A method for increasing the yield of 2-hydroxyphenazine

Abstract

This invention relates to the field of genetic engineering technology and provides a method for increasing the yield of 2-hydroxyphenazine, using strain Qlu-1ΔHΔPΔO as the starting strain, knocking out ppkA Genes were used to obtain strain Qlu-1ΔHΔPΔOΔP; ppkA The gene sequence is shown in SEQ ID NO. 1. This invention uses *Pseudomonas aeruginosa* Qlu-1ΔHΔPΔO from ZL 202210461035.X as the starting strain, and employs a scarless knockout method... ppkA Genes were used to obtain the engineered strain Qlu-1ΔHΔPΔOΔP. After fermentation and yield testing, it was found that the 2-hydroxyphenazine yield of Qlu-1ΔHΔPΔOΔP was increased to 293.2 mg / L, thus achieving an increase in the yield of 2-hydroxyphenazine by the strain.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to a method for increasing the yield of 2-hydroxyphenazine. Background Technology

[0002] The information disclosed in the background section of this invention is intended only to enhance the 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] The growth of the world's population is driving up global food demand. It is estimated that pests and diseases cause approximately 40% of the world's annual total production loss, resulting in significant economic losses. Chemical pesticides are widely used to prevent disease and pest damage; however, due to environmental and mammalian safety concerns, the increasing use of agricultural chemicals has become a global concern and has been widely criticized. Minimizing the use of chemical pesticides and finding alternatives has spurred the use of microbial-based methods, an effective and environmentally sensitive approach. Microbial-derived pesticides have become an important component of agricultural sustainability initiatives. Phenyrazines are nitrogen-containing heterocyclic compounds, a class of bioactive substances secreted by microorganisms such as Pseudomonas and Streptomyces, playing a crucial role in interactions between strains and other organisms. Currently, phenazine-1-carboxylic acid (PCA) and 2-hydroxyphenazine (2-OH-PHZ) are widely used, with 2-OH-PHZ showing good efficacy in plant biocontrol and broad application potential. *Pseudomonas aeruginosa* (…) Pseudomonas chlororaphis Qlu-1 can secrete phenazine compounds with antifungal activity, such as phenazine-1-carboxylic acid (PCA) and 2-hydroxy-phenazine (2-OH-PHZ). However, the production of 2-hydroxy-phenazine in wild-type Pseudomonas aeruginosa Qlu-1 is low, only about 6.5 mg / L. It is of great significance to increase the production of 2-hydroxy-phenazine in Pseudomonas aeruginosa through genetic engineering.

[0004] The inventor's previous patent, ZL 202210461035.X, disclosed a *Pseudomonas aeruginosa* strain that produces 2-hydroxyphenazine. Using Qlu-1 as the starting strain, genetic engineering was employed to increase the 2-hydroxyphenazine yield to 189 mg / L. However, to reduce production costs and enhance product market competitiveness, the yield of the engineered strain still needs further improvement. Summary of the Invention

[0005] In view of this, the present invention provides a method for increasing the yield of 2-hydroxyphenazine. The present invention uses Qlu-1ΔHΔPΔO (a derivative strain of Qlu-1) as the starting strain and, through genetic engineering operations, increases the yield of 2-hydroxyphenazine in the strain to 293.2 mg / L.

[0006] To achieve the above objectives, the present invention adopts the following technical solution: In a first aspect, the present invention provides a genetically engineered bacterium that increases the yield of 2-hydroxyphenazine, using strain Qlu-1ΔHΔPΔO as the starting strain, and knocking out... ppkA Genes were used to obtain strain Qlu-1ΔHΔPΔOΔP; ppkA The gene sequence is shown in SEQ ID NO. 1.

[0007] The strain Qlu-1ΔHΔPΔO of the present invention is a derivative strain of Qlu-1, disclosed in ZL 202210461035.X.

[0008] ppkA Gene knockout primers (primers for obtaining the ppkA-UD fusion fragment) include: ppkA-F1 / ppkA-R1, ppkA-F2 / ppkA-R2.

[0009] The sequence of ppkA-F1 is shown in SEQ ID NO. 3.

[0010] The sequence of ppkA-R1 is shown in SEQ ID NO. 4.

[0011] The sequence of ppkA-F2 is shown in SEQ ID NO. 5.

[0012] The sequence of ppkA-R2 is shown in SEQ ID NO. 6.

[0013] A second aspect of the present invention provides a method for preparing a genetically engineered bacterium that increases the yield of 2-hydroxyphenazine, using strain Qlu-1ΔHΔPΔO as the starting strain, and knocking out... ppkA Genes were extracted to obtain strain Qlu-1ΔHΔPΔOΔP.

[0014] In some embodiments, the preparation method specifically includes the following steps: PCR amplification of *Pseudomonas aeruginosa* (… Pseudomonas chlororaphis Qlu-1 ppkA Gene and upstream and downstream sequence fragments ppkA-UD were used to construct the plasmid pk18mobsacB. ppkA The gene knockout plasmid pK18-ppkA-UD was introduced into Escherichia coli via heat shock transformation and co-cultured with Pseudomonas aeruginosa Qlu-1ΔHΔPΔO to screen for knockout. ppkA The strain is strain Qlu-1ΔHΔPΔOΔP.

