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A kind of genetically engineered bacteria for transforming cephalosporin c and its preparation method

A technology of genetically engineered bacteria and cephalosporins, applied in genetic engineering, biochemical equipment and methods, botany equipment and methods, etc., can solve the problem of low transformation efficiency of strains, achieve stable transformation efficiency and improve efficiency

Inactive Publication Date: 2016-03-30
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

[0005] The technical problem to be solved in the present invention is to provide a genetically engineered bacterium with a higher efficiency of transforming cephalosporin C and a preparation method thereof for the low conversion efficiency of existing cephalosporin C into methoxycephalosporin C strains

Method used

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  • A kind of genetically engineered bacteria for transforming cephalosporin c and its preparation method
  • A kind of genetically engineered bacteria for transforming cephalosporin c and its preparation method
  • A kind of genetically engineered bacteria for transforming cephalosporin c and its preparation method

Examples

Experimental program
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Effect test

Embodiment 1

[0034] Example 1: Cloning of erythromycin resistance gene promoter and cephalosporin C methylase gene coding sequence

[0035] First, the total genome of the erythromycin-producing strain S.erythraeaHL3168E3 (purchased from ATCC) was used as a template to clone the erythromycin resistance gene promoter, the upstream primer: 5'-AAAAGATCTTCTAGAAGCCCGACCCGAGCA-3'; the downstream primer: 5'-AAAGAATTCTCCGGAGGTCGCACC- 3', PCR was carried out, and the obtained PCR product was purified and ligated to plasmid pSP72 (purchased from Promega Company) to obtain plasmid pHJJ-9, and sequenced, the sequence showed 100% homology with online sequence alignment. The plasmid pHJJ-9 was directly digested to obtain the erythromycin resistance gene promoter (KpnI / SacI).

[0036] Primers were designed according to the cmcJ gene and its sequence in Streptomyces clavulatus in GenBank, upstream: 5'-AAAGAGCTCCAGTAGCCACCCCAGG-3'; downstream: 5'-TTTGGATCCTCGAAGCTGTCCTGGC-3'. Using the total genome of Strept...

Embodiment 2

[0038] Example 2: Construction of specific site integration plasmid, conjugative transfer

[0039] The fragment (BamHI / EcoRI) containing the promoter of the erythromycin resistance gene and the coding sequence of the cmcJ gene was ligated into the plasmid pSET152 (BamHI / EcoRI) purchased from Takara Company to obtain the plasmid pHJJ-2. This recombinant plasmid is an integrated plasmid containing the erythromycin resistance gene promoter and the cmcJ gene coding sequence. This recombinant plasmid is used to transform large intestine DH5α, and the transformant is picked and cultured in LB. The plasmid is extracted for enzyme digestion and PCR (polymerase Chain reaction) verification, finally constructed into the specific site integration plasmid pHJJ-2. The schematic diagram of the above plasmid construction process is shown in figure 2 .

[0040] Cultivate Streptomyces clavuligerus ATCC27064 on slant. Pick an appropriate amount of bacteria from the slope and culture it in 5...

Embodiment 3

[0041] Example 3: Screening of Specific Site Homologous Recombination Engineering Bacteria

[0042] The zygotes were picked and cultured in TSB containing abramycin (50 μg / ml), and then the bacterial solution was applied to ISP2 medium containing abramycin (50 μg / ml), and cultured at 30°C. Because pSET152 contains phage The integration site (attP), which can be specifically homologously integrated with the attB site in the actinomycete genome, integrates the abramycin resistance gene carried by pHJJ-2 and the erythromycin resistance gene promoter and cmcJ gene The coding sequence is inserted into the attB site in the chromosome of Streptomyces clavulicularis and amplified synchronously with chromosome replication, and the zygote can stably express the abramycin resistance gene and the cmcJ gene. The zygote site-specific integration method, the schematic diagram is shown in image 3 . The zygote was picked and placed in a 4ml TSB small test tube (50 μg / ml abramycin, 2-3 gla...

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Abstract

The invention discloses a genetically engineered bacterium for converting cephalosporin C and a preparation method thereof. The genetically engineered bacterium integrates an expression cassette of cmcJ gene into the genome of a wild strain of Streptomyces clavuligerus (ATCC27064). It is verified by cephalosporin C conversion that the genetically engineered bacterium is advantaged in that the conversion rate from cephalosporin C to hydroxy cephalosporin C is improved from 57.0% of a wild strain to 67.4% of a recombinant mutant, and the conversion rate of methoxy cephalosporin C is improved from 43.1% of the wild strain to 51.9% of the recombinant mutant.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to a genetically engineered bacterium for efficiently transforming cephalosporin C and a preparation method thereof. Background technique [0002] In recent years, cephalosporins have occupied more and more market shares in the global anti-infective drug market due to their high growth and good clinical performance. my country's cephalosporin industry has made considerable progress since the 1980s, and the development in recent years has been changing with each passing day. As important β-lactam antibiotics, cephalosporin antibiotics have been developed from the first generation to the fourth generation now. In the early 1970s, 7α-methoxycephalosporin was discovered, which marked that the research on β-lactam drugs has entered a new era. [0003] Methoxycephalosporin refers to a class of Cephamycin C (Cephamycin C) produced by the fermentation of Streptomyces clavuligerus, which is s...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/31C12N15/63C12P35/06C12R1/465
Inventor 邵雷李继安黄娟娟郭兆霞陈代杰
Owner SHANGHAI INST OF PHARMA IND CO LTD
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