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Polyoxin synthesized regulate and control protein, encoding gene and application thereof

A polyoxin, coding gene technology, applied in the fields of application, genetic engineering, plant genetic improvement, etc., can solve the problems of no pathogenic bacteria resistance found, no polyoxin gene, etc.

Inactive Publication Date: 2008-06-11
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

2) Although polyoxin has been used for decades, no large-scale resistance of pathogenic bacteria has been found so far
[0006] Since polyoxin was first isolated from Streptomyces cocoa in 1965, the research group headed by Isono speculated the synthesis pathway o

Method used

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  • Polyoxin synthesized regulate and control protein, encoding gene and application thereof
  • Polyoxin synthesized regulate and control protein, encoding gene and application thereof
  • Polyoxin synthesized regulate and control protein, encoding gene and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Obtainment of polyoxomycin synthesis regulatory protein and its encoding gene (polR)

[0034] In the present invention, the structural gene sanX in the nikkomycin biosynthesis pathway in Streptomyces chromogenes is used as a probe to screen the Streptomyces cocoa cosmid library, the screened positive clone cosmid 9A is subcloned and sequenced, and the A total of 34 open reading frames were found by sequence determination and analysis, among which the polR gene was located on the flank of the polyoxin biosynthesis gene cluster. The specific method for obtaining the polyoxomycin synthesis regulatory protein and its encoding gene (polR) of the present invention is as follows.

[0035] 1. Preparation of probes

[0036] Degenerate primers were designed according to the sequence information of Streptomyces chromogenes sanX gene (dxf: 5'-GTGACCCTSATCCCSGACCTSATCGAG-3'; dxr:

[0037] 5'-GACCGCGGCGGCGCGCAGRTCSGTSGC-3'), using the total genome of Streptomyces cacaoi va...

Embodiment 2

[0043] Example 2. Functional verification of polR gene

[0044] 1. Construct polR blocking mutants and verify the effect of the mutation on polyoxomycin synthesis

[0045] 1) Construction of polR gene disruption vector (pDR101)

[0046] Two primers (P1 and P2) were designed inside polR; P1: 5'-GAACGCTCCTGGTCGCTCATC-3'; P2: 5'-ACGCCACCCTCCAGACCTACA-3'.

[0047] The total DNA of Streptomyces cacaoi var.asoensis strain (AS4.1602) was used as the template, and P1 and P2 were used as primers for PCR amplification to obtain a DNA fragment of 1885 bp, which was sequenced. The nucleotide sequence of the 5'-end 643-2527th of the sequence 2 in the list, the fragment is named polR-1.

[0048] The above amplified fragment was inserted into plasmid pKC1139 with blunt ends (Bierman M, Logan R, OBrien K, Seno ET, Rao RN, Schoner BE (1992) Plasmid cloning vectors for the conjugal transfer of DNA from Escherichia coli to Streptomyces spp.Gene 116:43-49) EcoRV site, obtain a recombinant vect...

Embodiment 3

[0080] Example 3. Application of the polyoxomycin synthesis regulation gene polR of the present invention in improving the yield of polyoxomycin

[0081] 1. Obtaining the engineering strain (transgenic pSET152::polR engineering strain) into which the polR gene was transferred

[0082] The complementary plasmid pSET152::polR can be used to add a copy of polR to the genome of Streptomyces cocoa var. asso (AS4.1602), since pSET152::polR can be integrated into the genome of Streptomyces cocoa var. asso (AS4.1602) , replicated together with the chromosome, so the polyoxomycin high-yield engineering strain (transformed pSET152::polR engineering strain) constructed with this plasmid has genetic stability. The specific method is as follows:

[0083] The pSET152::polR was transferred into E.coli ET12567 to remove the restriction modification by the method described in the method of PEG1000-mediated Streptomyces protoplast transformation (the method of protoplast transformation is the ...

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Abstract

The invention discloses a polyoxin synthesized regulatory protein, a relative encoding gene and application thereof, wherein the protein is represented as (a) or (b), (a) is the protein composed of amino acid residue sequences of sequence 1 in a sequence table, (b) is the protein derived from (a) via substituting and/or deleting and/or adding one or several amino acid residues on the amino acid residue sequences of sequence 1 in a sequence table, with polyoxin synthesis function. The inventive polyoxin synthesized regulatory protein and relative encoding gene has significant value for polyoxin production and high value for improving Polyoxin yield.

Description

technical field [0001] The present invention relates to a polyoxomycin synthesis regulation protein and its encoding gene and application. Background technique [0002] Polyoxomycin belongs to peptidyl nucleoside antibiotics. In addition to polyoxomycin, this class of antibiotics also includes new polyoxomycin, leukomycin, aminopuromycin, triprotomycin and nikkomycin. Polyoxomycin has been commercially produced in Japan and is widely used in agriculture as an antifungal pesticide. Polyoxomycin is produced by Streptomyces cacaoi var. asoensis. It is the first nucleoside antibiotic discovered to inhibit the biosynthesis of fungal cell wall chitin. It is mainly used to prevent and treat plant pathogenic fungal infections. The pyridine base nucleosides of polyoxomycin mainly include uridine, thymine, HMU (5-hydroxymethyluracil) and 5-carboxyuracil. Structurally, polyoxomycin is a structural analog of UDP-GlcNAc that competitively inhibits the activity of chitin synthase (Endo,...

Claims

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Application Information

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IPC IPC(8): C07K14/36C12N15/31C12N15/63C12P21/00C12R1/465
Inventor 谭华荣李睿谢周杰刘钢
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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