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Method for improving yield of ECO-0501 produced by fermenting Amycolatopsis orientalis

A mycobacterial pseudomycobacterium and yield technology, which is applied in the field of genetic engineering to achieve the beneficial effects of increasing yield and separating steps

Active Publication Date: 2013-03-06
SHANGHAI LAIYI BIOMEDICAL RES & DEV CENT +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At the same time, it was found that the compound has strong antibacterial activity against methicillin-resistant Staphylococcus aureus and vancomycin-resistant Enterococcus faecium, and thus has a good application prospect. The compound is present in very low amounts in the fermentation product of Amycolatopsisorientalis

Method used

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  • Method for improving yield of ECO-0501 produced by fermenting Amycolatopsis orientalis
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  • Method for improving yield of ECO-0501 produced by fermenting Amycolatopsis orientalis

Examples

Experimental program
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Effect test

Embodiment 1

[0053] Example 1 , Disruption of the vancomycin synthase gene

[0054] The vancomycin synthase genes vcm2, vcm3 and vcm4 encode non-ribosomal peptide synthases, namely Vcp1, Vcp2 and Vcp3, respectively. These three non-ribosomal peptide synthetases contain 7 modules and 24 domains, responsible for Biosynthesis of the vancomycin heptapeptide skeleton; this example takes blocking the vancomycin synthase gene vcm4 as an example, and blocks the synthesis of vancomycin by blocking the synthesis of the vancomycin heptapeptide skeleton.

[0055] This example uses the principle of gene homologous recombination to construct the gene blocking vector pKOA; then, the constructed gene blocking vector is introduced into Amycolatopsis orientalis through conjugative transfer to obtain conjugative transferons. According to the Apra resistance on the carrier, Sex genes were screened to obtain single crossover mutants. Continuous subculture was carried out on the single exchange mutant strain...

Embodiment 2

[0108] Example 2 Knockout of the vancomycin precursor synthetase gene that catalyzes the synthesis of β-hydroxytyrosine

[0109] There are 16 vancomycin precursor synthetase genes, among which vcm14, vcm15 and vcm16 encode hydrolase, peptide synthase VCP4, and P450 oxidase respectively, and these three enzymes are responsible for catalyzing the synthesis of β-hydroxytyrosine; this example Taking the vancomycin precursor synthetase gene vcm14 as an example to block the synthesis of vancomycin.

[0110] According to this embodiment figure 2 The principle of PCR-targeting shown is to replace the vcm14 gene to be knocked out with the Aprar gene cassette in vitro, and construct the gene blocking vector pLY26. The specific steps are as follows:

[0111] 2.1. Primer design and synthesis

[0112] The primer pair Dv14sense and Dv14anti were designed according to the gene sequence of A. orientalis ATCC 43491:

[0113] Dv14sense (SEQ ID NO.1):

[0114]GCGCCGTGGCTGTGCGGTGAGCGGGGAAG...

Embodiment 3

[0143] Example 3 Knockout of the vancomycin precursor synthetase gene that catalyzes the synthesis of hydroxyphenylglycine

[0144] There are 16 vancomycin precursor synthetase genes, among which vcm1, vcm17, vcm18 and vcm13 encode prephenate dehydrogenase, hydroxymandelate synthase Hmas, hydroxymandelate oxidase Hmo and hydroxyphenylglycine Aminotransferase HpgT, these five enzymes are responsible for catalyzing the synthesis of hydroxyphenylglycine (HPG, a non-natural amino acid in the vancomycin heptapeptide backbone); this example knocks out the vancomycin precursor synthetase gene vcm13 For example, blocking the synthesis of vancomycin.

[0145] With the PCR-Targeting method described in Example 2, the gene blocking vector pLY27 was constructed, and the vcm13 gene was knocked out, wherein the position of the replacement sequence of the apramycin resistance gene cassette in cos311 was 42432-42753.

[0146] Among them, the primers used for amplification are Dvcm13sense a...

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Abstract

The invention discloses a method for improving the yield of ECO-0501 produced by fermenting Amycolatopsis orientalis, which is achieved by interdicting the Amycolatopsis orientalis to synthesize vancomycin, wherein the synthesis of the interdicted vancomycin comprises interdicting or knocking out vancomycin synthetase genes or vancomycin precursor synthetase genes. The method for improving the yield of the compound ECO-0501 produced by fermenting the Amycolatopsis orientalis interdicts the synthesis of the vancomycin while improving the yield of the ECO-0501, thus the method is particularly favorable for the downstream separation steps.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a method for increasing the yield of ECO-0501 fermented by Amycolatopsisorientalis. Background technique [0002] Amycolatopsis orientalis (formerly known as Streptomycesorientalis or Nocardia orientalis) was screened from Indonesian soil by Micormick et al. in 1956, and its fermentation product was identified as Vangu Vancomycin is a glycopeptide antibiotic that is resistant to Gram-positive bacteria. It was developed by Eli Lilly and Company of the United States, and was approved by the FDA in 1958. Recent studies have shown that Amycolatopsis orientalis produces at least 10 secondary metabolites in addition to vancomycin. Banskota et al. analyzed the genome of the fungus by DECIPHER technology, found that one of the biosynthetic gene clusters encoded type I polyketide synthases (polyketide synthases), and predicted that it could directly synthesize a ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12P19/44C12R1/465
Inventor 阮林高郦伟翔黄鹤杨晟朱丽陈代杰姜卫红戈梅罗敏玉魏维
Owner SHANGHAI LAIYI BIOMEDICAL RES & DEV CENT
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