075]

[0076] The primer sequences from top to bottom in Table 1 correspond to SEQ ID No: 2-SEQ ID No: 17, wherein SEQ ID No: 2-5; 10-13 are primers for amplifying the upstream and downstream homologous arms of the core gene; SEQ ID No: 6-9; 14-17 are primers for verifying whether the gene knockout is successful.

[0077] 5. HPLC analysis

[0078] The crude extract of the fermentation broth was filtered through a 0.22 μm filter membrane and transferred to a sample bottle. HPLC analysis used an Agilent Technologies 1260i nfinity column with a particle size of 3.5 μm, 4.6 mm × 100 mm, chromatographically pure acetonitrile-water solution as the mobile phase, a mobile phase flow rate of 1.0 mL / min, elution for 30 min, a detection wavelength of 210 nm, and an injection volume of 5 μL. Set the method and sequence according to the instrument manual and run the sequence program. The peak area of ​​the nitropyrrolidone standard was compared with that of the sample to be tested to obtain the nitropyrrolidone production of JP2ΔBGC1, JP2ΔBGC16, JP2ΔBGC1 / 16 and wild-type JP2-4390. Figure 3 As shown, the results showed that after fermentation, the production of nitropyrrolidone in JP2ΔBGC1, JP2ΔBGC16 and JP2ΔBGC1 / 16 was increased by 46.7%, 70.3% and 91.7% compared with the wild type JP2-4390, which were 247 mg / L, 287 mg / L and 324 mg / L, respectively.

[0079] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Those skilled in the art may make some simple modifications, equivalent changes or modifications using the technical contents disclosed above, which all fall within the protection scope of the present invention.

Claims

1. A Pseudomonas protegens for producing nitropyrrolidone, characterized in that: It is deposited in China Center for Type Culture Collection with the accession number CCTCC M 20241968.

2. An engineered bacterium for producing nitropyrrolidone, characterized in that: The engineered bacteria is a mutant strain that blocks the Pseudomonas bypass metabolic pathway described in claim 1.

3. The engineered bacteria for producing nitropyrrolidine according to claim 2, characterized in that: The engineered bacteria is a mutant strain that uses the Pseudomonas as a target and knocks out the core gene in the BGC1 biosynthetic gene cluster, or knocks out the core gene in the BGC16 biosynthetic gene cluster, or knocks out the core genes in both the BGC1 and BGC16 biosynthetic gene clusters at the same time through a gene knockout method; The Pseudomonas contains 16 biosynthetic gene clusters, which are named BGC1 to BGC16 according to their locations in the genome.

4. The engineered bacteria for producing nitropyrrolidine according to claim 3, characterized in that: The core gene in the BGC1 biosynthetic gene cluster is located at 533,163bp-534,359bp; the core gene in the BGC16 biosynthetic gene cluster is located at 7,079,833bp-7,080,678bp.

5. A method for constructing an engineered bacterium for producing nitropyrrolidine according to any one of claims 2 to 4, characterized in that: The Pseudomonas is used as a target, and the core genes in the biosynthetic gene cluster are knocked out by a gene knockout method, including: Knockout of core genes in the BGC1 biosynthetic gene cluster; or knocking out the core genes in the BGC16 biosynthetic gene cluster; Or knock out the core genes in BGC1 and BGC16 simultaneously.

6. The method for constructing an engineered bacterium for producing nitropyrrolidine according to claim 5, characterized in that: The core genes in the biosynthetic gene cluster of the Pseudomonas sp. are knocked out by homologous recombination gene knockout method, including: Amplify the upstream and downstream homology arms of the core gene; connect the upstream and downstream homology arms by overlap PCR; insert the connected homology arms into the suicide plasmid pK18mobsacB; then introduce the suicide plasmid into the Pseudomonas; and screen the mutant strains.

7. The method for constructing an engineered bacterium for producing nitropyrrolidine according to claim 6, characterized in that: When knocking out the core genes in the BGC1 biosynthetic gene cluster: Primers for amplifying the upstream homology arms of the core gene include Pk18-BGC1-upF and Pk18-BGC1-upR, the sequences of which are shown in SEQ ID NO: 2 and SEQ ID NO: 3, respectively; Primers for amplifying the downstream homology arms of the core gene include Pk18-BGC1-dwF and Pk18-BGC1-dwR, the sequences of which are shown in SEQ ID NO: 4 and SEQ ID NO: 5, respectively; When knocking out the core genes in the BGC16 biosynthetic gene cluster: Primers for amplifying the upstream homology arms of the core gene include Pk18-BGC16-upF and Pk18-BGC16-upR, the sequences of which are shown in SEQ ID NO: 10 and SEQ ID NO: 11, respectively; Primers for amplifying the downstream homology arms of the core gene include Pk18-BGC16-dwF and Pk18-BGC16-dwR, and their sequences are shown in SEQ ID NO: 12 and SEQ ID NO: 13, respectively.

8. A biocontrol agent, characterized in that: The invention relates to an engineered bacterium comprising the Pseudomonas for producing nitropyrrolidine according to claim 1, or the engineered bacterium for producing nitropyrrolidine according to any one of claims 2 to 4, or an engineered bacterium constructed by the construction method of the engineered bacterium for producing nitropyrrolidine according to any one of claims 5 to 7.

9. Use of the Pseudomonas producing pyrrolidone according to claim 1, the engineered bacteria producing pyrrolidone according to any one of claims 2-4, or the engineered bacteria constructed by the construction method of the engineered bacteria producing pyrrolidone according to any one of claims 5-7, or the biocontrol agent according to claim 8, characterized in that: Used for preparing preparations for controlling rice sheath blight pathogen or for controlling rice sheath blight pathogen.

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