A method for constructing *Pseudomonas aeruginosa* to produce 2-hydroxyphenazine and its application

Abstract

This invention belongs to the field of bioengineering and provides a method for constructing *Pseudomonas aeruginosa* strains that produce 2-hydroxyphenazine and its applications. This invention uses Qlu-1-3 from patent ZL202011026637.X as the starting strain, and achieves this by knocking out a gene that inhibits phenazine synthesis in the genome. mdtA , gspM The gene enabled the engineered strain to produce 2-hydroxyphenazine at concentrations of 156.3 mg / L and 216.4 mg / L, laying the foundation for the subsequent production of 2-OH-PHZ using Pseudomonas aeruginosa.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering, and specifically relates to a method for constructing Pseudomonas aeruginosa to produce 2-hydroxyphenazine and its application. 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] 2-Hydroxyphenazine (2-OH-PHZ) exhibits strong antagonistic effects against many fungi and bacteria, making it a promising biological pesticide. However, chemical synthesis easily produces toxic and harmful substances, while biosynthesis results in very low yields.

[0004] The inventor's previous research (patent ZL202011026637.X) disclosed an engineered strain for the production of 2-hydroxyphenazine and its applications. This was achieved by knocking out negative regulatory factors. psrA and parS Methods for treating Pseudomonas aeruginosa ( Pseudomonas chlororaphis Qlu-1 was modified to produce strain Qlu-1-3, which produces 2-hydroxyphenazine. However, further research revealed that the yield of 2-hydroxyphenazine produced using strain Qlu-1-3 still needs to be improved. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for constructing *Pseudomonas aeruginosa* strains that produce 2-hydroxyphenazine and its applications. This invention uses Qlu-1-3 from patent ZL202011026637.X as the starting strain, and eliminates strains that inhibit phenazine synthesis in the genome. mdtA , gspM The gene enabled the engineered strain to produce 2-hydroxyphenazine at 156.3 mg / L and 216.4 mg / L, laying the foundation for the subsequent production of 2-OH-PHZ using Pseudomonas aeruginosa.

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

[0007] A first aspect of the present invention provides a method for constructing *Pseudomonas aeruginosa* for producing 2-hydroxyphenazine, comprising:

[0008] Starting with the 2-hydroxyphenazine-producing strain Qlu-1-3, the genome was knocked out mdtA Genes and / or gspM Genes were used to obtain corresponding genetically engineered strains;

[0009] The genetically engineered strain was inoculated into a fermentation medium and cultured to produce 2-hydroxyphenazine.

[0010] The 2-hydroxyphenazine-producing strain Qlu-1-3 is *Pseudomonas aeruginosa*. (Pseudomonas chlororaphis Qlu-1 was the starting strain, and negative regulatory genes were continuously knocked out. psrA and parS And thus;

[0011] The preservation number of the *Pseudomonas aeruginosa* Qlu-1 is CCTCCNO: M2020108, and it has been disclosed in patent ZL202011026637.X.

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

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

[0014] In some embodiments, the knockout genome mdtA The specific steps involved in gene generation include:

[0015] Searching within the already sequenced Qlu-1 genome sequence mdtA The gene sequence and its upstream and downstream sequences were used to obtain the mdtA-UD fragment via fusion PCR.

[0016] The mdtA-UD fragment was ligated to plasmid pk18moBsacB using enzyme digestion and ligation technology to construct... mdtA Gene knockout plasmid pK18-mdtA-UD;

[0017] The mdtA The gene knockout plasmid pK18-mdtA-UD was introduced into E. coli S17-1 (λpir), and then subjected to parent hybridization culture with the 2-hydroxyphenazine-producing strain Qlu-1-3. Positive clones were screened to obtain the knockout plasmid. mdtA The gene is from strain Qlu-1-3△M.

[0018] In some implementations... mdtA 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 mdtAThe gene knockout primers include: mdtA-F1, mdtA-R1, mdtA-F2, and mdtA-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 gspM The specific steps involved in gene generation include:

[0021] Searching within the already sequenced Qlu-1 genome sequence gspM Gene sequences and their upstream and downstream sequences were obtained using fusion PCR. gspM -UD fragment;

[0022] The gspM-UD fragment was ligated to plasmid pk18moBsacB using enzyme digestion and ligation technology to construct... gspM Gene knockout plasmid pK18-gspM-UD;

[0023] The gspM The gene knockout plasmid pK18-gspM-UD was introduced into E. coli S17-1 (λpir), and then subjected to parent-to-parent hybridization culture with strain Qlu-1-3ΔM. Positive clones were screened to obtain the knockout plasmid. gspM The gene is from strain Qlu-1-3△MG.

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

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

[0026] In some embodiments, the fermentation medium may be KB medium.

