A method for promoting production of phenazine-1-carboxylic acid using an enhanced synthetic pathway

Abstract

The application provides a genetically engineered bacterium for promoting production of phenazine-1-carboxylic acid by enhancing a synthesis pathway, and uses a green needle Pseudomonas aeruginosa genetically engineered bacterium QPCA Delta DTH as a starting strain, introduces an aroC gene into a site where a hppA gene is knocked out in the green needle Pseudomonas aeruginosa genetically engineered bacterium QPCA Delta DTH to obtain a strain QPCA Delta DTH:C; and / or introduces a phzIR gene into a Detg gene site of the strain QPCA Delta DTH:C to obtain a QPCA Delta DTH:C:IR genetically engineered bacterium. The aroC gene and the phzIR gene are integrated into the genome to enhance a phenazine synthesis precursor pathway, a shikimic acid pathway and a phenazine synthesis pathway, so that the yield of phenazine-1-carboxylic acid of the initial strain is increased from 4787.2 mg / L to 7746 mg / L.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering, and specifically relates to a method for promoting the production of phenazine-1-carboxylic acid by enhancing the synthetic pathway. Background Technology

[0002] The information disclosed in this background section is intended only to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Phenazine-1-carboxylic acid is a bioactive substance. Due to its good control effect on crop diseases, it was granted a pesticide certificate by the Ministry of Agriculture of the People's Republic of China in 2011 and officially named "Shenzinmycin". Phenazine-1-carboxylic acid can be produced by biosynthesis other than chemical synthesis, such as fermentation with Pseudomonas aeruginosa. However, Pseudomonas aeruginosa is an opportunistic pathogen. Therefore, the relatively safe Pseudomonas aeruginosa Qlu-1 is used as the production strain. However, the current production strain has the problem of low yield of phenazine-1-carboxylic acid, with a yield of only about 270 mg / L, which does not meet the application level. Increasing the yield of engineered strains has become an urgent need.

[0004] Previously, the inventors increased the yield of phenazine-1-carboxylic acid in the strain to 4780 mg / L through genetic engineering. However, the production cost of phenazine-1-carboxylic acid remains high, and it is still necessary to increase the yield of the engineered strain to reduce the cost of phenazine-1-carboxylic acid and prepare for its widespread application. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for promoting the production of phenazine-1-carboxylic acid using an enhanced synthetic pathway, by... aroC Gene, phzIR The gene integration into the genome enhanced the phenazine synthesis precursor pathway—the shikimic acid pathway—and the phenazine synthesis pathway, increasing the yield of phenazine-1-carboxylic acid in the initial strain from 4787.2 mg / L to 7746 mg / L.

[0006] To achieve the above objectives, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a genetically engineered bacterium that promotes the production of phenazine-1-carboxylic acid by enhancing the synthetic pathway, using *Pseudomonas aeruginosa* genetically engineered strain QPCAΔDTH as the starting strain, in which *Pseudomonas aeruginosa* genetically engineered strain QPCAΔDTH... hppA Introducing the gene knockout site aroC Genes were used to obtain strain QPCA△DTH:C;

[0008] And / or, will phzIR Genes were introduced into strain QPCA△DTH:C. Detg Gene loci were identified, resulting in the QPCA△DTH:C:IR genetically engineered bacteria.

[0009] In some implementations, the aroC The base sequence of the gene is shown in SEQ ID NO.1.

[0010] In some implementations, the phzIR The base sequence of the gene is shown in SEQ ID NO.9.

[0011] In some embodiments, the *Pseudomonas aeruginosa* genetically engineered strain QPCAΔDTH is a knockout strain of *Pseudomonas aeruginosa* (…). Pseudomonas chlororaphis The Qlu-1 genome phzO Gene, you Gene, tctB Genes and hppA The gene was obtained, and among them, Pseudomonas aeruginosa Qlu-1 has been deposited at the China Center for Type Culture Collection, with accession number CCTCCNO: M2020108, and has been disclosed in patent ZL202210712676.8.

[0012] In some implementations, the introduction aroC The specific steps involved in gene generation include:

[0013] Searching within the already sequenced Qlu-1 genome sequence aroC Gene sequence and hppA The upstream and downstream sequences of the gene were extracted using PCR. aroC Gene, hppA upstream fragment hppA-U of the gene hppA The downstream fragment hppA-D of the gene was ligated together by fusion PCR to obtain the aroC-hppA-UD fusion fragment.

