Method for improving production efficiency of phenazine-1-formamide

Abstract

The invention belongs to the field of gene engineering, and provides a method for improving the production efficiency of phenazine-1-formamide, which comprises the following steps: by taking a strain QPCN-1 as an original strain, knocking out shlA gene, Aapj gene and punA gene to obtain a strain QPCN [delta] SAP. According to the invention, QPCN-1 derived from pseudomonas chlororaphis Qlu-1 in ZL202011024026.1 is used as an original strain, shlA gene is knocked out by using a traceless knockout method to obtain QPCN delta S, and the yield of PCN reaches 1173.5 mg / L through HPLC detection after fermentation; according to the present invention, shlA and punA are successively knocked out on the basis of QPCN [delta] S to obtain strains QPCN [delta] SA and QPCN [delta] SAP, such that the yield of phenazine-1 formamide of the initial strain is increased to 6035.6 mg / L from 3579.3 mg / L.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering and relates to a method for improving the production efficiency of phenazine-1-carboxamide. 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 indication in any form that this information constitutes the prior art already known to those of ordinary skill in the art.

[0003] Using pesticides has always been an important means for stable agricultural production and increased yields. However, most of the pesticides currently on the market are produced by chemical synthesis methods under harsh conditions. Currently, chemical synthetic pesticides are indispensable for ensuring crop production and increasing yields. However, their production processes are often energy-intensive and produce toxic and harmful substances, and the pesticides applied to farmland are often not easily degraded, resulting in serious pesticide residues and ecological environmental pollution. Therefore, finding alternatives to chemical pesticides has become an important research content for current scientific and technological workers. Biogenic pesticides are a class of bioactive substances naturally synthesized by organisms, which not only have the same control effect on crop pests and diseases as chemical pesticides, but also often have the characteristic of being easily biodegradable by organisms. Therefore, biogenic pesticides are important alternatives to future chemical synthetic pesticides.

[0004] Phenazine-1-carboxamide (PCN) is a bioactive substance existing in nature and can be produced by Pseudomonas spp., Streptomyces spp., etc. in the form of stimulatory metabolites. Similar to phenazine mycin (the active ingredient is 1% phenazine-1-carboxylic acid), which is also a phenazine substance, it has good control effects on wheat scab, rice blast, rice sheath blight, watermelon fusarium wilt, pepper blight, etc. It is one of the biopesticides for future development.

[0005] Currently, the research results in the academic community show that PCN is mainly produced by Pseudomonas spp. such as Pseudomonas chlororaphis and Pseudomonas aeruginosa. It is obtained by the catalysis of phenazine-1-carboxylic acid in the strain by the PhzH enzyme.

[0006] The inventor's previous patent ZL202011024026.1 disclosed a genetically engineered strain that can produce PCN. Starting from the strain Qlu-1, the yield of phenazine-1-carboxamide of the strain was increased to 5787.2 mg / L through genetic engineering operations. However, its yield still needs to be improved to better reduce the production cost of phenazine-1-carboxamide and provide a firm foundation for the popularization and application of phenazine-1-carboxamide. Summary of the Invention

[0007] To solve the above problems, the present invention provides a method for improving the production efficiency of phenazine-1-carboxamide. The present invention uses QPCN-1 (a derivative strain of Qlu-1) as the starting strain, and through genetic engineering operations, the phenazine-1-carboxamide production of the strain has been increased to 6035.6 mg / L.

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] In the first aspect of the present invention, there is provided a genetically engineered bacterium for improving the production efficiency of phenazine-1-carboxamide. Using the strain QPCN-1 as the starting strain, the shlA gene, Aapj gene, and punA gene are knocked out to obtain the strain QPCNΔSAP.

[0010] The strain QPCN-1 of the present invention is a derivative strain of Qlu-1 and is publicly disclosed in ZL202011024026.1.

[0011] In some embodiments, the sequence of the shlA gene is as shown in SEQ ID NO.1.

[0012] The shlA gene knockout primers (primers for obtaining the shlA-UD fusion fragment) include: shlA-F1 / shlA-R1, shlA-F2 / shlA-R2.