[0027] A second aspect of the present invention provides a genetically engineered strain for producing 2-hydroxyphenazine, said strain being a 2-hydroxyphenazine-producing strain Qlu-1-3 whose genome has been knocked out. mdtA Genes and / or gspM Inherited by genes;

[0028] The 2-hydroxyphenazine-producing strain Qlu-1-3 is *Pseudomonas aeruginosa* (…). Pseudomonas chlororaphis Qlu-1 was the starting strain, and negative regulatory genes were continuously knocked out. psrA andparS And thus;

[0029] The preservation number of the *Pseudomonas aeruginosa* Qlu-1 is CCTCCNO: M2020108.

[0030] A third aspect of the invention provides the use of the above-described genetically engineered strain in the production of 2-hydroxyphenazine.

[0031] Beneficial effects of the present invention

[0032] (1) This invention selects a negative regulator of phenazine synthesis in *Pseudomonas aeruginosa* and promotes the increase of 2-hydroxyphenazine production in *Pseudomonas aeruginosa* by knocking it out. This invention uses the 2-hydroxyphenazine-producing strain Qlu-1-3 from patent ZL202011026637.X as the starting strain, and achieves a scarless knockout... mdtA The gene was used to obtain an engineered strain named Qlu-1-3△M. Fermentation yield testing showed that Qlu-1-3△M increased the yield of 2-hydroxyphenazine to 156.3 mg / L. This invention, based on Qlu-1-3△M, knocks out... gspM Genes were extracted to obtain strain Qlu-1-3△MG, and yield testing showed that its 2-hydroxyphenazine production increased to 216.4 mg / L.

[0033] (2) The present invention utilizes KB medium for fermentation, and the yield reaches 216.4 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 mutant plasmid pK18-mdtA-ud;

[0036] (A) mdtA Amplification of upstream and downstream homologous arm fragments of the gene: 1, mdtA 1. Amplification of upstream homologous arms of the gene; 2. DNALadder DL5000; 3. mdtA Amplification of downstream homologous arm fragments of the gene;

[0037] (B) mdtA Amplification of upstream and downstream homologous arm fusion fragments: 1. DNA Ladder DL5000; 2. mdtA Gene upstream and downstream homologous arm fusion fragments; 3, mdtA Gene upstream and downstream homologous arm fusion fragment.

[0038] Figure 2 mdtA PCR verification of gene knockout strains;

[0039] External primer detection: 1. DNA Ladder DL2000; 2. ... mdtA 3. Amplified fragment using the genome of gene knockout strain Qlu-1-3△M as template; 4. Blank control;

[0040] Internal primer detection: 1. DNA Ladder DL2000; 2. ... mdtA 3. Amplified fragment using the genome of the gene knockout strain Qlu-1-3△M as a template; 4. Blank control.

[0041] Figure 3 Yield of 2-hydroxyphenazine after 48 hours of fermentation by different strains. Detailed Implementation

[0042] 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.

[0043] 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.

[0044] In the following examples, the fermentation method, extraction method and HPLC detection method of the genetically engineered strains Qlu-1-3△M and Qlu-1-3△MY are exactly the same as the relevant methods in Example 1 of patent ZL202011026637.X.

[0045] Example 1

[0046] 1. Inoculate Pseudomonas aeruginosa Qlu-1-3 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.

[0047] 2. Search within the sequenced genome data of *Pseudomonas aeruginosa* Qlu-1. mdtA Gene and upstream / downstream sequences were amplified using the genome of *Pseudomonas aeruginosa* Qlu-1-3 as a template and primers F1 / R1 and F2 / R2, respectively. mdtA upstream gene sequence mdtA-U , mdtAThe downstream sequence mdtA-D of the gene was used as a template. Using mdtA-U and mdtA-D as templates and F1 / R2 as primers, the mdtA-U and mdtA-D fragments were ligated by fusion PCR to obtain the fusion fragment mdtA-UD.

[0048] 3. The fusion fragment mdtA-UD was ligated with the knockout plasmid pk18moBsacB to construct the recombinant plasmid pk18-mdtA-UD.

[0049] 4. The recombinant plasmid pk18-mdtA-UD was introduced into Escherichia coli S17-1 (λpir) by heat shock transformation.

[0050] 5. Perform a biparental hybridization culture between Escherichia coli S17-1 (λpir) and Pseudomonas aeruginosa Qlu-1-3, and introduce the recombinant plasmid pk18-mdtA-UD into Pseudomonas aeruginosa Qlu-1-3.

[0051] 6. The mutant strain Qlu-1-3△M was obtained through sucrose plate screening and photocopy screening.

[0052] 7. Validate Qlu-1-3 using PCR verification. mdtA Gene knockout strain Qlu-1-3△M ( Figure 2 ).

[0053] 8. After fermentation, 2-hydroxyphenazine was extracted from the fermentation broth. HPLC analysis showed that the yield of 2-hydroxyphenazine from the strain increased to 156.3 mg / L after 48 hours.

