Method for high-yield production of phenazine-1-formamide by using pseudomonas

Abstract

The invention belongs to the field of genetic engineering, and provides a genetic engineering strain for high yield of phenazine-1-formamide by using pseudomonas, and the genetic engineering strain is obtained by taking a strain QPCN-6 as an original strain and knocking out a gspM gene or continuously knocking out at least one of an araG gene and a pstA gene. According to the invention, a derivative strain QPCN-6 of Qlu-1 in ZL 202011024026.1 is used as an original strain, and the strain is modified through molecular biology and genetic engineering methods, so that the synthesis efficiency of the strain PCN is improved.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering and relates to a method for highly producing phenazine-1-carboxamide using Pseudomonas. Background Art

[0002] Disclosing the information of this background art section is only intended to enhance the understanding of the overall background of the present invention, and it is not necessarily regarded as an admission or an implication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Chemically synthesized pesticides that have been used since modern times are crucial for the high and stable yields of agricultural crops. However, the extensive use of chemically synthesized pesticides that are not easily degraded easily causes pesticide residues in grains, fruits, and vegetables, as well as ecological environmental pollution, which does not conform to the trend of sustainable development in today's society. Biopesticides are a type of bioactive substance derived from organisms or produced by organisms. Compared with the currently widely used chemically synthesized pesticides, biopesticides have obvious advantages such as low toxicity, environmental friendliness, easy degradation, and low resistance generation, and have attracted the attention of more and more scholars and are gradually applied to biological control practices. Among them, phenazine substances produced by Pseudomonas and Streptomyces are representatives of a typical type of biopesticide.

[0004] Phenazine-1-carboxamide is a nitrogen-containing heterocyclic bioactive substance and has good control effects on fungal diseases of different crops. For example, previous experiments have shown that phenazine-1-carboxamide can be used for wheat scab, wheat root rot, rice sheath blight, rice blast, pepper blight, melon gummy stem blight, etc., and its control efficacy for some diseases is better than that of the similar phenazine-1-carboxylic acid (the active ingredient of which is phenazine-1-carboxylic acid), so it is a potential new biopesticide.

[0005] At present, although there are chemical methods for producing phenazine-1-carboxamide, the conditions are harsh and the synthesis efficiency is low. The inventor's previous patent ZL 202011024026.1 prepared a strain QPCN-6 with high production of phenazine-1-carboxamide. On this basis, in order to reduce production costs and make the product more competitive in the market, the yield of the engineered strain still needs to be improved. Summary of the Invention

[0006] To solve the above problems, the present invention provides a method for highly producing phenazine-1-carboxamide using Pseudomonas. The present invention uses the derivative strain QPCN-6 of Qlu-1 in ZL 202011024026.1 as the starting strain, and modifies the strain through molecular biology and genetic engineering methods to improve the synthesis efficiency of strain PCN.

[0007] To achieve the above object, the present invention adopts the following technical solutions:

[0008] In the first aspect of the present invention, a genetically engineered strain for high-yield production of phenazine-1-carboxamide using Pseudomonas is provided. Starting from strain QPCN-6, the gspM gene is knocked out, or at least one of the araG gene and the pstA gene is further knocked out to obtain the strain.

[0009] Qlu-1 is a Pseudomonas chlororaphis strain screened from rhizosphere soil of plants, which has a phzABCDEFG gene cluster for synthesizing phenazine substances and can produce phenazine-1-carboxylic acid, a precursor substance of phenazine-1-carboxamide. In the present invention, the derivative strain QPCN-6 of Qlu-1 is used as the starting strain and is transformed by genetic engineering and molecular biology methods to improve the yield of phenazine-1-carboxamide, laying a foundation for the future promotion of phenazine-1-carboxamide.

[0010] In some embodiments, the sequence of the gspM gene is as shown in SEQ ID NO.1.

[0011] The primers for knocking out the gspM gene (primers for obtaining the gspM-UD fusion fragment) include: gspM-F1 / gspM-R1, gspM-F2 / gspM-R2.

[0012] Among them, the sequence of gspM-F1 is as shown in SEQ ID NO.4.

[0013] The sequence of gspM-R1 is as shown in SEQ ID NO.5.

[0014] The sequence of gspM-F2 is as shown in SEQ ID NO.6.

[0015] The sequence of gspM-R2 is as shown in SEQ ID NO.7.

[0016] The sequence of the araG upstream and downstream fusion fragment is as shown in SEQ ID NO.8.

