A method for promoting production of phenoxazine-1-carboxylic acid by limiting a competing pathway for phenoxazine synthesis

Abstract

The application belongs to the technical field of genetic engineering, and provides a method for promoting production of phenoxazine-1-carboxylic acid by limiting competition pathways of phenoxazine synthesis. The application takes high-yield strain QPCA-7 as a starting strain, limits the synthesis pathways of salicylic acid, siderophore and the like of phenoxazine, and realizes the improvement of the yield of phenoxazine-1-carboxylic acid of the strain. ycaC The application obtains an engineering strain by non-scarring knockout of the gene for limiting the synthesis of siderophore in Pseudomonas, and the strain is named QPCA-8, and the yield of phenoxazine-1-carboxylic acid of the strain is improved. pabC The application knocks out the gene for limiting the production of salicylic acid in Pseudomonas on the basis of QPCA-8, and the yield of phenoxazine-1-carboxylic acid of the strain is improved.

Description

Technical Field

[0001] This invention belongs to the field of genetic engineering technology, and specifically relates to a method for promoting the production of phenazine-1-carboxylic acid by restricting competitive pathways in phenazine synthesis. 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] Chemical pesticides are crucial for high and stable crop yields, but the extensive use of non-degradable chemical pesticides easily causes environmental pollution and hinders sustainable development. Biopesticides offer a good alternative. Compared to chemical pesticides, biopesticides have significant advantages such as low toxicity, environmental friendliness, easy degradation, and low likelihood of developing resistance, attracting increasing attention from scholars and being gradually applied in biological control practices. Phenrazines produced by Pseudomonas and Streptomyces are a typical example of biopesticides. Phenazine-1-carboxylic acid, due to its broad-spectrum, high-efficiency, safe, and effective control of fungal root and stem rot, is mainly used to control rice sheath blight, wheat scab, cucumber and watermelon wilt, melon vine blight, and pepper root rot. In 2011, it was granted a pesticide certificate by the Ministry of Agriculture and Rural Affairs of the People's Republic of China and named "Shenzinmycin".

[0004] Currently, although there are chemical methods for producing phenazine-1-carboxylic acid, the conditions are harsh and it releases toxic and harmful substances into the environment. Therefore, it is mainly produced by Pseudomonas bacteria. However, the production potency of Pseudomonas bacteria is currently low, resulting in high production costs of phenazine-1-carboxylic acid, which limits its widespread application. Improving the production potency of phenazine-1-carboxylic acid from Pseudomonas bacteria is an important task at present.

[0005] The production of phenazine-1-carboxylic acid can be carried out through biosynthesis other than chemical synthesis. The relatively safe Pseudomonas aeruginosa Qlu-1 is used as the production strain. However, the current production strain has a low yield, which leads to high production costs of phenazine-1-carboxylic acid. Therefore, increasing the yield of engineered bacteria has become an urgent need.

[0006] The inventor's previous patent ZL202011026544.7 disclosed a high-yield strain QPCA-7, but the yield of its phenazine-1-carboxylic acid still needs to be improved. Summary of the Invention

[0007] To address the aforementioned problems, this invention provides a method for promoting the production of phenazine-1-carboxylic acid by restricting competitive pathways in phenazine synthesis. Phenazine synthesis in Pseudomonas utilizes the shikimic acid pathway as a precursor pathway. This shikimic acid pathway is an important synthetic pathway in Pseudomonas and also a precursor pathway for the synthesis of aromatic amino acids, siderophores, salicylic acid, coenzyme Q, etc. This invention discovers that, among the numerous competitive pathways for phenazine synthesis, restricting the synthesis of siderophores, salicylic acid, etc., and guiding more carbon flux into the phenazine synthesis pathway, can significantly improve the synthesis efficiency of phenazine-1-carboxylic acid. This invention uses the high-yielding strain QPCA-7 from patent ZL202011026544.7 as the starting strain, and improves the synthesis efficiency of strain PCA by restricting the synthesis of siderophores, salicylic acid, etc.