[0015] Among them, Pseudomonas aeruginosa ( Pseudomonas chlororaphisQlu-1 is the *Pseudomonas aeruginosa* described in patent ZL202210461035.X. Pseudomonas chlororaphis Qlu-1.

[0016] In some implementations, the ppkA The gene sequence is shown in SEQ ID NO.1, and the ppkA-UD sequence is shown in SEQ ID NO:2.

[0017] In some embodiments, the preparation method specifically includes the following steps: PCR amplification of *Pseudomonas aeruginosa* (… Pseudomonas chlororaphis Qlu-1 ppkA The gene and its upstream and downstream sequence fragments ppkA-UD were amplified using the Qlu-1ΔHΔPΔO genome as a template and ppkA-F1 / ppkA-R1 and ppkA-F2 / ppkA-R2 as primers. ppkA The upstream and downstream fragments ppkA-U and ppkA-D of the gene were used as templates. Using ppkA-F1 / ppkA-R2 as primers, fusion PCR was employed to ligate the ppkA-U and ppkA-D fragments, yielding the fusion fragment ppkA-UD. The ppkA-UD fragment was then ligated into the plasmid pk18mobsacB to construct the gene. ppkA The plasmid pK18-ppkA-UD was knocked out, and then introduced into *E. coli* via heat shock transformation. It was then co-cultured with *Pseudomonas aeruginosa* Qlu-1ΔHΔPΔO. The knockout was screened and verified by PCR. ppkA Strain Qlu-1ΔHΔPΔOΔP.

[0018] The sequence of ppkA-F1 is shown in SEQ ID NO. 3.

[0019] The sequence of ppkA-R1 is shown in SEQ ID NO. 4.

[0020] The sequence of ppkA-F2 is shown in SEQ ID NO. 5.

[0021] The sequence of ppkA-R2 is shown in SEQ ID NO. 6.

[0022] In a third aspect, the present invention provides a strain Qlu-1ΔHΔPΔOΔP prepared by the above method.

[0023] A fourth aspect of the present invention provides the application of the above-mentioned strain Qlu-1ΔHΔPΔOΔP in the production of 2-hydroxyphenazine, comprising: inoculating strain Qlu-1ΔHΔPΔOΔP into a fermentation medium to produce 2-hydroxyphenazine.

[0024] A fifth aspect of the present invention provides a method for producing 2-hydroxyphenazine using the above-mentioned strain Qlu-1ΔHΔPΔOΔP, comprising: inoculating strain Qlu-1ΔHΔPΔOΔP into a fermentation medium to produce 2-hydroxyphenazine; Preferably, the fermentation medium is selected from KB medium.

[0025] Compared with the prior art, the present invention has achieved the following beneficial effects: (1) The genetically engineered Pseudomonas aeruginosa of the present invention can produce 293.2 mg / L of 2-hydroxyphenazine in 72 h, which improves the production efficiency of the strain and provides a solid foundation for the industrialization of subsequent engineered strains. Attached Figure Description

[0026] The accompanying drawings, which form part of this specification, are used to provide a further understanding of the invention. The illustrative embodiments and descriptions of the invention are used to explain the invention and do not constitute an undue limitation thereof. Obviously, those skilled in the art can obtain other drawings based on these drawings without any inventive effort.

[0027] Figure 1 Electrophoresis diagram of the mutant plasmid pK18-ppkA-UD in Example 1; where, (A) ppkA Amplification of upstream and downstream homologous arm fragments: 1, ppkA Upstream homologous arm amplification; 2, ppkA 3. Upstream homologous arm amplification; 4. DNA Ladder DL5000; ppkA Downstream homologous arm fragment amplification; 5, ppkA Downstream homologous arm fragment amplification; (B) ppkA Amplification of upstream and downstream homologous arm fusion fragments: 1, ppkA Upstream and downstream homologous arm fusion fragments; 2, ppkA Upstream and downstream homologous arm fusion fragments; 3, DNA LadderDL5000; 4, ppkA Upstream and downstream homologous arm fusion fragments; 5, ppkA Upstream and downstream homologous arm fusion fragment.

[0028] Figure 2 In Example 1 ppkA PCR validation diagram of the knockout strain; External primer detection: 1. Blank control; 2. DNALadder DL5000; 3. Fragment amplified using Qlu-1ΔHΔPΔO genome as template; 4. ppkA The gene knockout strain Qlu-1ΔHΔPΔOΔP genome was used as a template for amplification of the fragment; internal primer detection: 1, using the Qlu-1ΔHΔPΔO genome as a template for amplification of the fragment; 2, using...ppkA 3. Gene knockout strain Qlu-1ΔHΔPΔOΔP genome as template for amplification fragment; 4. Blank control; 5. DNA Ladder DL5000.