[0017] In some embodiments, the sequence of the araG gene is as shown in SEQ ID NO.2.

[0018] The primers for knocking out the AraG gene (primers for obtaining the AraG-UD fusion fragment) include: AraG-F1 / AraG-R1, AraG-F2 / AraG-R2.

[0019] Among them, the sequence of AraG-F1 is as shown in SEQ ID NO.9.

[0020] The sequence of AraG-R1 is as shown in SEQ ID NO.10.

[0021] The sequence of AraG-F2 is as shown in SEQ ID NO.11.

[0022] The sequence of AraG-R2 is shown in SEQ ID NO.12.

[0023] The sequence of the fusion fragment upstream and downstream of gspM is shown in SEQ ID NO.13.

[0024] In some embodiments, the sequence of the pstA gene is shown in SEQ ID NO.3.

[0025] The pstA gene knockout primers (primers for obtaining the pstA-UD fusion fragment) include: pstA-F1 / pstA-R1, pstA-F2 / pstA-R2.

[0026] Among them, the sequence of pstA-F1 is shown in SEQ ID NO.14.

[0027] The sequence of pstA-R1 is shown in SEQ ID NO.15.

[0028] The sequence of pstA-F2 is shown in SEQ ID NO.16.

[0029] The sequence of pstA-R2 is shown in SEQ ID NO.17.

[0030] The sequence of the fusion fragment upstream and downstream of pstA is shown in SEQ ID NO.18.

[0031] In the second aspect of the present invention, a method for constructing a genetically engineered strain of Pseudomonas for high-yield production of phenazine-1-carboxamide is provided, including:

[0032] Using strain QPCN-6 as the starting strain, knocking out the gspM gene to obtain strain QPCN-6-1;

[0033] Or, continuously knocking out the araG gene to obtain strain QPCN-6-2;

[0034] Or, continuously knocking out the pstA gene to obtain strain QPCN-6-3, thus obtaining the strain.

[0035] In some embodiments, the step of knocking out the gspM gene includes:

[0036] i. Amplifying the upstream and downstream homologous arms of the gspM gene fragment; connecting the upstream and downstream homologous arms by fusion PCR and inserting them into a plasmid to obtain a gspM gene recombinant plasmid;

[0037] ii. Introducing the gspM gene recombinant plasmid into Escherichia coli and performing parental hybridization culture with strain QPCN-6, thereby introducing the gspM gene recombinant plasmid into strain QPCN-6;

[0038] iii. Screen positive clones to obtain strain QPCN-6-1.

[0039] In some embodiments, the step of knocking out the araG gene includes:

[0040] i. Amplify the upstream and downstream homologous arms of the araG gene fragment; connect the upstream and downstream homologous arms by fusion PCR and insert them into a plasmid to obtain an araG gene recombinant plasmid;

[0041] ii. After introducing the araG gene recombinant plasmid into Escherichia coli, perform biparental hybridization culture with strain QPCN-6-1, thereby introducing the araG gene recombinant plasmid into strain QPCN-6-1;

[0042] iii. Screen positive clones to obtain strain QPCN-6-2.

[0043] In some embodiments, the step of knocking out the pstA gene includes:

[0044] i. Amplify the upstream and downstream homologous arms of the pstA gene fragment; connect the upstream and downstream homologous arms by fusion PCR and insert them into a plasmid to obtain a pstA gene recombinant plasmid;

[0045] ii. After introducing the pstA gene recombinant plasmid into Escherichia coli, perform biparental hybridization culture with strain QPCN-6-2, thereby introducing the pstA gene recombinant plasmid into strain QPCN-6-2;

[0046] iii. Screen positive clones to obtain strain QPCN-6-3.

[0047] The third aspect of the present invention provides strain QPCN-6-1, strain QPCN-6-2 or strain QPCN-6-3 prepared by the above method.

[0048] The fourth aspect of the present invention provides a method for highly producing phenazine-1-carboxamide using Pseudomonas, including:

[0049] Inoculate the above strain QPCN-6-1, strain QPCN-6-2 or strain QPCN-6-3 into a fermentation medium to produce phenazine-1-carboxamide.

[0050] The present invention ferments using KB medium, and the yield reaches 10936 mg / L, greatly improving the production capacity of the strain and providing a solid foundation for the industrialization of subsequent engineering strains.

