Construction method of phenazine-1-carboxylic acid high-yield engineering strain

Abstract

This invention belongs to the field of bioengineering technology and provides a method for constructing a high-yield engineered strain of phenazine-1-carboxylic acid. Using QPCA-7, a high-yield strain of phenazine-1-carboxylic acid, as the starting strain, the production capacity of phenazine-1-carboxylic acid was increased to 10859.3 mg / L by successively knocking out recently discovered genes such as GspC and Algb, which have negative regulatory effects on phenazine-1-carboxylic acid.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology and relates to a method for constructing an engineered strain that produces a high bioactive substance, phenazine-1-carboxylic acid. 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] With the rapid growth of the global population, food supply faces immense pressure. Simultaneously, food production is severely threatened by various pathogens and pests, forcing the use of large quantities of pesticides to control these pests. Currently, most pesticides used are chemically synthesized. While they offer good control, they are characterized by environmental pollution and slow degradation, contradicting the principles of sustainable development in modern society. Biopesticides, a class of natural antibacterial and insecticidal agents derived from nature, offer significant advantages over chemical pesticides, including lower toxicity, environmental friendliness, and reduced likelihood of resistance development. They have attracted increasing attention from scholars and are gradually being applied in biological control practices. Phenazine-1-carboxylic acid is an excellent example of a biopesticide discovered in recent years. A 1% suspension of phenazine-1-carboxylic acid shows good control effects against rice sheath blight, rice false smut, rice blast, wheat scab, cucumber downy mildew, cucumber gray mold, watermelon wilt, and pepper blight. Furthermore, it is biodegradable in the environment. Due to the above characteristics, phenazine-1 carboxylic acid was granted a pesticide certificate by the Chinese Ministry of Agriculture in 2011 and named "Shenzinmycin".

[0004] Currently, although there are chemical methods for producing phenazine-1-carboxylic acid, these methods require harsh conditions and release toxic and harmful substances into the environment. Therefore, they are mainly produced by *Pseudomonas aeruginosa*, a type of Pseudomonas. However, *Pseudomonas aeruginosa* is a relatively common opportunistic pathogen in hospitals and is not very suitable as a production strain for widespread application. In the inventor's previous work (patent CN112126611A), a genetically engineered strain, QPCA-7, was obtained that could produce 7854 mg / L of phenazine-1-carboxylic acid in 48 hours. However, the fermentation titer was still not high. Summary of the Invention

[0005] To address the aforementioned problems, this invention provides a method for constructing a high-yield engineered strain of phenazine-1-carboxylic acid. Using QPCA-7, a high-yield strain of phenazine-1-carboxylic acid, as the starting strain, the phenazine-1-carboxylic acid production capacity of the strain was increased to 10859.3 mg / L by successively knocking out recently discovered genes such as GspC and Algb, which have negative regulatory effects on phenazine-1-carboxylic acid.

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

[0007] In a first aspect, the present invention provides a high-yield engineered strain of phenazine-1-carboxylic acid, said engineered strain being obtained by knocking out the GspC gene and / or Algb gene in the genome of engineered strain QPCA-7;

[0008] The engineered strain QPCA-7 was obtained by knocking out one or more of the phzO, lon, rsmE, psrA, parS, rpeA, and pykF genes in the genome of Pseudomonas aeruginosa Qlu-1.

[0009] The preservation number of the *Pseudomonas aeruginosa* Qlu-1 is CCTCC NO: M 2020108.

[0010] This invention improves the fermentation potency of phenazine-1-carboxylic acid strain and reduces the production cost of phenazine-1-carboxylic acid through genetic engineering.

[0011] A second aspect of the present invention provides a method for constructing a high-yield engineered strain of phenazine-1-carboxylic acid, comprising:

[0012] The QPCA-7 strain is obtained by knocking out the GspC gene and / or Algb gene in its genome.