[0014] The aroC-hppA-UD fusion fragment was linked to plasmid pk18moBsacB to construct... aroC Recombinant genome plasmid pk18-aroC-hppA-UD;

[0015] The aroC The genome integration plasmid pK18-aroC-hppA-UD was introduced into Escherichia coli S17-1 (λpir).

[0016] The mutant strain QPCA△DTH was then subjected to parental hybridization culture with the aforementioned Pseudomonas aeruginosa QPCA△DTH, and positive clones were screened to obtain the mutant strain QPCA△DTH:C.

[0017] In some embodiments, the base sequence of the aroC-hppA-UD fusion fragment is shown in SEQ ID NO.2.

[0018] In some implementations, the aroC The primers for gene transfer to the genome include: aroC-F1, aroC-R1, aroC-F2, aroC-R2, aroC-F3, and aroC-R3, with sequences shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively.

[0019] In some implementations, the import phzIR The specific steps involved in gene generation include:

[0020] Searching within the already sequenced Qlu-1 genome sequence phzIR Gene sequence and Detg The upstream and downstream sequences of the gene were extracted using PCR. phzIR Gene, Detg upstream gene fragment Detg-U, Detg The downstream fragment Detg-D of the gene was used to link the three fragments together by fusion PCR to obtain the phzIR-Detg-UD fusion fragment.

[0021] The phzIR-Detg-UD fusion fragment was linked to plasmid pk18moBsacB to construct... phzIR Recombinant genome plasmid pk18-phzIR-Detg-UD;

[0022] The phzIR The genome integration plasmid pk18-phzIR-Detg-UD was introduced into Escherichia coli S17-1 (λpir).

[0023] The mutant strain QPCA△DTH:C was then subjected to parental hybridization culture, and positive clones were screened to obtain the mutant strain QPCA△DTH:C:IR.

[0024] In some embodiments, the base sequence of the phzIR-Detg-UD fusion fragment is shown in SEQ ID NO.10;

[0025] In some implementations, the phzIRThe primers for gene transfer into the genome include: phzIR-F1, phzIR-R1, phzIR-F2, phzIR-R2, phzIR-F3, and phzIR-R3, with sequences shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16, respectively.

[0026] A second aspect of the present invention provides a method for promoting the production of phenazine-1-carboxylic acid using an enhanced synthetic pathway, comprising:

[0027] Construct the above-mentioned genetically engineered bacterium QPCA△DTH:C:IR;

[0028] The phenazine-1-carboxylic acid is produced by fermentation using the genetically engineered strain QPCA△DTH:C:IR.

[0029] In some embodiments, the fermentation medium may be KB medium.

[0030] Beneficial effects of the present invention

[0031] (1) This invention uses the engineered strain QPCA△DTH derived from Pseudomonas aeruginosa Qlu-1 in patent ZL202210712676.8 as the starting strain, and integrates the key enzyme gene in the shikimic acid pathway, the precursor pathway for phenazine synthesis. aroC Entering the genome promotes the synthesis of more branched acids, products of the shikimic acid pathway. aroC The integration site of the gene is selected in this invention from strain QPCA△DTH, where the gene has been knocked out. hppA By identifying the gene locus and enhancing the precursor pathway for phenazine synthesis, a mutant strain QPCA△DTH:C was obtained. After fermentation, the yield of phenazine-1-carboxylic acid was significantly increased to 5696 mg / L.

[0032] The synthesis of phenazine, besides phzABCDEF In addition to gene involvement, phzIR Genes also participate in the synthesis of phenazines by producing signaling molecules; this invention relates to this method... phzIphzR Genes (two adjacent genes in *Pseudomonas aeruginosa*) are introduced into ΔDTH:C. Detg The gene locus promotes the phenazine synthesis pathway of the strain, thereby enhancing phenazine synthesis. The present invention obtained the mutant strain QPCA△DTH:C:IR, which, after fermentation, increased the phenazine-1-carboxylic acid yield to 7746 mg / L.

