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Genetic engineering bacteria for producing high-purity cephalosporin C and application thereof

A technology of genetically engineered bacteria and gene fusion, applied in the field of genetically engineered bacteria producing high-purity cephalosporin C and its application, can solve the problems of affecting product purity and impurity removal

Inactive Publication Date: 2011-09-07
INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

When CPC is used as a raw material in the downstream chemical synthesis of cephalosporins, DAOC will also become an impurity that cannot be removed, affecting the purity of the product

Method used

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  • Genetic engineering bacteria for producing high-purity cephalosporin C and application thereof
  • Genetic engineering bacteria for producing high-purity cephalosporin C and application thereof
  • Genetic engineering bacteria for producing high-purity cephalosporin C and application thereof

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Embodiment 1

[0029] Embodiment 1, the genetically engineered bacterium that produces high-purity cephalosporin and its application

[0030] Acremonium chrysogenum CGMCC 3.3795 was purchased from China General Microorganism Collection Center (CGMCC);

[0031] Streptomyces clavuligerus ATCC27064 was purchased from the American Type Culture Collection (ATCC).

[0032] 1. Construction of recombinant plasmid pAg-H3-cefF

[0033] 1. PCR amplification of promoter PpcbC

[0034] Using Acrernonium chrysogenum CGMCC 3.3795 genomic DNA as a template, a primer pair was designed for the promoter region of the gene pcbC:

[0035] The upstream primer is: 5'-G Actagt GTGGATGGCACCTTTTGGG-3′, the lowercase letter is the enzyme cutting site SpeI,

[0036] The downstream primer is: 5′-C ggcgcgcc GGTGACGGTTTGTCCTGCC-3′, the lowercase letter is the enzyme cutting site AscI.

[0037] A DNA fragment of 1188bps in size was obtained by PCR amplification, which was digested with SpeI and AscI to recover a 1....

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Abstract

The invention discloses genetic engineering bacteria for producing high-purity cephalosporin and application of the genetic engineering bacteria. The genetic engineering bacteria is obtained by introducing the following fusion gene into host bacteria: the fusion gene formed by a DNA fragment shown by nucleotides from the 7th site to the 1194th site in a sequence 1 in a sequence table and an encoding gene of a protein cefF. One of the obtained genetic engineering bacteria is called Acremonium chrysogenum cffF genetic engineering bacterium with the preserving number of CGMCC No.4515. In the invention, a Streptomyces clavuligerus sourced gene cefF with an Acremonium chrysogenum promoter PpcbC is transferred to the Acremonium chrysogenum so that the gene cefF is transcribed and expressed in vivo to enhance a catalytic reaction in which DAOC (Deacetoxycephalosporin C) is hydroxylated into DAC (Deacetylcephalosporin C), thereby reducing the content of the DAOC in a finished product CPC (Cephalosporin C), and improving the purity of industrial products.

Description

technical field [0001] The invention relates to genetically engineered bacteria and applications thereof, in particular to genetically engineered bacteria for producing high-purity cephalosporin C and applications thereof. Background technique [0002] Cephalosporin C (CPC) is the raw material for industrial production of cephalosporins. Cephalosporins belong to β-lactam antibiotics and are a class of broad-spectrum antibiotics with important application value. The industrial producer of cephalosporin C is Acremonium chrysogenum. The biosynthetic pathway of cephalosporin C in Cephalosporium acremonium has been studied very clearly, which has laid a good foundation for the transformation of Cephalosporium acremonium by means of genetic engineering. Deacetoxycephalosporin C (Deacetoxycephalosporin C, DAOC) is an intermediate product in the biosynthesis of cephalosporin C. In biosynthesis, penicillin N (penicillin N) is catalyzed by the ring expansion / hydroxylation bifunction...

Claims

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Application Information

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IPC IPC(8): C12N1/15C12N15/62C12N15/63C12P35/06C12R1/645
Inventor 刘钢安洋
Owner INST OF MICROBIOLOGY - CHINESE ACAD OF SCI
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