[0013] In some embodiments, the specific steps for knocking out the GspC gene in the genome of engineered strain QPCA-7 include:

[0014] The upstream and downstream fragments of the GspC gene were extracted and ligated by PCR to obtain the GspC-ud fragment;

[0015] The GspC-ud fragment was ligated into plasmid pk18moBsacB to construct the GspC gene knockout plasmid pK18-GspC-ud;

[0016] The GspC gene knockout plasmid pK18-GspC-ud was introduced into Escherichia coli S17-1(λ), ​​and then subjected to parental hybridization culture with Pseudomonas aeruginosa QPCA-7.

[0017] Positive clones were screened to obtain strain QPCA-8, which had the GspC gene knocked out.

[0018] In some embodiments, the specific steps for knocking out the Algb gene in the genome of engineered strain QPCA-7 include:

[0019] The upstream and downstream fragments of the Algb gene were extracted and ligated by PCR to obtain the Algb-ud fragment;

[0020] The Algb-ud fragment was ligated into plasmid pk18moBsacB to construct the Algb gene knockout plasmid pK18-Algb-ud.

[0021] The Algb knockout plasmid pK18-Algb-ud was introduced into Escherichia coli S17-1(λ), ​​and then subjected to parental hybridization culture with Pseudomonas aeruginosa QPCA-7.

[0022] Positive clones are screened to obtain strains with the Algb gene knocked out.

[0023] Beneficial effects of the present invention

[0024] (1) This invention uses QPCA-7, which produces high levels of phenazine-1-carboxylic acid, as the starting strain. By successively knocking out newly discovered genes such as GspC and Algb that have a negative regulatory effect on phenazine-1-carboxylic acid, the production capacity of the strain of phenazine-1-carboxylic acid is increased to 10859.3 mg / L.

[0025] (2) The present invention utilizes KB medium for fermentation, and the yield reaches 10859.3 mg / L, which greatly improves the production capacity of the strain and provides a solid foundation for the industrialization of subsequent engineered strains.

[0026] (3) The construction method of the present invention is simple, practical and easy to promote. Attached Figure Description

[0027] 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.

[0028] Figure 1 Electrophoresis diagram of the mutant plasmid pK18-GspC-ud constructed in Example 1 of this invention;

[0029] Figure 2 This refers to the screening of GspC gene double-resistance plate hybridization between parents in Example 1 of the present invention;

[0030] Figure 3 This is the GspC gene double-crossover positive monoclonal sample screened using the photocopying method in Example 1 of this invention;

[0031] Figure 4 The yield of phenazine-1 carboxylic acid by different strains in Example 1 of the present invention. Detailed Implementation

[0032] 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.

[0033] A method for constructing a high-yield engineered strain of phenazine-1-carboxylic acid includes:

[0034] Using QPCA-7, a strain that produces high levels of phenazine-1-carboxylic acid, as the starting strain, the production capacity of phenazine-1-carboxylic acid was increased to 10859.3 mg / L by successively knocking out newly discovered genes such as GspC and Algb, which have negative regulatory effects on phenazine-1-carboxylic acid.

[0035] This paper introduces gene manipulation using the scarless knockout of the GspC gene as an example.

[0036] The QPCA-7 genome was extracted using a kit;

[0037] Search for the GspC gene and its upstream and downstream sequences in the sequenced Qlu-1 genome, and use PCR to retrieve and ligate the upstream and downstream fragments of the GspC gene: GspC-ud;

[0038] The GspC gene knockout plasmid pK18-GspC-ud was constructed by ligating the GspC-ud fragment to plasmid pk18moBsacB using restriction endonuclease digestion and ligation technology.

[0039] pK18-GspC-ud was introduced into Escherichia coli S17-1(λ) via heat shock transformation;

[0040] After co-culturing Pseudomonas aeruginosa QPCA-7 and Escherichia coli S17-1(λ), ​​the mixture was plated on KB(A + K + Single colonies were selected after the double-antibiotic plate was applied.

[0041] Single colonies were plated on 15% sucrose KB plates containing ampicillin, and strain QPCA-8 with the GspC gene knocked out was screened and verified by PCR.