[0033] (2) This invention utilizes KB medium for fermentation, achieving a yield of 7746 mg / L, which greatly improves the production capacity of the strain and provides a solid foundation for the industrialization of subsequent engineered strains. Attached Figure Description

[0034] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. Exemplary embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0035] Figure 1 Electrophoresis diagram of the integrated plasmid pk18-aroC-hppA-UD;

[0036] (A) hppA Upstream and downstream homologous arms of genes and aroC Gene amplification: 1, hppA Upstream homologous arm amplification; 2, hppA 3. Amplification of downstream homologous arms of the gene; 4. DNA Ladder DL2000; aroC Gene fragment amplification;

[0037] (B)1, DNA Ladder DL5000; 2, aroC-hppA-UD fusion fragment amplification; 3, aroC-hppA-UD fusion fragment amplification.

[0038] Figure 2 aroC Genome PCR validation of gene insertion;

[0039] 1. DNA Ladder DL5000; 2. aroC 3. Amplify the fragment using the QPCA△DTH:C gene insert as a template; 4. Amplify the fragment using the QPCA△DTH:C genome as a template.

[0040] Figure 3 Yields of phenazine-1-carboxylic acid by different strains after 48 hours of fermentation. Detailed Implementation

[0041] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0042] The present invention will be further described in detail below with reference to specific embodiments. It should be noted that the specific embodiments are explanations of the present invention and not limitations thereof.

[0043] In the following examples, the fermentation conditions of strains QPCA△DTH:C and QPCA△DTH:C:IR are the same as those of strain QPCA△DTH in the verification example of patent ZL202210712676.8, and the yield testing methods are also the same.

[0044] Example 1

[0045] 1. Inoculate Pseudomonas aeruginosa Qlu-1 into KB medium and culture overnight at 30°C with shaking at 180 rpm. Extract the genome of Pseudomonas aeruginosa Qlu-1 using a prokaryotic genome extraction kit and store at -20°C for later use.

[0046] 2. Search within the sequenced genome data of *Pseudomonas aeruginosa* Qlu-1. aroC Genes and hppA The upstream and downstream sequences of the gene were amplified using the genome of *Pseudomonas aeruginosa* Qlu-1 as a template and primers F1 / R1, F2 / R2, and F3 / R3, respectively. hppA The upstream sequence of the gene is hppA-U. aroC gene sequence ,hppA The downstream sequence of the gene, hppA-D, was obtained; using hppA-U, aroC, and hppA-D fragments as templates, and F1 / R3 as primers, fusion PCR was performed. aroC Fragments and hppA Connect the upstream and downstream fragments to obtain the fused fragment aroC-hppA-UD ( Figure 1 ).

[0047] 3. The fusion fragment aroC-hppA-UD was ligated with the integration plasmid pk18moBsacB to construct the recombinant plasmid pk18-aroC-hppA-UD.

[0048] 4. The recombinant plasmid pk18-aroC-hppA-UD was introduced into Escherichia coli S17-1 (λpir) by heat shock transformation.

[0049] 5. Perform a two-parent hybridization culture between Escherichia coli S17-1 (λpir) and Pseudomonas aeruginosa QPCA△DTH, and introduce the recombinant plasmid pk18-aroC-hppA-UD into Pseudomonas aeruginosa QPCA△DTH.

[0050] 6. Gene-introduced strains were obtained through sucrose plate screening and photocopy screening.

[0051] 7. Verify QPCAΔDTH using PCR validation. aroC The gene insert strain QPCA△DTH:C ( Figure 2 ).

[0052] 8. After fermentation, phenazine-1-carboxylic acid was extracted from the fermentation broth. HPLC analysis revealed that the yield of phenazine-1-carboxylic acid increased to 5696 mg / L after 48 hours of fermentation. Figure 3 ).

[0053] Example 2

[0054] Using the same method as in Example 1, phzIR Gene integration into QPCA△DTH:C Detg At the gene locus, a mutant strain QPCA△DTH:C:IR was obtained, and its yield increased to 7746 mg / L after 48 h of fermentation. Figure 3 ).