[0051] Advantages of the present invention

[0052] (1) The present invention obtains an engineered strain named QPCN-6-1 by seamlessly knocking out the gspM gene. After fermentation and production measurement, it is found that the PCN production of QPCN-6-1 is increased to 7414 mg / L. On the basis of QPCN-6-1, the present invention knocks out the araG gene to obtain the strain QPCN-6-2. After production measurement, its phenazine-1-carboxamide production is increased to 8948 mg / L at 60 h. In the strain QPCN-6-2, the pstA gene is knocked out to obtain the strain QPCN-6-3. After production measurement, the strain PCN production reaches 10936 mg / L. The present invention achieves the improvement of the strain phenazine-1-carboxamide production by modifying the genome of the strain.

[0053] (2) The method of the present invention is simple, practical and easy to promote. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention, and the exemplary embodiments of the present invention and their description are used to explain the present invention and do not constitute improper limitations on the present invention.

[0055] Figure 1 The yield of phenazine-1-carboxamide during fermentation of different strains.

[0056] Figure 2 Electrophoresis diagram for mutant plasmid pK18-gspM-ud, (A) Amplification of gspM upstream and downstream homology arm fragments: 1, gspM upstream homology arm amplification; 2, DNALadder DL5000; 3, gspM downstream homology arm fragment amplification; (B) Amplification of gspM upstream and downstream homology arm fusion fragments: 1, gspM upstream and downstream homology arm fusion fragments; 2, DNALadder DL5000; 3, gspM upstream and downstream homology arm fusion fragments.

[0057] Figure 3 This is the PCR verification diagram of the gspM knockout strain. External primer detection: 1, using the genome of the gspM gene knockout strain QPCN-6-1 as a template to amplify the fragment; 2, DNA Ladder DL2000; 3, using the QPCN-6 genome as a template to amplify the fragment; 4, blank control; internal primer detection: 1, DNALadder DL2000; 2, using the QPCN-6 genome as a template to amplify the fragment; 3, using the genome of the gspM gene knockout strain QPCN-6-1 as a template to amplify the fragment; 4, blank control. DETAILED DESCRIPTION

[0058] It should be noted that the following detailed description is exemplary and aims to provide further explanation of the present invention. Unless otherwise specified, all technical and scientific terms used in the present invention have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0059] The present invention will be further described in detail below in conjunction with specific embodiments. It should be noted that the specific embodiments are interpretations rather than limitations of the present invention.

[0060] Example 1

[0061] Taking the introduction of the gspM gene into the genome as an example, gene manipulation will be introduced.

[0062] 1. Inoculate Pseudomonas chlororaphis QPCN-6 into KB medium (pH 7.0) at an inoculation amount of 1%, and perform shaking culture overnight at 30 °C with a shaking speed of 180 rpm. Extract the genome of Pseudomonas chlororaphis QPCN-6 using a prokaryotic genome extraction kit and store it at -20 °C for later use.

[0063] 2. Search for the gspM gene and its upstream and downstream sequences in the sequenced genome data of Pseudomonas chlororaphis Qlu-1. Using the genome of Pseudomonas chlororaphis QPCN-6 as a template, and using gspM-F1 / gspM-R1 and gspM-F2 / gspM-R2 as primers, amplify the gspM upstream sequence gspM-U and the gspM downstream sequence gspM-D respectively; using gspM-U and gspM-D as templates and gspM-F1 / gspM-R2 as primers, connect the gspM-U and gspM-D fragments by fusion PCR to obtain the fusion fragment gspM-UD.

[0064] 3. Connect the fusion fragment gspM-UD with the knockout plasmid pk18moBsacB by enzymatic digestion and ligation to construct the recombinant plasmid pk18-gspM-UD.

[0065] 4. Introduce the recombinant plasmid pk18-gspM-UD into Escherichia coli S17-1(λpir) by heat shock transformation.

[0066] 5. Perform parental hybridization culture on Escherichia coli S17-1(λpir) and Pseudomonas chlororaphis QPCN-6, and introduce the recombinant plasmid pk18-gspM-UD into Pseudomonas chlororaphis QPCN-6.

[0067] 6. Obtain the gene mutant strain QPCN-6-1 through sucrose plate screening and replica screening.

[0068] 7. Verify the gspM knockout strain QPCN-6-1 of QPCN-6 by PCR verification method.

[0069] 8. After fermentation, phenazine-1-carboxamide in the fermentation broth was extracted. It was found by HPLC detection that the yield of phenazine-1-carboxamide of the strain increased to 7414 mg / L.

