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Genetically engineered bacterium for producing A82846B as well as preparation method and application of genetically engineered bacterium

A genetically engineered bacteria and gene technology, which is applied in the field of genetically engineered bacteria for the production of A82846B and its construction/preparation, can solve the problems of increased time and economic costs for separation and purification, and achieve the advantages of reducing production costs, improving production efficiency, and reducing environmental pollution. Effect

Active Publication Date: 2018-11-02
SHANGHAI INST OF PHARMA IND CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The technical problem to be solved by the present invention is to overcome the above-mentioned A82846B fermentation process in the prior art, the by-products A82846A and A82846C are produced, which increases time and economic costs for the production of A82846B and subsequent separation and purification; provide a genetic engineering method for the production of A82846B Bacteria and its construction method and application can use genetic engineering technology to improve the efficiency of producing A82846B and reduce costs

Method used

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  • Genetically engineered bacterium for producing A82846B as well as preparation method and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing A82846B as well as preparation method and application of genetically engineered bacterium
  • Genetically engineered bacterium for producing A82846B as well as preparation method and application of genetically engineered bacterium

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Effect test

Embodiment 1

[0035] Amplification, Cloning and Expression of Endogenous Halogenase Gene chaA

[0036](1) Genome extraction of Amycolatopsis orientalis strain NRRL 18099 (purchased from NRRL) and amplification of endogenous halogenase gene chaA:

[0037] NRRL 18099 was inoculated in 25ml of TSB (Shanghai Yuanju Biological Co., Ltd.) medium, and cultured at 28°C and 220rpm shaking (Shanghai Zhichu Instrument Co., Ltd.) for 48h. The culture solution was centrifuged (Hunan Xiangyi Instrument Development Co., Ltd.) at 12,000 rpm for 5 minutes, the supernatant was poured out, and the bacteria were recovered. Using the Bacterial Genomic DNA Rapid Extraction Kit, the genome was extracted according to the extraction method provided by the supplier (Shanghai Jierui Bioengineering Co., Ltd.).

[0038] The primers were synthesized by Shanghai Bailige Biotechnology Co., Ltd., and the design is as follows:

[0039] ChaA-F: 5'-CATATGATGTCGGTCGAAGACTTCGATGTGG-3' (see SEQ ID NO.6);

[0040] ChaA-R: 5'-G...

Embodiment 2

[0062] Amplification, cloning and expression of exogenous halogenase gene

[0063] (1) Total gene synthesis of exogenous halogenase gene

[0064] Exogenous halogenase genes include the genes of halogenases with high homology to their own halogenase obtained by Blast amino acid sequence alignment: vancomycin halogenase synthesis gene vcm8, balhimycin halogenase gene bhaA, teicoplanin halogenase synthesis gene thaA, etc. Listed in Table 3 below.

[0065] Table 3. The source and number of strains of exogenous halogenase gene

[0066]

[0067] Splice the gene vcm8 sequence in the above table with the terminator sequence (SEQ ID No.10), add an NdeI restriction site at the beginning of the target gene sequence, and add a BamHI restriction site at the end of the terminator sequence to form NdeI The sequence of -vcm8-terminator-BamHI sequence was sent to Beijing Weishang Lide Biotechnology Co., Ltd. for whole gene synthesis, and connected to PWSV plasmid to obtain PWSV-vcm8. In ...

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Abstract

The invention discloses a genetically engineered bacterium for producing A82846B. The genetically engineered bacterium is an engineering bacterium which integrates an endogenous halogenase gene chaA or an exogenous halogenase gene whose homology with the endogenous halogenase gene chaA is not less than 84% into a genome of amycolatopsis orientalis. The invention further discloses a preparation method of the genetically engineered bacterium, and a method for fermenting the genetically engineered bacterium and obtaining the A82846B from a fermentation liquid. According to the invention, the genetically engineered bacterium over-expresses the introduced halogenase gene and produces the A82846B, and the yield (mg / L) of the A82846B or the proportion of a target product in total products is remarkably improved; the production efficiency is improved, the production cost is reduced, and the environmental pollution is reduced.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular to a genetically engineered bacterium producing A82846B and its construction / preparation method and application. Background technique [0002] With the increasing use of antibacterial drugs, the problem of bacterial resistance is becoming more and more serious, and the proportion of Methicillin-resistant Staphylococcus aureus (MRSA) in skin infections is increasing year by year. Glycopeptide antibiotics such as vancomycin and teicoplanin are commonly used clinically to treat severe infections caused by MRSA. However, the rapid emergence of vancomycin-resistant strains makes it urgent to find new anti-MRSA antibacterial drugs. Oritavancin is a second-generation glycopeptide antibiotic approved by the US FDA in August 2014 for the treatment of acute bacterial skin and structural infections caused by sensitive Gram-positive bacteria (including MRSA). It is a new and effective antibiotic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P21/02C12R1/01
CPCC07K9/00C12N9/14C12Y308/01001
Inventor 陈少欣王伟艳马巧慧沈晓放杨松柏
Owner SHANGHAI INST OF PHARMA IND CO LTD
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