[0055] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A genetically engineered bacterium that promotes the production of phenazine-1-carboxylic acid by enhancing the synthetic pathway, characterized in that, Using the genetically engineered *Pseudomonas aeruginosa* strain QPCAΔDTH as the starting strain, in the genetically engineered *Pseudomonas aeruginosa* strain QPCAΔDTH... hppA Introducing the gene knockout site aroC Gene, obtained strain QPCA△DTH:C; the described aroC The gene's base sequence is shown in SEQ ID NO.1; And / or, will phzIR Genes were introduced into strain QPCA△DTH:C. Detg Gene loci were used to obtain QPCA△DTH:C:IR genetically engineered bacteria; the aforementioned phzIR The gene's base sequence is shown in SEQ ID NO.9; The genetically engineered *Pseudomonas aeruginosa* strain QPCAΔDTH is a knockout strain of *Pseudomonas aeruginosa*. Pseudomonas chlororaphis The Qlu-1 genome phzO Gene, degU Gene, tctB Genes and hppA The strain was obtained through genetic testing. Among them, Pseudomonas aeruginosa Qlu-1 has been deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M2020108.

2. The genetically engineered bacterium that promotes the production of phenazine-1-carboxylic acid by enhancing the synthetic pathway as described in claim 1, characterized in that, The introduction aroC The specific steps involved in gene generation include: Searching within the already sequenced Qlu-1 genome sequence aroC Gene sequence and hppA The upstream and downstream sequences of the gene were extracted using PCR. aroC Gene, hppA upstream gene fragment hppA-U, hppA The downstream fragment hppA-D of the gene was ligated together by fusion PCR to obtain the aroC-hppA-UD fusion fragment. The aroC-hppA-UD fusion fragment was linked to plasmid pk18moBsacB to construct... aroC Recombinant genome plasmid pk18-aroC-hppA-UD; The aroC The genome integration plasmid pK18-aroC-hppA-UD was introduced into Escherichia coli S17-1 (λpir). The mutant strain QPCA△DTH was then subjected to parental hybridization culture with the aforementioned Pseudomonas aeruginosa QPCA△DTH, and positive clones were screened to obtain the mutant strain QPCA△DTH:C.

3. The genetically engineered bacterium that promotes the production of phenazine-1-carboxylic acid by enhancing the synthetic pathway as described in claim 1, characterized in that, The base sequence of the aroC-hppA-UD fusion fragment is shown in SEQ ID NO.

2.

4. The genetically engineered bacterium that promotes the production of phenazine-1-carboxylic acid by enhancing the synthetic pathway as described in claim 1, characterized in that, The aroC The primers for gene transfer to the genome include: aroC-F1, aroC-R1, aroC-F2, aroC-R2, aroC-F3, and aroC-R3, with sequences shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, SEQ ID NO.6, SEQ ID NO.7, and SEQ ID NO.8, respectively.

5. The genetically engineered bacterium that promotes the production of phenazine-1-carboxylic acid by enhancing the synthetic pathway as described in claim 1, characterized in that, The import phzIR The specific steps involved in gene generation include: Searching within the already sequenced Qlu-1 genome sequence phzIR Gene sequence and Detg The upstream and downstream sequences of the gene were extracted using PCR. phzIR Gene, Detg upstream gene fragment Detg-U, Detg The downstream fragment Detg-D of the gene was used to link the three fragments together by fusion PCR to obtain the phzIR-Detg-UD fusion fragment. The phzIR-Detg-UD fusion fragment was linked to plasmid pk18moBsacB to construct... phzIR Recombinant genome plasmid pk18-phzIR-Detg-UD; The phzIR The genome integration plasmid pk18-phzIR-Detg-UD was introduced into Escherichia coli S17-1 (λpir). The mutant strain QPCA△DTH:C was then subjected to parental hybridization culture, and positive clones were screened to obtain the mutant strain QPCA△DTH:C:IR.

6. The genetically engineered bacterium that promotes the production of phenazine-1-carboxylic acid by enhancing the synthetic pathway as described in claim 1, characterized in that, The base sequence of the phzIR-Detg-UD fusion fragment is shown in SEQ ID NO.10; Or, the stated phzIR The primers for gene transfer into the genome include: phzIR-F1, phzIR-R1, phzIR-F2, phzIR-R2, phzIR-F3, and phzIR-R3, with sequences shown in SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO.14, SEQ ID NO.15, and SEQ ID NO.16, respectively.

7. A method for promoting the production of phenazine-1-carboxylic acid using an enhanced synthetic pathway, characterized in that, include: Construct the genetically engineered bacterium QPCA△DTH:C:IR as described in any one of claims 1-6; The phenazine-1-carboxylic acid is produced by fermentation using the genetically engineered strain QPCA△DTH:C:IR.

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