Construction method and use of Streptomyces roseosporus gene engineering bacteria

A technology of Streptomyces roseospora and genetically engineered bacteria, applied in the field of genetic engineering and microbial fermentation, can solve the problems of low daptomycin yield and high production cost

Active Publication Date: 2011-08-10
NANTONG YIKAI MEDICAL DEVICES CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although domestic strains producing daptomycin have already been produced in order to initially realize pilot-scale production, the strains are primitive, t

Method used

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  • Construction method and use of Streptomyces roseosporus gene engineering bacteria
  • Construction method and use of Streptomyces roseosporus gene engineering bacteria
  • Construction method and use of Streptomyces roseosporus gene engineering bacteria

Examples

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Effect test

Embodiment 1

[0027] Construction of Genetic Engineering Bacteria HP-EF01 (CGMCC 3734)

[0028] Primers were designed to PCR clone dptE and dptF (dptEF) located upstream of NRPS. (Upstream and downstream primers are respectively: Upper primer: GGAGTG CATATG GTGAGTGAG (the underline is the NdeI restriction site); Citation: CATCTA TCTAGA ATCCCCTCAGG (the underline is the XbaI restriction site). The PCR product and the expression vector pIB139 were digested with NdeI / XbaI respectively. After recovery, dptEF and pIB139 were ligated with T4 ligase at a ratio of 3:1 to 1:1 at 16°C for 2 hours to construct the recombinant expression vector pEF139 (such as figure 2 ), and then introduce ET12567 Escherichia coli competent cells by the heat shock method, and spread them on the ampicillin-resistant plate after activating and culturing in LB medium for one hour. Then pick the transformant to extract the plasmid and strictly verify it. Then the recombinant expression vector pEF139 was introduced ...

Embodiment 2

[0043] Construction of Genetic Engineering Bacteria HP-GJ01 (CGMCC 3733)

[0044] Design primers to PCR clone dptG, dptH, dptI and dptJ (dptGHIJ) located downstream of NRPS. The upstream and downstream primers are respectively: Top primer: TCCGTG CATATG AGGAAACAT (the underline is the NdeI restriction site); Citation: CGTCAG TCTAGA GACGCTTGC (XbaI restriction site is underlined). The PCR product and expression vector pIB139 were double-digested with NdeI / XbaI respectively, after gel cutting and recovery, dptGHIJ and pIB139 were ligated with T4 ligase at a ratio of 3:1 to 1:1 at 16°C for 2 hours to construct the recombinant expression vector pGJ139 (like figure 2 ), and then introduce ET12567 Escherichia coli competent cells by the heat shock method, and spread them on the ampicillin-resistant plate after activating and culturing in LB medium for one hour. Then pick the transformant to extract the plasmid and strictly verify it. Then, the recombinant expression vector p...

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Abstract

The invention provides a construction method and use of Streptomyces roseosporus gene engineering bacteria. The construction method comprises: cloning upstream and downstream accessory key genes dptE, dptF, dptG, dptH, dptI and dptJ of a nonribosomal peptide synthetases (NRPS) gene in a daptomycin gene cluster; and constructing recombinant plasmids by using an Escherichia coli-Streptomyces shuttle expression vector. The recombinant plasmids are transferred into Escherichia coli ET12567 to culture the ET12567 and the Streptomyces roseosporus together, the Streptomyces roseosporus is transformed by a combined transfer method, a transformant is screened by brulamycin resistance and polymerase chain reaction (PCR) is performed for further confirmation. The Streptomyces roseosporus gene engineering bacteria constructed by the invention can be directly used in the industrial production of daptomycin and can improve yield and reduce cost.

Description

technical field [0001] The invention relates to the technical field of genetic engineering and microbial fermentation, and specifically relates to a construction method of Streptomyces roseospore genetically engineered bacteria and its application in the production of daptomycin. Background technique [0002] Daptomycin is a calcium ion-dependent antibiotic. In the presence of calcium ions, daptomycin will bind to cell membrane proteins in the form of non-covalent bonds, and the daptomycin-binding protein ( DBPs) as its target site, daptomycin can disrupt the transport of amino acids in the cell membrane, thereby hindering the biosynthesis of the teichoic acid lipid (LTA) of the bacterial cell wall peptidoglycan and changing the properties of the plasma membrane; in addition, it also It can achieve the purpose of killing bacteria by destroying the cell membrane of bacteria and causing the contents to leak out. It is also reported that its combination with the cell membrane ...

Claims

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Application Information

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IPC IPC(8): C12N15/52C12N15/76C12N1/21C12P21/02C12R1/465
Inventor 闻建平宇光海贾晓强
Owner NANTONG YIKAI MEDICAL DEVICES CO LTD
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