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Gene recombination mediated by phiC312 and inheritance reformation for erythrocin producing bacterium

A technology for producing erythromycin and bacteria, which is applied in the field of genetic transformation, and can solve problems such as the inability to directly apply the genetic transformation of Saccharopolyspora rubrum

Active Publication Date: 2012-11-21
SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the gene recombination system based on ΦC31 integrase cannot be directly applied to the genetic transformation of erythromycin-producing strain Saccharopolyspora rubrum

Method used

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  • Gene recombination mediated by phiC312 and inheritance reformation for erythrocin producing bacterium
  • Gene recombination mediated by phiC312 and inheritance reformation for erythrocin producing bacterium
  • Gene recombination mediated by phiC312 and inheritance reformation for erythrocin producing bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Constructing the red Saccharopolyspora containing the bacterial attachment site attB target sequence in the chromosome

[0037] According to the method described in the literature ("Streptomyces Genetics Manual" Norwich, England: The John Innes Foundation; 2000), the polynucleotide containing the attB sequence of the bacterial attachment site was inserted into the red sugar polynucleotide by homologous recombination double exchange structure in the spore chromosome. In this example, we chose to insert the attB sequence into a NRPS gene cluster with a loss of function in the chromosome of Saccharopolyspora rubrum. In order to increase the integration efficiency mediated by ΦC31 integrase and carry out the second or multiple recombinations on the basis of the first site-specific recombination, we selected the attB sequence containing 8 copies as an insert to integrate into the red sugar in polyspora chromosomes. The insertion of this fragment will not have any...

Embodiment 2

[0056] Example 2 The 12-carbon hydroxylase EryK gene and the mycaminose methylase EryG gene of the macrocyclic skeleton in the biosynthesis of erythromycin were integrated into Saccharopolyspora red (S.erythraea E3-attB) attB target sequence

[0057] column

[0058] This example is a further modification based on the inventor's application for a Chinese patent titled 'A Strain, Construction Method and Use of an Optimized Erythromycin Fermentation Component' to construct a stable and enhanced expression of erythromycin During the biosynthesis process, the carbon 12 hydroxylase EryK and mycaminose methylase EryG production strains of erythromycin can improve the impurity components and yield of erythromycin.

[0059] Plasmid construction:

[0060] In order to construct an integrated plasmid based on ΦC31 integrase-mediated gene insertion, select the plasmid pSET152 ( Figure 5 ) as the plasmid parent. Digest containing ermE with EcoRI / SpeI * -eryK-eryG-eryK structure of the...

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Abstract

The invention discloses gene recombination mediated on the basis of phiC312 and inheritance reformation for an erythrocin producing bacterium. A target gene can be stably integrated on a locus pre-constructed by a host cell genome by a method of gene recombination mediated by a phiC312 locus specificity integration mechanism. The invention also relates to an application of the method on the inheritance reformation of the erythrocin producing bacterium.

Description

technical field [0001] The invention belongs to the field of biotechnology engineering, and more specifically, the invention relates to a method, a composition and a system for expressing a target gene in Saccharopolyspora rubrum cells. The invention also relates to the genetic modification of erythromycin producing strains by this method. Background technique [0002] Since erythromycin entered clinical practice as a broad-spectrum antibiotic drug, conventional genetic breeding and strain selection for the purpose of improving the fermentative unit of its production strain have never stopped. In recent years, the development of genetic engineering technology has obviously involved the field of strain improvement for erythromycin production. Using these technologies, genes beneficial to erythromycin production can be introduced into strains, and improved strains can be obtained quickly, thereby avoiding the long process of traditional conventional breeding methods. However...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/80C12P19/62C12R1/19C12R1/645
Inventor 刘文张嗣良吴杰群张庆林邓玮
Owner SHANGHAI INST OF ORGANIC CHEM CHINESE ACAD OF SCI
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