[0054] Example 2

[0055] Using the same method as in Example 1 gspM The gene was knocked out from Qlu-1-3△M to obtain the mutant strain Qlu-1-3△MY. After fermentation, the yield of 2-hydroxyphenazine reached 216.4 mg / L after 48 h.

[0056] 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 method for constructing Pseudomonas chlororaphis for producing 2-hydroxyphenazine, characterized by, include: Starting with the 2-hydroxyphenazine-producing strain Qlu-1-3, the genome was knocked out mdtA In genes or knockout genomes mdtA Genes and gspM Genes were used to obtain corresponding genetically engineered strains; The 2-hydroxyphenazine-producing strain Qlu-1-3 is *Pseudomonas aeruginosa* (…). Pseudomonas chlororaphis Qlu-1 was the starting strain, and negative regulatory genes were continuously knocked out. psrA and parS And thus; The preservation number of the *Pseudomonas aeruginosa* Qlu-1 is CCTCCNO: M2020108.

2. The method for constructing *Pseudomonas aeruginosa* for producing 2-hydroxyphenazine as described in claim 1, characterized in that, The mdtA The gene sequence is shown in SEQ ID NO.

1.

3. The method for constructing *Pseudomonas aeruginosa* for producing 2-hydroxyphenazine as described in claim 1, characterized in that, The gspM The gene sequence is shown in SEQ ID NO.

7.

4. The method for constructing *Pseudomonas aeruginosa* for producing 2-hydroxyphenazine as described in claim 1, characterized in that, The knockout genome mdtA The specific steps involved in gene generation include: Searching within the already sequenced Qlu-1 genome sequence mdtA The gene sequence and its upstream and downstream sequences were used to obtain the mdtA-UD fragment via fusion PCR. The mdtA-UD fragment was ligated to plasmid pk18moBsacB using enzyme digestion and ligation technology to construct... mdtA Gene knockout plasmid pK18-mdtA-UD; The mdtA The gene knockout plasmid pK18-mdtA-UD was introduced into E. coli S17-1 (λpir), and then subjected to parent hybridization culture with the 2-hydroxyphenazine-producing strain Qlu-1-3. Positive clones were screened to obtain the knockout plasmid. mdtA The gene is from strain Qlu-1-3△M.

5. The method for constructing *Pseudomonas aeruginosa* for producing 2-hydroxyphenazine as described in claim 4, characterized in that, mdtA The base sequences of the upstream and downstream fusion fragments of the gene are shown in SEQ ID NO.

2.

6. The method for constructing *Pseudomonas aeruginosa* for producing 2-hydroxyphenazine as described in claim 4, characterized in that, The mdtA The gene knockout primers include: mdtA-F1, mdtA-R1, mdtA-F2, and mdtA-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 method for constructing *Pseudomonas aeruginosa* for producing 2-hydroxyphenazine as described in claim 4, characterized in that, Knockout genome gspM The specific steps involved in gene generation include: Searching within the already sequenced Qlu-1 genome sequence gspM Gene sequences and their upstream and downstream sequences were obtained using fusion PCR. gspM -UD fragment; The gspM-UD fragment was ligated to plasmid pk18moBsacB using enzyme digestion and ligation technology to construct... gspM Gene knockout plasmid pK18-gspM-UD; The gspM The gene knockout plasmid pK18-gspM-UD was introduced into E. coli S17-1 (λpir), and then subjected to parent-to-parent hybridization culture with strain Qlu-1-3ΔM. Positive clones were screened to obtain the knockout plasmid. gspM The gene is from strain Qlu-1-3△MG.

8. The method for constructing *Pseudomonas aeruginosa* for producing 2-hydroxyphenazine as described in claim 7, characterized in that, gspM The base sequences of the upstream and downstream fusion fragments of the gene are shown in SEQ ID NO. 8; or, gspM The gene knockout primers include: gspM-F1, gspM-R1, gspM-F2, and gspM-R2, with sequences shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively.

9. A genetically engineered strain for producing 2-hydroxyphenazine, characterized in that, The engineered strain is the Qlu-1-3 strain whose genome has been knocked out of its 2-hydroxyphenazine-producing genome. mdtA In the genome of the 2-hydroxyphenazine-producing strain Qlu-1-3, the gene or knockout gene is present. mdtA Genes and gspM Inherited by genes; The 2-hydroxyphenazine-producing strain Qlu-1-3 is *Pseudomonas aeruginosa* (…). Pseudomonas chlororaphis Qlu-1 was the starting strain, and negative regulatory genes were continuously knocked out. psrA and parS And thus; The preservation number of the *Pseudomonas aeruginosa* Qlu-1 is CCTCCNO: M2020108.

10. The use of the genetically engineered strain according to claim 9 in the production of 2-hydroxyphenazine.

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