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Construction method and application of streptomyces roseosporus gene engineering bacteria HP-ORF53-55

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: 2014-03-26
NANTONG YIKAI MEDICAL DEVICES CO LTD
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  • Claims
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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, the yield of daptomycin is low, and the production cost is high. Improving the production of daptomycin has important economic value and social significance

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  • Construction method and application of streptomyces roseosporus gene engineering bacteria HP-ORF53-55
  • Construction method and application of streptomyces roseosporus gene engineering bacteria HP-ORF53-55
  • Construction method and application of streptomyces roseosporus gene engineering bacteria HP-ORF53-55

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Embodiment 1

[0028] Construction of Genetic Engineering Bacteria HP-ORF53-55 (CGMCC 4097)

[0029] Design primers for PCR clone ORF53-55. (Upstream and downstream primers are respectively: Upper primer: GAAGGAAA CATATG GTGCTCGGCT (the underline is the NdeI restriction site); Citation: GTACAA TCTAGA TCACTTGACGCTCCCTG (XbaI restriction site is underlined). The PCR product and the expression vector pIB139 were digested with NdeI / XbaI respectively, and after recovery, the ORF53-55 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 pAB139 (Such as figure 2 ), and then introduce ET12567 Escherichia coli competent cells by the heat shock method, and apply them to the apramycin selection plate after being activated and cultivated in LB medium for one hour. Then pick the transformant to extract the plasmid and strictly verify it. Then, the recombinant expression vector pAB139 was introduced into Streptomyces ros...

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Abstract

The invention provides a construction method and application of streptomyces roseosporus gene engineering bacteria. The construction method comprises the following steps of: cloning three key genes ORF53-55 for coding ATP-binding transport protein at the downstream part of a non-ribosomal protein synthase (NRPS) gene in a daptomycin synthetic gene cluster, and constructing recombinant plasmids by using an Escherichia coli-streptomyces shuttle plasmid expression vector; leading the recombinant plasmids into Escherichia coli ET12567; culturing the ET12567 together with the streptomyces roseosporus; converting the streptomyces roseosporus by a combining and transferring method; then, screening converters through the resistance of apramycin; and making a further verification on the gene engineering bacteria through polymerase chain reaction (PCR). The streptomyces roseosporus gene engineering bacteria constructed by the invention can be directly applied to the industrialized production of daptomycin, the yield can be improved, and the cost can be lowered.

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 strain HP-ORF53-55 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...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/76C12N15/66C12P21/02C12N1/21C12R1/465
Inventor 闻建平宇光海贾晓强
Owner NANTONG YIKAI MEDICAL DEVICES CO LTD
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