[0013] Among them, the sequence of shlA-F1 is as shown in SEQ ID NO.4.

[0014] The sequence of shlA-R1 is as shown in SEQ ID NO.5.

[0015] The sequence of shlA-F2 is as shown in SEQ ID NO.6.

[0016] The sequence of shlA-R2 is as shown in SEQ ID NO.7.

[0017] The sequence of the fusion fragment (shlA-UD fusion fragment) used for knockout is as shown in SEQ ID NO.8.

[0018] In some embodiments, the sequence of the Aapj gene is as shown in SEQ ID NO.2.

[0019] The Aapj gene knockout primers (primers for obtaining the Aapj-UD fusion fragment) include: Aapj-F1 / Aapj-R1, Aapj-F2 / Aapj-R2.

[0020] Among them, the sequence of Aapj-F1 is as shown in SEQ ID NO.9.

[0021] The sequence of Aapj-R1 is shown in SEQ ID NO.10.

[0022] The sequence of Aapj-F2 is shown in SEQ ID NO.11.

[0023] The sequence of Aapj-R2 is shown in SEQ ID NO.12.

[0024] The sequence of the fusion fragment (AapJ-UD fusion fragment) for importing into the genome is shown in SEQ ID NO.13.

[0025] In some embodiments, the sequence of the punA gene is shown in SEQ ID NO.3.

[0026] The punA gene knockout primers (primers for obtaining the punA-UD fusion fragment) include: punA-F1 / punA-R1, punA-F2 / punA-R2.

[0027] Among them, the sequence of punA-F1 is shown in SEQ ID NO.14.

[0028] The sequence of punA-R1 is shown in SEQ ID NO.15.

[0029] The sequence of punA-F2 is shown in SEQ ID NO.16.

[0030] The sequence of punA-R2 is shown in SEQ ID NO.17.

[0031] The sequence of the fusion fragment (punA-UD fusion fragment) for importing into the genome is shown in SEQ ID NO.18.

[0032] In the second aspect of the present invention, a method for constructing a genetically engineered bacterium for improving the production efficiency of phenazine-1-carboxamide is provided, including:

[0033] Using strain QPCN-1 as the starting strain, knocking out the shlA gene to obtain strain QPCNΔS;

[0034] Continuing to knock out the Aapj gene to obtain strain QPCNΔSA;

[0035] Continuing to knock out the punA gene to obtain strain QPCNΔSAP, thus obtained.

[0036] In some embodiments, the specific steps for knocking out the shlA gene include:

[0037] i. Amplify the upstream and downstream homologous arms of the shlA gene fragment; use fusion PCR to connect the upstream and downstream homologous arms and insert them into a plasmid to obtain the shlA gene recombinant plasmid;

[0038] ii. After introducing the shlA gene recombinant plasmid into Escherichia coli, perform a biparental hybridization culture with strain QPCN-1 to introduce the shlA gene recombinant plasmid into strain QPCN-1.

[0039] iii. Screen for positive clones to obtain strain QPCNΔS.

[0040] In some embodiments, the specific steps for knocking out the Aapj gene include:

[0041] i. Amplify the upstream and downstream homologous arms of the Aapj gene fragment; use fusion PCR to ligate the upstream and downstream homologous arms and insert them into a plasmid to obtain the Aapj gene recombinant plasmid.

[0042] ii. After introducing the Aapj gene recombinant plasmid into Escherichia coli, perform a biparental hybridization culture with strain QPCNΔS to introduce the Aapj gene recombinant plasmid into strain QPCNΔS.

[0043] iii. Screen for positive clones to obtain strain QPCNΔSA.

[0044] In some embodiments, the specific steps for knocking out the punA gene include:

[0045] i. Amplify the upstream and downstream homologous arms of the punA gene fragment; use fusion PCR to ligate the upstream and downstream homologous arms and insert them into a plasmid to obtain the punA gene recombinant plasmid.

[0046] ii. After introducing the punA gene recombinant plasmid into Escherichia coli, perform a biparental hybridization culture with strain QPCNΔSA to introduce the punA gene recombinant plasmid into strain QPCNΔSA.

