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Genetic engineering strain high in yield of phenazine-1-formamide and construction method thereof

The technology of a genetically engineered strain and construction method is applied in the field of genetically engineered strains and its construction for high-yielding phenazine-1-carboxamide, which can solve the problems of low yield and efficiency of phenazine-1-carboxamide, and achieve increased yield, The effect of effective biological control

Pending Publication Date: 2017-05-10
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] Aiming at the defects in the prior art, the purpose of the present invention is to provide a genetically engineered strain of high-yield phenazine-1-carboxamide and its construction method, to overcome the low yield and efficiency of the production of phenazine-1-carboxamide by existing bacterial strains Defects, providing a genetically engineered strain for producing phenazine-1-carboxamide and uses thereof

Method used

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  • Genetic engineering strain high in yield of phenazine-1-formamide and construction method thereof
  • Genetic engineering strain high in yield of phenazine-1-formamide and construction method thereof
  • Genetic engineering strain high in yield of phenazine-1-formamide and construction method thereof

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Effect test

Embodiment 1

[0050] Embodiment 1, the construction of lon gene in vitro mutant

[0051] Primers were designed according to the lon gene and its upstream and downstream sequences in the Pseudomonas chloropinus HT66 genome (see Table 1):

[0052] Table 1

[0053]

[0054] Using the genomic DNA as a template, the corresponding fragment in the genome is amplified.

[0055] The upstream and downstream PCR products were ligated by fusion PCR, and the fusion PCR product and the pK18mobsacB vector were respectively digested with EcoRI and XbaI, recovered through the column, and ligated with T4 ligase to obtain in vitro mutant plasmids.

[0056] Escherichia coli S17 was transformed with the in vitro mutant plasmid. The S17 strain carrying the recombinant plasmid was fully activated, inoculated in LB medium containing 50 mg / L kanamycin, and cultured at 37°C and 180 rpm for 12 hours. The fully activated HT66 strain was inoculated in LB medium and cultured at 28°C and 180 rpm for 12h. Collect...

Embodiment 2

[0058] Embodiment 2, construction of parS gene in vitro mutant

[0059] Primers were designed according to the parS and upstream and downstream sequences in the Pseudomonas chloropinus HT66 genome (see Table 2):

[0060] Table 2

[0061]

[0062] Using the genomic DNA as a template, the corresponding fragment in the genome is amplified.

[0063] The upstream and downstream PCR products were ligated by fusion PCR, and the fusion PCR product and the pK18mobsacB vector were respectively digested with BamHI and HindIII, recovered by column, and ligated with T4 ligase to obtain in vitro mutant plasmids.

[0064] Escherichia coli S17 was transformed with the obtained in vitro mutant plasmid. The S17 strain carrying the recombinant plasmid was fully activated, inoculated in LB medium containing 50 mg / L kanamycin, and cultured at 37°C and 180 rpm for 12 hours. The fully activated HT66 strain was inoculated in LB medium and cultured at 28°C and 180 rpm for 12 hours. Collect an...

Embodiment 3

[0065] Example 3, Construction of lon and parS double mutant strain HT66ΔlonΔparS

[0066] The S17 strain carrying the parS recombinant plasmid was fully activated, inoculated in LB medium containing 50 mg / L kanamycin, and cultured at 180 rpm at 37°C for 12 hours. The fully activated HT66Δlon strain was inoculated in LB medium and cultured at 28°C and 180 rpm for 12 hours. Collect and wash the two kinds of bacteria at 5000 rpm, resuspend in LB medium and mix with EP tube at a concentration ratio of HT66Δlon:S17 of 3:1, let it stand for 1 hour; take the mixed bacteria solution and spot it on the LB plate, Cultivate at 28°C for 24 hours; scrape and resuspend the mixed colonies grown in LB, dilute and spread on LB plates with kanamycin 50mg / L and ampicillin 100mg / L, culture at 28°C for 2-3 days and then pick Coat the single clone on a 10% sucrose plate, culture at 28°C for 1-2 days; pick the single clone on the sucrose plate, spot it on the kanamycin 50 mg / L resistant LB plate...

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Abstract

The invention discloses a genetic engineering strain high in yield of phenazine-1-formamide and a construction method thereof. The genetic engineering strain is a genetic engineering strain obtained by knocking out one or two of lon gene and parS gene of HT66CCTCC NO: M 2013467 genome of Pseudomonas chlororaphis, or a genetic engineering strain obtained by knocking out one or two of lon gene and parS gene of HT66CCTCC NO: M 2013467 rpeA and psrA single mutation strain or amphimutation strain genome of Pseudomonas chlororaphis, wherein single mutation strain is HT66 Delta rpeA and HT66 Delta psrA, and amphimutation strain is HT66 Delta rpeA Delta psrA. Compared with the prior art, the genetic engineering strain has the advantages that after lon and parS genes are delected from the genome of Pseudomonas chlororaphis, fermentation yield of phenazine-1-formamide is up to 2425mg / L, and the genetic engineering strain can be used for industrial production and agricultural application of phenazine-1-formamide.

Description

technical field [0001] The invention belongs to the field of genetic engineering, and relates to a pseudomonas genetically engineered strain and a construction method thereof, in particular to a genetically engineered strain with high production of phenazine-1-carboxamide and a construction method thereof. Background technique [0002] There are 1,600 kinds of common crop disease organisms in my country, and the annual loss is about 100 billion yuan, which seriously affects the development of my country's agriculture. In recent years, the large-scale use of chemical pesticides has led to the increasing resistance of pests and diseases. In addition, the application of high-concentration and toxic chemical pesticides has also caused great pollution to farmland and water environment, seriously threatening my country's food security and life and health. Therefore, biopesticides with low toxicity, low residue and less resistance to pesticides have attracted great attention from ...

Claims

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Application Information

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IPC IPC(8): C12N1/21C12N15/78C12P17/12C12R1/38
CPCC12P17/12C07K14/21
Inventor 彭华松张雪洪谭剑
Owner SHANGHAI JIAO TONG UNIV
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