[0070] Example 2

[0071] By the same method, the araG gene and the pstA gene were successively knocked out in the QPCN-6 genome to obtain strain QPCN-6-2 and strain QPCN-6-3 respectively.

[0072] Example 3

[0073] Strains QPCN-6-1, QPCN-6-2, and QPCN-6-3 were respectively inoculated into KB medium (pH 7.0) at an inoculation amount of 1%, cultured and fermented at 30 °C, extracted with ethyl acetate and detected by HPLC. It was found that: the PCN yield of strain QPCN-6-1 increased to 7414 mg / L at 60 h, the PCN yield of strain QPCN-6-2 increased to 8948 mg / L at 60 h, and strain QPCN-6-3 could produce 10936 of phenazine-1-carboxamide at 60 h.

[0074] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A genetically engineered strain for high production of phenazine-1-carboxamide using Pseudomonas sp., characterized in that: The strain QPCN-6 is used as the starting strain, and the gspM gene is knocked out, or at least one of the araG gene and the pstA gene is further knocked out.

2. The genetically engineered strain for high production of phenazine-1-carboxamide using Pseudomonas as claimed in claim 1, characterized in that: The sequence of the gspM gene is shown in SEQ ID NO.

1.

3. The genetically engineered strain for high production of phenazine-1-carboxamide using Pseudomonas as claimed in claim 1, characterized in that: The sequence of the araG gene is shown in SEQ ID NO.

2.

4. The genetically engineered strain for high production of phenazine-1-carboxamide using Pseudomonas as claimed in claim 1, characterized in that: The sequence of the pstA gene is shown in SEQ ID NO.

3.

5. A method for constructing a genetically engineered strain for high production of phenazine-1-carboxamide using Pseudomonas sp., characterized in that: include: Using strain QPCN-6 as the starting strain, the gspM gene was knocked out to obtain strain QPCN-6-1; Or, continue to knock out the araG gene to obtain strain QPCN-6-2; Alternatively, continue to knock out the pstA gene to obtain strain QPCN-6-3.

6. The method for constructing a genetically engineered strain for high production of phenazine-1-carboxamide using Pseudomonas as claimed in claim 5, characterized in that: The step of knocking out the gspM gene comprises: i. Amplify the upstream and downstream homology arms of the gspM gene fragment; connect the upstream and downstream homology arms by fusion PCR and insert them into the plasmid to obtain a gspM gene recombinant plasmid; ii. After the gspM gene recombinant plasmid is introduced into Escherichia coli, the recombinant plasmid is cultured with the strain QPCN-6 to introduce the gspM gene recombinant plasmid into the strain QPCN-6; iii. Screen the positive clones to obtain strain QPCN-6-1.

7. The method for constructing a genetically engineered strain for high production of phenazine-1-carboxamide using Pseudomonas as claimed in claim 5, characterized in that: The step of knocking out the araG gene comprises: i. Amplify the upstream and downstream homology arms of the araG gene fragment; connect the upstream and downstream homology arms by fusion PCR and insert them into the plasmid to obtain the araG gene recombinant plasmid; ii. After the araG gene recombinant plasmid is introduced into Escherichia coli, the recombinant plasmid is cultured with the strain QPCN-6-1 to introduce the araG gene recombinant plasmid into the strain QPCN-6-1; iii. Screen the positive clones to obtain strain QPCN-6-2.

8. The method for constructing a genetically engineered strain for high production of phenazine-1-carboxamide using Pseudomonas as claimed in claim 5, characterized in that: The step of knocking out the pstA gene comprises: i. Amplify the upstream and downstream homologous arms of the pstA gene fragment; connect the upstream and downstream homologous arms by fusion PCR and insert them into the plasmid to obtain a pstA gene recombinant plasmid; ii. After the pstA gene recombinant plasmid is introduced into Escherichia coli, the pstA gene recombinant plasmid is cultured with the strain QPCN-6-2 to introduce the pstA gene recombinant plasmid into the strain QPCN-6-2; iii. Screen the positive clones to obtain strain QPCN-6-3.

9. Strain QPCN-6-1, strain QPCN-6-2 or strain QPCN-6-3 prepared by the method according to any one of claims 5 to 8.

10. A method for high production of phenazine-1-carboxamide using Pseudomonas sp., characterized in that: include: The strain QPCN-6-1, strain QPCN-6-2 or strain QPCN-6-3 according to claim 9 is inoculated into a fermentation medium to produce phenazine-1-carboxamide.

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