[0047] iii. Screen for positive clones to obtain strain QPCNΔSAP.

[0048] In the third aspect of the present invention, there is provided strain QPCNΔSAP prepared by the above method.

[0049] In the fourth aspect of the present invention, there is provided the application of the above strain QPCNΔSAP in the production of phenazine-1-carboxamide, including:

[0050] Inoculate strain QPCNΔSAP into a fermentation medium to produce phenazine-1-carboxamide.

[0051] Advantages of the present invention

[0052] (1) The present invention uses strain QPCNΔSAP to ferment in KB medium, and the yield reaches 6035.6 mg / L, improving the production efficiency of the strain and providing a solid foundation for the industrialization of subsequent engineering strains.

[0053] (2) The present invention uses QPCN-1 derived from Pseudomonas chlororaphis Qlu-1 in ZL202011024026.1 as the starting strain, and obtains QPCNΔS by knocking out the shlA gene using the scarless knockout method. After fermentation, the PCN yield reaches 1173.5 mg / L detected by HPLC; successively, on the basis of QPCNΔA, shlA and punA are knocked out to obtain strains QPCNΔSA and QPCNΔSAP, increasing the yield of phenazine-1-carboxamide of the initial strain from 3579.3 mg / L to 6035.6 mg / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] The specification drawings forming a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention.

[0055] Figure 1 It is the yield of phenazine-1-carboxamide when different strains are fermented for 60 h.

[0056] Figure 2 It is the electrophoresis diagram of the construction of the mutant plasmid pK18-shlA-ud. (A) Amplification of the upstream and downstream homologous arms of shlA: 1, amplification of the upstream homologous arm of shlA; 2, amplification of the upstream homologous arm of shlA; 3, DNA Ladder DL5000; 4, amplification of the downstream homologous arm fragment of shlA; 5, amplification of the downstream homologous arm fragment of shlA; (B) Amplification of the fusion fragment of the upstream and downstream homologous arms of shlA: 1, fusion fragment of the upstream and downstream homologous arms of shlA; 2, fusion fragment of the upstream and downstream homologous arms of shlA; 3, DNA Ladder DL5000; 4, fusion fragment of the upstream and downstream homologous arms of shlA; 5, fusion fragment of the upstream and downstream homologous arms of shlA.

[0057] Figure 3 It is the PCR verification diagram of the shlA knockout strain. External primer detection: 1, amplification of the fragment using the QPCN-1 genome as a template; 2, DNA Ladder DL5000; 3, amplification of the fragment using the genome of the shlA gene knockout strain QPCN△S as a template; 4, blank control; Internal primer detection: 1, amplification of the fragment using the genome of the shlA gene knockout strain QPCN△S as a template; 2, amplification of the fragment using the QPCN-1 genome as a template; 3, DNA Ladder DL5000; 4, blank control. DETAILED DESCRIPTION OF THE INVENTION

[0058] It should be noted that the following detailed description is exemplary and is intended to provide further illustration 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 pertains.

[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 explanations rather than limitations of the present invention.

[0060] Example 1

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

[0062] 1. Inoculate Pseudomonas chlororaphis Qlu-1 into KB medium (pH = 7) at an inoculation amount of 1%, and culture it overnight with shaking at 180 rpm at 30°C. Extract the genome of Pseudomonas chlororaphis Qlu-1 using a prokaryotic genome extraction kit and store it at -20°C for later use.

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

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

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

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

[0067] 6. Obtain the gene knockout strain QPCN△S through sucrose plate screening and replica screening.

[0068] 7. Verify the shlA knockout strain QPCN△S of QPCN-1 by PCR verification method.

[0069] 8. Inoculate strain QPCN△S into KB medium (pH 7.0) at an inoculation amount of 1%, culture and ferment at 30°C, extract phenazine-1-carboxamide from the fermentation broth, and detect by HPLC. It is found that the yield of phenazine-1-carboxylic acid of the strain after 60 h is increased to 1173.5 mg / L.