[0042] In some embodiments, the sequence of the GspC gene is shown in SEQ ID NO.1.

[0043] In some embodiments, the base sequences of the upstream and downstream fusion fragments of the GspC gene are shown in SEQ ID NO.2.

[0044] In some embodiments, the GspC gene knockout primers include: GspC-F1, GspC-R1, GspC-F2, and GspC-R2, with sequences as shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

[0045] In some embodiments, the sequence of the Algb gene is shown in SEQ ID NO.7.

[0046] In some embodiments, the base sequences of the upstream and downstream fusion fragments of the Algb gene are shown in SEQ ID NO.8.

[0047] In some embodiments, the Algb gene knockout primers include Algb-F1, Algb-R1, Algb-F2, and Algb-R2, with sequences as shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively.

[0048] In some embodiments, the high-producing PCA strain QPCA-9 (a double knockout strain of GspC and Algb genes) was obtained by successively knocking out the negative regulatory genes GspC and Algb in the QPCA-7 genome using the same method. After fermentation, HPLC detection using ethyl acetate extraction showed that QPCA-9 could produce 10859.3 mg / L of phenazine-1-carboxylic acid at 48 h, laying the foundation for further large-scale fermentation production.

[0049] 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.

[0050] Qlu-1 is a strain of *Pseudomonas aeruginosa* capable of producing phenazine-1-carboxylic acid and 2-hydroxyphenazine. In the inventor's previous work (patent CN112126611A), starting with Qlu-1, the strain QPCA was obtained by knocking out the modified gene phzO, thus inactivating the phzO enzyme that catalyzes the conversion of PCA to 2-hydroxyphenazine. The strain then specifically accumulated PCA. Subsequently, based on strain QPCA, genes with negative regulatory effects, such as lon, rsmE, psrA, parS, rpeA, and pykF, were knocked out in its genome to obtain the engineered strain QPCA-7, which can produce 7854 mg / L of phenazine-1-carboxylic acid in 48 hours.

[0051] In the following examples, the engineered strain QPCA-7 obtained in patent CN112126611A was used as the starting strain. By successively knocking out the newly discovered genes such as GspC and Algb that have a negative regulatory effect on phenazine-1-carboxylic acid, the production capacity of the strain of phenazine-1-carboxylic acid was increased to 10859.3 mg / L.

[0052] Example 1

[0053] The specific steps for GspC gene knockout.

[0054] 1. Inoculate LB (A) with Pseudomonas aeruginosa strain QPCA-7, derived from Pseudomonas aeruginosa Qlu-1. +The culture medium was used to extract the genome of QPCA-7 overnight at 30°C and 180 rpm on a shaker. The genome was then extracted using a genome extraction kit and stored at -20°C for later use.

[0055] 2. Search for the GspC gene and its upstream and downstream sequences in the sequenced Qlu-1 genome data. Using the QPCA-7 strain genome as a template, amplify the upstream fragment GspC-U and the downstream fragment GspC-D of the GspC gene using primers GspC-F1 / GspC-R1 and GspC-F2 / GspC-R2, respectively. Using GspC-U and GspC-D as templates, and GspC-F1 / GspC-R1 as a template, amplify the upstream and downstream fusion fragment GspC-UD. Figure 1 );

[0056] 3. The fusion fragment GspC-UD was ligated with the knockout plasmid pk18moBsacB to construct the recombinant plasmid pk18-GspC-UD;

[0057] 4. The recombinant plasmid pk18-GspC-UD was introduced into Escherichia coli S17-1(λ) by heat shock transformation;

[0058] 5. Perform a two-parent hybridization culture between Escherichia coli S17-1(λ) and Pseudomonas QPCA-7, and introduce the recombinant plasmid pk18-GspC-UD into Pseudomonas QPCA-7;

[0059] 6. Screening via sucrose plate ( Figure 2 ), photocopy screening ( Figure 3 QPCA-7GspC knockout strains were screened using methods such as PCR screening, i.e., the GspC gene was knocked out (QPCA-7△GspC) to obtain strain QPCA-8.