[0029] Figure 3 The yield of 2-hydroxyphenazine after 72 hours of fermentation by different strains. Detailed Implementation

[0030] 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 herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0031] Example 1 (1) Inoculate Pseudomonas aeruginosa Qlu-1ΔHΔPΔO into KB medium, and culture overnight at 30°C with a shaker at 180 rpm. Extract the genome of Pseudomonas aeruginosa Qlu-1ΔHΔPΔO using a prokaryotic genome extraction kit and store at -20°C for later use.

[0032] (2) Searching for genome data of the sequenced Pseudomonas aeruginosa Qlu-1 ppkA Gene and upstream / downstream sequences were amplified using the genome of *Pseudomonas aeruginosa* Qlu-1ΔHΔPΔO as a template and ppkA-F1 / ppkA-R1 and ppkA-F2 / ppkA-R2 as primers. ppkA Upstream sequence ppkA-U, ppkA The downstream sequence ppkA-D was used as a template with ppkA-U and ppkA-D as templates and ppkA-F1 / ppkA-R2 as primers. Fusion PCR was then used to ligate the ppkA-U and ppkA-D fragments to obtain the fusion fragment ppkA-UD. Figure 1 ).

[0033] (3) The fusion fragment ppkA-UD was ligated with the knockout plasmid pk18mobsacB to construct the recombinant plasmid pk18-ppkA-UD.

[0034] (4) The recombinant plasmid pk18-ppkA-UD was introduced into Escherichia coli S17-1 (λpir) by heat shock transformation.

[0035] (5) Escherichia coli S17-1(λpir) and Pseudomonas aeruginosa Qlu-1ΔHΔPΔO were hybridized to each parent and the recombinant plasmid pk18-ppkA-UD was introduced into Pseudomonas aeruginosa Qlu-1ΔHΔPΔO.

[0036] (6) The mutant strain Qlu-1ΔHΔPΔOΔP was obtained by screening with sucrose plates and photocopy screening.

[0037] The Qlu-1ΔHΔPΔO was verified using PCR. ppkA Knockout strain Qlu-1ΔHΔPΔOΔP. Figure 2 ) Strains Qlu-1ΔHΔPΔOΔP, Qlu-1, and Qlu-1ΔHΔPΔO were inoculated into KB medium and fermented at 30°C. 2-hydroxyphenazine was extracted from the fermentation broth. HPLC analysis revealed that strain Qlu-1ΔHΔPΔOΔP produced 293.2 mg / L of 2-hydroxyphenazine after 72 hours. Figure 3 As shown.

[0038] 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 increases the yield of 2-hydroxyphenazine, characterized in that, With Pseudomonas aeruginosa ( Pseudomonas chlororaphis Qlu-1ΔHΔPΔO was the starting strain, and the knockout was... ppkA Genes were used to obtain strain Qlu-1ΔHΔPΔOΔP; ppkA The gene sequence is shown in SEQ ID NO.

1.

2. A method for preparing a genetically engineered bacterium that increases the yield of 2-hydroxyphenazine, characterized in that, With Pseudomonas aeruginosa ( Pseudomonas chlororaphis Qlu-1ΔHΔPΔO was the starting strain, and the knockout was... ppkA Genes were used to obtain strain Qlu-1ΔHΔPΔOΔP; ppkA The gene sequence is shown in SEQ ID NO.

1.

3. The method for preparing the genetically engineered bacteria for increasing 2-hydroxyphenazine yield as described in claim 2, characterized in that, The preparation method specifically includes the following steps: PCR amplification of *Pseudomonas aeruginosa* (… Pseudomonas chlororaphis Qlu-1 ppkA Gene and upstream and downstream sequence fragments ppkA-UD were used to construct the plasmid pk18mobsacB. ppkA The gene knockout plasmid pK18-ppkA-UD was introduced into *E. coli* S17-1λpir via heat shock transformation and co-cultured with *Pseudomonas aeruginosa* Qlu-1ΔHΔPΔO to screen for knockout. ppkA The strain is strain Qlu-1ΔHΔPΔOΔP; The ppkA-UD sequence is shown in SEQ ID NO:

2.

4. The strain Qlu-1ΔHΔPΔOΔP prepared by the method according to any one of claims 2-3.

5. The use of the strain Qlu-1ΔHΔPΔOΔP according to any one of claims 1 and 4 in the production of 2-hydroxyphenazine, characterized in that, include: The strain Qlu-1ΔHΔPΔOΔP was inoculated into a fermentation medium to produce 2-hydroxyphenazine; The fermentation medium is selected from KB medium.

6. The method for producing 2-hydroxyphenazine using the strain Qlu-1ΔHΔPΔOΔP according to any one of claims 1 and 4, characterized in that, include: The strain Qlu-1ΔHΔPΔOΔP was inoculated into a fermentation medium to produce 2-hydroxyphenazine; The fermentation medium is selected from KB medium.

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