[0070] Example 2

[0071] Knock out Aapj and punA successively in the QPCN-1 genome by the same method to obtain QPCNΔSA and QPCNΔSAP in sequence. Inoculate strains QPCNΔSA and QPCNΔSAP into KB medium (pH 7.0) at an inoculation amount of 1% respectively, culture and ferment at 30°C, extract PCN with ethyl acetate and detect by HPLC. It is found that the yields of phenazine-1-carboxamide of strains QPCNΔSA and QPCNΔSAP are 3579.3 mg / L and 6035.6 mg / L respectively.

[0072] The above are only the preferred embodiments of the present invention and are not used 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 bacterium for improving the production efficiency of phenazine-1-carboxamide, characterized in that: Using strain QPCN-1 as the starting strain, the shlA gene, Aapj gene, and punA gene were knocked out to obtain strain QPCNΔSAP.

2. The genetically engineered bacteria for improving the production efficiency of phenazine-1-carboxamide according to claim 1, characterized in that: The sequence of the shlA gene is shown in SEQ ID NO.

1.

3. The genetically engineered bacteria for improving the production efficiency of phenazine-1-carboxamide according to claim 1, characterized in that: The sequence of the Aapj gene is shown in SEQ ID NO.

2.

4. The genetically engineered bacteria for improving the production efficiency of phenazine-1-carboxamide according to claim 1, characterized in that: The sequence of the punA gene is shown in SEQ ID NO.

3.

5. A method for constructing a genetically engineered bacterium for improving the production efficiency of phenazine-1-carboxamide, characterized in that: include: Using strain QPCN-1 as the starting strain, the shlA gene was knocked out to obtain strain QPCNΔS; The Aapj gene was further knocked out to obtain the strain QPCNΔSA; Continue to knock out the punA gene to obtain the strain QPCNΔSAP.

6. The method for constructing a genetically engineered bacterium for improving the production efficiency of phenazine-1-carboxamide according to claim 5, characterized in that: The specific steps of knocking out the shlA gene include: i. Amplifying the upstream and downstream homology arms of the shlA gene fragment; connecting the upstream and downstream homology arms by fusion PCR and inserting them into the plasmid to obtain the shlA gene recombinant plasmid; ii. After the shlA gene recombinant plasmid is introduced into Escherichia coli, the recombinant plasmid is cultured with the strain QPCN-1 to introduce the shlA gene recombinant plasmid into the strain QPCN-1; iii. Screen the positive clones to obtain the strain QPCNΔS.

7. The method for constructing a genetically engineered bacterium for improving the production efficiency of phenazine-1-carboxamide according to claim 5, characterized in that: The specific steps of knocking out the Aapj gene include: i. Amplify the upstream and downstream homology arms of the Aapj gene fragment; connect the upstream and downstream homology arms by fusion PCR and insert them into the plasmid to obtain the Aapj gene recombinant plasmid; ii. After the Aapj gene recombinant plasmid is introduced into Escherichia coli, biparental hybridization is performed with the strain QPCNΔS, thereby introducing the Aapj gene recombinant plasmid into the strain QPCNΔS; iii. Screen the positive clones to obtain the strain QPCNΔSA.

8. The method for constructing a genetically engineered bacterium for improving the production efficiency of phenazine-1-carboxamide according to claim 5, characterized in that: The specific steps of knocking out the punA gene include: i. Amplify the upstream and downstream homology arms of the punA gene fragment; connect the upstream and downstream homology arms by fusion PCR and insert them into the plasmid to obtain the punA gene recombinant plasmid; ii. After the punA gene recombinant plasmid is introduced into Escherichia coli, biparental hybridization culture is performed with the strain QPCNΔSA, thereby introducing the punA gene recombinant plasmid into the strain QPCNΔSA; iii. Screen the positive clones to obtain the strain QPCNΔSAP.

9. The strain QPCNΔSAP prepared by the method according to any one of claims 5 to 8.

10. Use of the strain QPCNΔSAP according to any one of claims 1 to 4 and 9 in the production of phenazine-1-carboxamide, characterized in that: include: The strain QPCNΔSAP was inoculated into the fermentation medium to produce phenazine-1-carboxamide.

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