[0008] To achieve the above objectives, the present invention adopts the following technical solution:

[0009] In a first aspect, the present invention provides a genetically engineered strain that promotes the production of phenazine-1-carboxylic acid by restricting a competitive pathway in phenazine synthesis, said engineered strain being a knockout strain of the QPCA-7 genome. ycaC Genes and / or pabC Inherited by genes;

[0010] The engineered strain QPCA-7 is a knockout strain of *Pseudomonas aeruginosa*. Pseudomonas chlororaphis Qlu-1 phzO Gene, lon Gene, rsmE Gene, psrA Gene, parS Gene, rpeA Genes and pykF Inherited by genes;

[0011] The preservation number of the aforementioned Pseudomonas aeruginosa Qlu-1 is CCTCCNO: M2020108, and it has been disclosed in patent ZL202011026544.7.

[0012] In some implementations, the ycaC The gene sequence is shown in SEQ ID NO.1.

[0013] In some embodiments, the pabC The gene sequence is shown in SEQ ID NO.7.

[0014] In some implementations, knocking out the genome of engineered strain QPCA-7 ycaC The specific steps involved in gene generation include:

[0015] PCR fishing and ligation ycaC The upstream and downstream segments of the gene were used to obtain the ycaC-UD fragment;

[0016] The ycaC-UD fragment was ligated into plasmid pk18moBsacB to construct... ycaC Gene knockout plasmid pK18-ycaC-UD;

[0017] The ycaC The gene knockout plasmid pK18-ycaC-UD was introduced into Escherichia coli S17-1 (λpir), and then subjected to parental hybridization culture with Pseudomonas aeruginosa QPCA-7. Positive clones were screened to obtain the knockout plasmid. ycaC The gene is from strain QPCA-8.

[0018] In some implementations... ycaC The base sequences of the upstream and downstream fusion fragments of the gene are shown in SEQ ID NO.2.

[0019] In some embodiments, the ycaC The gene knockout primers include ycaC-F1, ycaC-R1, ycaC-F2, and ycaC-R2, with sequences shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

[0020] In some implementations, knocking out the genome of engineered strain QPCA-8 pabC The specific steps involved in gene generation include:

[0021] PCR fishing and ligation pabC The upstream and downstream segments of the gene were used to obtain the pabC-UD fragment;

[0022] The pabC-UD fragment was ligated into plasmid pk18moBsacB to construct... pabC Gene knockout plasmid pK18-pabC-UD;

[0023] The pabC The gene knockout plasmid pK18-pabC-UD was introduced into Escherichia coli S17-1 (λpir), and then subjected to parental hybridization culture with Pseudomonas aeruginosa QPCA-8. Positive clones were screened to obtain the knockout plasmid. pabC The gene is from strain QPCA-9.

[0024] In some implementations... pabC The base sequences of the upstream and downstream fusion fragments of the gene are shown in SEQ ID NO.8.

[0025] In some implementations... pabCThe gene knockout primers include: pabC-F1, pabC-R1, pabC-F2, and pabC-R2, with sequences shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively.

[0026] A second aspect of the present invention provides a method for promoting the production of phenazine-1-carboxylic acid by limiting competitive pathways in phenazine synthesis, comprising:

[0027] The above-mentioned genetically engineered strains are inoculated into a fermentation medium and cultured to obtain the final product.

[0028] The fermentation medium may be KB medium.

[0029] Beneficial effects of the present invention

[0030] (1) This invention improves the production potency of phenazine-1-carboxylic acid by restricting competitive pathways in phenazine synthesis. The invention uses the high-yielding strain QPCA-7 from ZL202011026544.7 as the starting strain, and achieves this through traceless knockout... ycaC By restricting the synthesis of siderophores in *Pseudomonas*, an engineered strain named QPCA-8 was obtained. Fermentation yield analysis revealed that QPCA-8 produced an increased PCA yield of 8914 mg / L. This invention, based on QPCA-8, knocks out... pabC Genes were used to restrict salicylic acid production in Pseudomonas aeruginosa, resulting in strain QPCA-9. After yield testing, the production of phenazine-1-carboxylic acid increased to 9948 mg / L at 48 h.

