Genetic engineering strain for producing 1-hydroxyphenazine as well as preparation method and applications of genetic engineering strain

A genetically engineered strain, the technology of hydroxyphenazine, applied in the field of genetic engineering, can solve the problems of biological safety of strains, difficulty in large-scale development and application, low intracellular content, etc., achieve good industrial application potential, and short fermentation cycle , nutritional requirements simple effect

Inactive Publication Date: 2017-02-15
SHANGHAI JIAO TONG UNIV
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  • Abstract
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  • Claims
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Problems solved by technology

[0006] Current technical defects: natural 1-hydroxyphenazine compounds mainly appear in human opportunistic pathogens - Pseudomonas aeruginosa, in which phenazine-1-carboxylic acid is used as substrate, in phzS The function of the gene is transformed, not only its intracellular content is very low, but also the strain has certain biological safety problems, making it difficult to carry out large-scale development and application

Method used

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  • Genetic engineering strain for producing 1-hydroxyphenazine as well as preparation method and applications of genetic engineering strain
  • Genetic engineering strain for producing 1-hydroxyphenazine as well as preparation method and applications of genetic engineering strain
  • Genetic engineering strain for producing 1-hydroxyphenazine as well as preparation method and applications of genetic engineering strain

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Embodiment 1

[0047] This embodiment relates to a method for preparing a genetically engineered strain for producing 1-hydroxyphenazine, comprising the following steps:

[0048] 1. Construction of recombinant plasmids

[0049] 1. Design primers F2 and R2, and use the Pseudomonas aeruginosa PAO1 genome as a template to amplify its phzS gene by PCR. See the electropherogram figure 2 a.

[0050] 2. Design the upstream homology arm primers F1 and R1 and the downstream homology arm primers F3 and R3 for amplifying phzH respectively, amplify the homology arms by PCR and recover and purify them. See the electrophoresis figure 2 b;

[0051] 3. Connect the plasmid pK18mobsacB through the Infusion system, and integrate the upstream and downstream fragments of the phzS gene and the phzH gene. The fusion fragment is 2.8 kb, see the electrophoresis figure 2 c;

[0052] 4. Transform the competent cells of Escherichia coli DH5α with the recombinant plasmid, and then extract the plasmid for sequenci...

Embodiment 2

[0073]This embodiment relates to a liquid chromatography analysis method utilizing the 1-hydroxyphenazine prepared in Example 1, comprising the following steps:

[0074] Take out 1mL of bacterial liquid from the fermentation broth, centrifuge at 12000rpm for 90s, take 400μL of supernatant and put it in another clean 1.5mL centrifuge tube; add 800μL of ethyl acetate to the centrifuge tube and shake fully; centrifuge the extraction mixture at 5000rpm 5min, take 400μL of the upper organic phase, and place it in another clean 1.5mL centrifuge tube; distill the extracted ethyl acetate phase under reduced pressure, and after it is completely evaporated, add 200μL of methanol, and perform HPLC detection after fully dissolving (if the yield Larger, need to be further diluted before injection). HPLC detection conditions: detection wavelength 254nm, C18 reverse phase column, flow rate 1mL / min, column temperature 30°C. Mobile phase: the aqueous phase is 5mM ammonium acetate solution, an...

Embodiment 3

[0079] This embodiment relates to a method for preparing 1-hydroxyphenazine using the genetically engineered strain obtained in Example 1, comprising the following steps:

[0080] The fully activated strains HT66WT and HT66-S were respectively inoculated into vials containing 5 mL of KB medium and cultured with shaking at 28°C and 180 rpm. After 12 hours, they were inoculated into a 250mL Erlenmeyer flask containing 100mL KB medium at an inoculation ratio of 1%, and cultured for 48h under the same conditions. Take out 1mL bacterial liquid from the fermentation broth at regular intervals, centrifuge at 12000rpm for 90s, and suck up the supernatant with a pipette gun. Use 1 mL of deionized water to resuspend the bacteria, use deionized water as a blank, and measure its optical density value at a wavelength of 600 nm in a spectrophotometer, that is, OD600. During the measurement, the bacterial solution needs to be properly diluted so that the final OD600 reading is between 0.4 a...

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Abstract

The invention provides a genetic engineering strain for producing 1-hydroxyphenazine as well as a preparation method and applications of the genetic engineering strain. The genetic engineering strain is obtained through the following method: the phzH gene in Pseudomonas chlororaphis HT66 CCTCC NO:M2013467 and the genome of the derivative of the phzH gene is replaced by the exogenous phzS gene. The preparation method comprises the following steps: construction of recombinant plasmids, biparental hybridization, and screening of gene substitution mutant strains. For the first time, the exogenous phzS gene replaces the phzH gene in the strain HT66, the obtained genetic engineering strain can produce high-concentration phenazine-1-carboxylic acid through fermentation by taking natural agricultural and sideline products as raw materials, the high-concentration phenazine-1-carboxylic acid is taken as a substrate and is converted, and thus the high-level 1-hydroxyphenazine is obtained. The genetic engineering strain is safe and reliable, is friendly to the environment, and can be used for preparing 1-hydroxyphenazine.

Description

technical field [0001] The invention relates to a genetic engineering bacterial strain for producing 1-hydroxyphenazine, a preparation method and application thereof, and belongs to the technical field of genetic engineering. Background technique [0002] Phenazine derivatives are a class of nitrogen-containing heterocyclic compounds, usually in different colors, mainly orange or yellow, and also green, red, etc. The difference in side chain substituents determines the difference in color of phenazine, and the difference in structure At the same time, the different physical properties and the biological activity of inhibiting pathogenic bacteria are determined. At present, a variety of phenazine compounds with similar basic structures have been found in nature. Some phenazine-producing strains can produce several different phenazine derivatives at the same time, and almost all phenazine compounds are biologically active to bacteria and fungi. active. The phenazine antibiot...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/03C12P17/12C12R1/38
CPCC12N9/0004C12N15/03C12P17/12
Inventor 彭华松张雪洪江耀祖
Owner SHANGHAI JIAO TONG UNIV
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