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A genetic engineering strain used for producing phenazine-1-carboxamide, a preparing method thereof and uses of the strain

A technology of genetically engineered strains and formamide, which is applied in the field of genetic engineering, can solve the problems of complex metabolic regulation network and difficulty in further improving the yield of secondary metabolites, etc., and achieves the effects of good stability, promoting industrial and agricultural applications, and increasing yield.

Inactive Publication Date: 2017-08-15
SHANGHAI JIAO TONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Due to the complexity of the metabolic regulatory network in microbial cells and the interaction of different regulatory genes, it is difficult to further increase the yield of secondary metabolites only by changing the regulatory genes.

Method used

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  • A genetic engineering strain used for producing phenazine-1-carboxamide, a preparing method thereof and uses of the strain
  • A genetic engineering strain used for producing phenazine-1-carboxamide, a preparing method thereof and uses of the strain
  • A genetic engineering strain used for producing phenazine-1-carboxamide, a preparing method thereof and uses of the strain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] This embodiment relates to a method of using the HT66 genome as a template to prepare the HT66ΔpykA genetically engineered strain by the insertion mutagenesis method, including the following steps:

[0039] 1. Primer Design

[0040] Using the pykA gene sequence as a reference, primers were designed and synthesized using primer 5. The primer sequences are as follows, and the underlines indicate the cleavage sites of restriction endonucleases Hind III and Sac I.

[0041] pykA1:CC AAGCTTATGTCCGTCCGTCGTACCAA (SEQ ID NO. 3)

[0042] pykA2:AC GAGCTC TCAGACCATTGGGTCGCCA (SEQ ID NO. 4)

[0043] Using the plasmid pBbB5k as a template, primers for the Kan resistance gene (Kan) were designed and synthesized. The sequences of the primers are as follows, and the underline indicates the cleavage site of the restriction endonuclease Xho I.

[0044] pBb-Kan-F:CC CTCGAG GGAATTGCCAGCTGGGGCGC SEQ ID NO.5

[0045] pBb-Kan-R:CC CTCGAG TCAGAAGAACTCGTCAAGAAG SEQ ID NO. 6

[0046] ...

Embodiment 2

[0053] This embodiment relates to the preparation of a high-yield PCN genetically engineered strain. The starting strain P3 is a high-yield PCN strain obtained by multiple mutations of Pseudomonas chloropinus HT66. Specifically include the following steps:

[0054] 1. Primer design:

[0055] Using the upstream and downstream sequences of the pykA gene as a reference, the knockout primers were designed and synthesized using primer 5. The primer sequences are as follows (the underline indicates the XbaI and HindIII restriction sites):

[0056] pykAF1:CCGGGGATCC TCTAGA AAGATCGTTACAACGCGGTCG (SEQ ID NO. 7)

[0057] pykAR1:TGGTACGACGGACGGACATG (SEQ ID NO. 8)

[0058] pykAF2:TGTCCGTCCGTCGTACCAACCCAATGGTCTGAGCCACC (SEQ ID NO. 9)

[0059] pykAR2:GGCCAGTGCC AAGCTT CGAGTTCGGTTCCAGCCTG (SEQ ID NO. 10)

[0060] 2. Recombinant plasmid construction:

[0061] Using the P3 genome as a template, the upstream and downstream homology arms of pykA were amplified by PCR using pykAF1, pykA...

Embodiment 3

[0066] This embodiment relates to a method for synthesizing PCN using the genetically engineered bacterial strain prepared in Example 1, comprising the following steps:

[0067] Bacterial strains HT66ΔpykA and HT66wt (wild strain HT66 as a control) were respectively activated twice on KB solid plates, then cultured in 5 mL KB liquid medium, cultured to logarithmic phase, and transferred to 60 mL KB culture medium at a ratio of 2%. The basic Erlenmeyer flask was used for fermentation. The fermentation conditions were 28° C., 180 rpm. Samples were taken regularly, and the PCN yield of the strain was determined by HPLC.

[0068] HPLC detection conditions:

[0069] The mobile phase is acetonitrile-25mM ammonium acetate, the chromatographic column is WondaSilC18-WRreversephasecolumn (5μm; 4.6×250mm, Shimadzu, Japan), the detection wavelength is 254nm, the detection conditions: 0-2min, 8% acetonitrile-92% 25mM ammonium acetate, 2- In 20 minutes, the concentration of acetonitrile ...

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Abstract

A genetic engineering strain used for producing phenazine-1-carboxamide (PCN), a preparing method thereof and uses of the strain are provided. The strain is prepared by knocking a pykA gene out from pseudomonas chlororaphis HT66 CCTCC NO:M 2013467 and derivatives thereof. The method includes steps of designing primers, constructing a recombinant plasmid and screening a mutant strain. Compared with the prior art, beneficial effects of the strain, the method and the uses are that a novel regulating strategy is adopted, flow directions of metabolic substances in cells are changed, the yield of the PCN is greatly increased, and the strain is safe, reliable and good in stability and can significantly promote agricultural and industrial applications of the PCN.

Description

technical field [0001] The invention relates to a genetically engineered bacterial strain with high yield of phenazine-1-carboxamide (PCN), a preparation method and an application thereof, belonging to the technical field of genetic engineering. Background technique [0002] So far, scientists have discovered more than 6,000 compounds containing phenazine structures, most of which are chemically synthesized, and less than 100 have been isolated from living organisms. However, most of the phenazine compounds isolated from living organisms have broad-spectrum antibacterial, fungal and other plant and animal pathogenic bacteria. For example, phenazine-1-carboxylic acid (PCA) can inhibit wheat take-all which causes root corrosion of wheat, phenazine-1-carboxamide has significant inhibitory effect on pathogens such as tomato wilt, and some phenazine derivatives It has shown its application in the field of medicine, and more and more scholars are studying its mechanism of action....

Claims

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Application Information

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IPC IPC(8): C12N1/21C12P17/12C12R1/38
CPCC12N1/20C12N9/1205C12P17/12C12Y207/0104
Inventor 彭华松张雪洪金雪洁
Owner SHANGHAI JIAO TONG UNIV
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