[0031] (2) By limiting the synthesis of salicylic acid and iron carriers in phenazine synthesis, this invention utilizes KB medium for fermentation, achieving a yield of 9948 mg / L, thus significantly increasing the yield of phenazine-1-carboxylic acid in the strain.

[0032] (3) The production method of the present invention is simple, practical and easy to promote. Attached Figure Description

[0033] 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.

[0034] Figure 1 Electrophoresis diagram of mutant plasmid pK18-ycaC-UD; 1, ycaC 1. Amplification of upstream and downstream fusion fragments of genes; 2. DNA Ladder DL2000; 3. ycaC Amplification of upstream homologous arm fragments of the gene; 4, ycaC Amplification of downstream homologous arm fragments of the gene.

[0035] Figure 2 for ycaC PCR verification of gene knockout strains.

[0036] External primer detection: 1, with ycaC 1. Amplified fragment using the genome of the gene knockout strain Qlu-8 as a template; 2. Blank control; 3. Blank control; 4. DNA Ladder DL5000; 5. Amplified fragment using the genome of Qlu-7 as a template.

[0037] Internal primer detection: 1. DNA Ladder DL5000; 2. Fragment amplified using Qlu-7 genome as template; 3. ycaC The genome of the gene knockout strain Qlu-8 was used as a template for amplification; 4, blank control.

[0038] Figure 3 The yield of phenazine-1-carboxylic acid after 48 hours of fermentation by different strains. Detailed Implementation

[0039] It should be noted that the following detailed descriptions are exemplary and intended to provide further explanation 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.

[0040] 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.

[0041] In the following examples, the fermentation conditions for strains QPCA-8 and QPCA-9 are the same as those for strain QPCA-7 in the example of patent ZL202011026544.7, and the yield testing methods are also the same.

[0042] Example 1

[0043] 1. Inoculate the engineered strain QPCA-7 into KB medium and culture overnight at 30°C with shaking at 180 rpm. Extract the genome of the engineered strain QPCA-7 using a prokaryotic genome extraction kit and store at -20°C for later use.

[0044] 2. Search within the sequenced genome data of the engineered strain QPCA-7 ycaC Gene and upstream / downstream sequences were amplified using the genome of engineered strain QPCA-7 as a template and F1 / R1 and F2 / R2 primers, respectively. ycaC The upstream sequence of the gene is ycaC-U. ycaCThe downstream sequence of the gene is ycaC-D; using ycaC-U and ycaC-D as templates and F1 / R2 as primers, the ycaC-U and ycaC-D fragments are ligated by fusion PCR to obtain the fusion fragment ycaC-UD.

[0045] 3. The fusion fragment ycaC-UD was ligated with the knockout plasmid pk18moBsacB to construct the recombinant plasmid pk18-ycaC-UD. Figure 1 ).

[0046] 4. The recombinant plasmid pk18-ycaC-UD was introduced into Escherichia coli S17-1 (λpir) by heat shock transformation.

[0047] 5. Perform a two-parent hybridization culture between Escherichia coli S17-1 (λpir) and Pseudomonas aeruginosa QPCA-7, and introduce the recombinant plasmid pk18-rsmE-UD into Pseudomonas aeruginosa QPCA-7.

[0048] 6. The mutant strain QPCA-8 was obtained through sucrose plate screening and photocopy screening.

[0049] 7. Verify QPCA-7 using PCR validation. ycaC gene knockout strain QPCA-8, ( Figure 2 ).

[0050] 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 8796 mg / L after 48 hours of fermentation. Figure 3 ).

[0051] Example 2

[0052] Using the same method as in Example 1, pabC The gene was knocked out from QPCA-8, and the mutant strain QPCA-9 was obtained. After 48 hours of fermentation, the yield of phenazine-1-carboxylic acid reached 9948 mg / L.