[0060] This invention further knocked out the Algb gene (QPCA-7△GspC△Algb) from QPCA-8 to obtain strain QPCA-9. HPLC analysis after fermentation showed an increase in the accumulation of phenazine-1-carboxylic acid in the fermentation broth. Figure 4 The strain QPCA-9 was able to produce 10859.3 mg / L of phenazine-1-carboxylic acid in 48 hours.

[0061] 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. An engineered strain producing phenazine-1-carboxylic acid, characterized in that, The engineered strain is a knockout engineered strain QPCA-7 whose genome contains... GspC The gene or the engineered strain is a knockout of the QPCA-7 engineered strain genome. GspC Genes and Algb Inherited by genes; The engineered strain QPCA-7 is a knockout strain of *Pseudomonas aeruginosa* (…). Pseudomonas chlororaphis Qlu-1 genome 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 CCTCC NO: M 2020108; The GspC The gene sequence is shown in SEQ ID NO.1; The Algb The gene sequence is shown in SEQ ID NO.

7.

2. The method for constructing a phenazine-1-carboxylic acid-producing engineered strain as described in claim 1, characterized in that, include: Knockout of the QPCA-7 engineered strain genome GspC In the genome of gene or knockout engineered strain QPCA-7 GspC Genes and Algb Genes, that is.

3. The method for constructing the phenazine-1-carboxylic acid-producing engineered strain as described in claim 2, characterized in that, Knockout of the QPCA-7 engineered strain genome GspC The specific steps involved in gene generation include: PCR fishing and ligation GspC The upstream and downstream segments of the gene are used to obtain the GspC-ud fragment; The GspC-ud fragment was ligated into plasmid pk18moBsacB to construct... GspC Knock out plasmid pK18-GspC-ud; The GspC The gene knockout plasmid pK18-GspC-ud was introduced into Escherichia coli S17-1(λ), ​​and then crossbred with Pseudomonas aeruginosa QPCA-7. Screening for positive clones yields the knockout. GspC The gene is from strain QPCA-8.

4. The method for constructing the phenazine-1-carboxylic acid-producing engineered strain as described in claim 2, characterized in that, GspC The base sequences of the upstream and downstream fusion fragments of the gene are shown in SEQ ID NO.

2.

5. The method for constructing the phenazine-1-carboxylic acid-producing engineered strain as described in claim 2, characterized in that, GspC The gene knockout primers include: GspC-F1, GspC-R1, GspC-F2, and GspC-R2, with sequences shown in SEQ ID NO.3, SEQ ID NO.4, SEQ ID NO.5, and SEQ ID NO.6, respectively.

6. The method for constructing the phenazine-1-carboxylic acid-producing engineered strain as described in claim 3, characterized in that, Knockout of the QPCA-8 engineered strain genome Algb The specific steps involved in gene generation include: PCR fishing and ligation Algb The upstream and downstream segments of the gene are used to obtain the Algb-ud fragment; The Algb-ud fragment was ligated into plasmid pk18moBsacB to construct... Algb Gene knockout plasmid pK18-Algb-ud; The Algb The knockout plasmid pK18-Algb-ud was introduced into Escherichia coli S17-1 (λ), and then crossbred with Pseudomonas aeruginosa QPCA-8. Screening for positive clones yields the knockout. Algb Strains of genes.

7. The method for constructing the phenazine-1-carboxylic acid-producing engineered strain as described in claim 2, characterized in that, Algb The base sequences of the upstream and downstream fusion fragments of the gene are shown in SEQ ID NO.

8.

8. The method for constructing the phenazine-1-carboxylic acid-producing engineered strain as described in claim 2, characterized in that, Algb The gene knockout primers include Algb-F1, Algb-R1, Algb-F2, and Algb-R2, with sequences shown in SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, and SEQ ID NO.12, respectively.

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