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Gene engineering strain capable of safely and efficiently producing Phenazino-1-carboxylic acid and application thereof

A technology of genetically engineered strains and sulfazinamycin, applied in microorganism-based methods, bacteria, microorganisms, etc., can solve the problems of low fermentation titer, increased metabolic flux, unfavorable for industrial production and large-scale promotion and use, etc. To achieve the effect of superior safety

Active Publication Date: 2015-09-30
周莲
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Problems solved by technology

[0005] Aiming at the defects of the existing technology, the present invention provides a genetically engineered strain for safe and efficient production of shenzamycin and its application; first, the present invention aims at the potential safety hazards of the existing strain PA1201, and systematically knocks out the main pathogenicity in the PA1201 genome related genes / gene clusters, thereby greatly improving the safety of the bacterial strain; secondly, the present invention aims at the problems of low fermentation titer of the existing bacterial strain PA1201, high production cost of Shenzimycin, which is unfavorable for industrialized production and large-scale popularization and use. Following the principle of "opening up sources of income and reducing expenditure", the original strain PA1201 was substantially genetically modified and metabolically modified by means of genetic engineering and protein engineering, so as to increase the production of shenzimycin, including: 1) preventing the production of phenazine-1- amide and pyocyanin conversion; 2) knock out the metabolism-related genes trpE, pabB and pch gene clusters of the synthetic precursor chorismate of shenazinmycin, and reduce the biological activity of chorismate metabolism proteins UbiC and PheA, and improve chorismic acid level; 3) by enhancing the expression levels of DAHP synthases PhzC and AroG, increasing the metabolic flux of carbon source to the shenazinamycin biosynthetic pathway in the bacteria; 4) enhancing two shenazinamycin biosynthetic gene clusters The expression levels of phzA1-G1 and phzA2-G2 can further increase the output of shenzimycin; 5) enhance the expression level of shenzimycin efflux pump MexGHI-OpmD, accelerate the speed of shenzimycin to in vitro discharge, and improve the output of shenzimycin. Azithromycin production

Method used

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  • Gene engineering strain capable of safely and efficiently producing Phenazino-1-carboxylic acid and application thereof
  • Gene engineering strain capable of safely and efficiently producing Phenazino-1-carboxylic acid and application thereof
  • Gene engineering strain capable of safely and efficiently producing Phenazino-1-carboxylic acid and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0125] This embodiment involves knocking out the pabB gene, comprising the following steps:

[0126] 1. Construction of recombinant suicide plasmid pEX-pabB:

[0127] 1.1 Design two pairs of primers (pabB-FOR-1 and pabB-REV-1, pabB-FOR-2 and pabB-REV-2) to amplify the upstream and downstream flanking sequences of the gene pabB respectively. The nucleotide sequences of the primers are shown in the table 2 shown in SEQ ID No.37 and SEQ ID No.38, SEQ ID No.39 and SEQ ID No.40.

[0128] 1.2 Using the genomic DNA of strain PA1201 as a template, use the high-fidelity polymerase KOD-plus-neo and the designed primers to amplify the two flanking sequences of the gene pabB. The PCR product is detected by 0.8% agarose gel electrophoresis and gelled by AxyPrep Gel Recovery Kit recovered two flanking fragments, the lengths were 466bp and 907bp ( Figure 5 ).

[0129] 1.3 Using the above two flanking fragments as templates, using pabB-FOR-1 and pabB-REV-2 as primers, use the high-fidelit...

Embodiment 2

[0142] In this example, by constructing the genome integration vector mini-Tn7T-Gm-phzC, inserting the full gene sequence of phzC at the attTn7 site of the genome of PA1201 and its derivative engineering strains to increase the expression copy number and expression level of phzC ( Figure 8 ), including the following steps:

[0143] 1. Construction of genome integration vector mini-Tn7T-Gm-phzC

[0144] 1.1 A pair of primers (iphzC-FOR and iphzC-REV) were designed to amplify the coding sequence of the phzC gene using the high-fidelity polymerase KOD-plus-neo using Pseudomonas aeruginosa PA1201 genomic DNA as a template. The obtained PCR product was detected by 0.8% agarose gel electrophoresis, and the fragment was recovered by using the AxyPrep gel recovery kit, and the length was 1640bp. The nucleotide sequences of the primers are shown in Table 2 as SEQ ID No.53 and SEQ ID No.54.

[0145] 1.2 Design a pair of primers (iptac-FOR and iptac-REV), use the pME6032 plasmid DNA a...

Embodiment 3

[0160] In this example, the PA1201 derived engineering strain PA1201MSHΔ 9pheA (W323L) Rv2949c (P tac -phzC) the aroG gene promoter on the genome was replaced by P tac The method for a strong promoter specifically comprises the following steps:

[0161] 1. Construction of recombinant suicide plasmid pEX-P tac -aroG

[0162] 1.1 Design 2 pairs of primers (r2020-FOR-1, r2020-REV-1 and raroG-FOR-3, raroG-REV-3), using the genomic DNA of Pseudomonas aeruginosa PA1201 as a template, using high-fidelity The polymerase KOD-plus-neo amplified the upstream flanking sequences of aroG and aroG, the PCR products were detected by 0.8% agarose gel electrophoresis, and the two flanking fragments were recovered by AxyPrep Gel Recovery Kit, the lengths were 680bp and 640bp, respectively. The nucleotide sequences of the primers are shown in Table 2 as SEQ ID No.61, SEQ ID No.62, SEQ ID No.63 and SEQ ID No.64.

[0163] 1.2 Design a pair of primers (rPtac-FOR-2 and rPtac-REV-2), use the pME60...

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Abstract

The invention discloses a gene engineering strain capable of safely and efficiently producing Phenazino-1-carboxylic acid. The gene engineering strain is Pseudomonas aeruginosa with a collection number of CCTCC No. M2015040. The gene engineering strain is prepared according to the following steps: performing pathogenic gene knockout on a strain PA1201; then, performing Shenqinmycin metabolizing gene knockout and metabolize metabolizing gene knockout; then, performing point mutation to reduce the bioactivity of PheA; performing isozyme substitution method to reduce the bioactivity of chorismic acid / pyruvic acid lyase; increasing a phzC gene copy number in a genome; replacing an aroG promoter with a strong promoter Ptac so as to enhance the gene expression level of DAHP synthase; and enhancing the Phenazino-1-carboxylic acid biosynthetic gene cluster expression level and Phenazino-1-carboxylic acid efflux pump MexGHI-OpmD expression level by a promoter replacement method. The obtained strain is capable of safely and economically producing Phenazino-1-carboxylic acid with superhigh yield.

Description

technical field [0001] The invention relates to a genetically engineered bacterial strain for safe and efficient production of shenzamycin and its application, in particular to a genetically engineered bacterial strain of safe, super-high-yielding green pesticide shenzamycin and its application. Background technique [0002] my country is a large agricultural country, and the high and stable yield of grain crops is the top priority for the people to live and work in peace and contentment and the country's economic development. Agricultural crop diseases caused by plant pathogenic bacteria can lead to crop yield reduction, resulting in huge economic losses. At present, the main methods used to prevent and control plant diseases include breeding disease-resistant varieties, cultivation control, chemical control and biological control. The rational application of pesticides can guarantee the sustainable development of agriculture in our country to a great extent. In recent ye...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N1/21C12N15/78C12P17/12C12R1/385
Inventor 周莲金凯明何亚文
Owner 周莲
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