[0053] 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 strain that promotes the production of phenazine-1-carboxylic acid by restricting the competitive pathway of phenazine synthesis, characterized in that, The engineered strain is a knockout engineered strain QPCA-7 whose genome contains... ycaC Gene or ycaC Genes and pabC Inherited by genes; The engineered strain QPCA-7 is a knockout strain of *Pseudomonas aeruginosa*. Pseudomonas chlororaphis Qlu-1 phzO Gene, lon Gene, rsmE Gene, psrA Gene, parS Gene, rpeA Genes and pykF Inherited by genes; The preservation number of the *Pseudomonas aeruginosa* Qlu-1 is CCTCCNO: M2020108.

2. The genetically engineered strain that promotes the production of phenazine-1-carboxylic acid by restricting the competitive pathway of phenazine synthesis as described in claim 1, characterized in that, The ycaC The gene sequence is shown in SEQ ID NO.

1.

3. The genetically engineered strain that promotes the production of phenazine-1-carboxylic acid by restricting the competitive pathway of phenazine synthesis as described in claim 1, characterized in that, The pabC The gene sequence is shown in SEQ ID NO.

7.

4. The genetically engineered strain that promotes the production of phenazine-1-carboxylic acid by restricting the competitive pathway of phenazine synthesis as described in claim 1, characterized in that, Knockout of the QPCA-7 engineered strain genome ycaC The specific steps involved in gene generation include: PCR fishing and ligation ycaC The upstream and downstream segments of the gene were used to obtain the ycaC-UD fragment; The ycaC-UD fragment was ligated into plasmid pk18moBsacB to construct... ycaC Gene knockout plasmid pK18-ycaC-UD; The ycaC The gene knockout plasmid pK18-ycaC-UD was introduced into Escherichia coli S17-1 (λpir), and then subjected to parental hybridization culture with Pseudomonas aeruginosa QPCA-7. Positive clones were screened to obtain the knockout plasmid. ycaC The gene is from strain QPCA-8.

5. The genetically engineered strain that promotes the production of phenazine-1-carboxylic acid by restricting the competitive pathway of phenazine synthesis as described in claim 1, characterized in that, ycaC The base sequences of the upstream and downstream fusion fragments of the gene are shown in SEQ ID NO.

2.

6. The genetically engineered strain that promotes the production of phenazine-1-carboxylic acid by restricting the competitive pathway of phenazine synthesis as described in claim 1, characterized in that, The ycaC The gene knockout primers include ycaC-F1, ycaC-R1, ycaC-F2, and ycaC-R2, with sequences shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

7. The genetically engineered strain that promotes the production of phenazine-1-carboxylic acid by restricting the competitive pathway of phenazine synthesis as described in claim 1, characterized in that, Knockout of the QPCA-8 engineered strain genome pabC The specific steps involved in gene generation include: PCR fishing and ligation pabC The upstream and downstream segments of the gene were used to obtain the pabC-UD fragment; The pabC-UD fragment was ligated into plasmid pk18moBsacB to construct... pabC Gene knockout plasmid pK18-pabC-UD; The pabC The gene knockout plasmid pK18-pabC-UD was introduced into Escherichia coli S17-1 (λpir), and then subjected to parental hybridization culture with Pseudomonas aeruginosa QPCA-8. Positive clones were screened to obtain the knockout plasmid. pabC The gene is from strain QPCA-9.

8. The genetically engineered strain that promotes the production of phenazine-1-carboxylic acid by restricting the competitive pathway of phenazine synthesis as described in claim 1, characterized in that, pabC The base sequences of the upstream and downstream fusion fragments of the gene are shown in SEQ ID NO.

8.

9. The genetically engineered strain that promotes the production of phenazine-1-carboxylic acid by restricting the competitive pathway of phenazine synthesis as described in claim 1, characterized in that, pabC The gene knockout primers include: pabC-F1, pabC-R1, pabC-F2, and pabC-R2, with sequences shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively.

10. A method for promoting the production of phenazine-1-carboxylic acid by limiting competitive pathways in phenazine synthesis, characterized in that, include: The genetically engineered strain according to any one of claims 1-9 is inoculated into a fermentation medium and cultured to obtain the product.

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