Preparation method of cephalosporin C and genetically engineered bacterium used therein

A technology of genetically engineered bacteria and cephalosporins, applied in the biological field, can solve the problem of low production of cephalosporin C, and achieve the effect of increasing the transcription level and CPC yield

Active Publication Date: 2016-01-06
SHANGHAI INST OF PHARMA IND +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Therefore the technical problem to be solved by the present invention is to provide a new preparation method of cephalosporin C and the genetically engineered bacterium used therein for the problem that the yield of cephalosporin C in the currently existing bacterial strains is not high.

Method used

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  • Preparation method of cephalosporin C and genetically engineered bacterium used therein
  • Preparation method of cephalosporin C and genetically engineered bacterium used therein
  • Preparation method of cephalosporin C and genetically engineered bacterium used therein

Examples

Experimental program
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Effect test

Embodiment 1

[0035] 1 strains and reagents

[0036] 1.1 Strains

[0037] Escherichia coli DH5α, plasmid pYG858 (For the construction method of plasmid pYG858, please refer to the literature: Zhang Piyan, Zhu Chunbao, Zhu Baoquan. Cloning and application of the bidirectional promoter region of Cephalosporium acremonium pcbAB-pcbC[J].Acta Microbiology,2004,44(2 ):255-257.). Acremonium chrysogenum CPCC480085 (China Pharmaceutical Culture Collection, CPCC), the strain was purchased from China Microbial Resources Network (Beijing Putian Tongchuang Biotechnology Co., Ltd.), resource platform number: 1511C0004000004213. CPC standard products were purchased from Sinopharm Weiqida Pharmaceutical Co., Ltd.

[0038] 1.2 Reagents

[0039] Restriction enzymes, T4DNA ligase, LATaq enzyme, cDNA reverse transcription kit and Real-time PCR kit were purchased from Dalian Takara Company, high-purity plasmid extraction kit was purchased from MN Company in Germany, LysingEnzyme and Bleomycin were all purchased From...

Embodiment 2

[0071] Example 2 Transformation and fermentation culture of Cephalosporium acremonium protoplasts

[0072] Using PEG-CaCl 2 Mediated protoplast transformation method transforms the constructed plasmid into Cephalosporium acremonium. The specific steps are as follows: Take 7μl of 2μg / μl high-purity plasmid pYG280 and add 100μl with a concentration greater than 10 8 Pcs / ml of protoplasts, ice bath for 20 minutes, add P buffer solution containing 10% PEG6000 (the formula of P buffer solution is: KCl4.47%, CaCl 2 0.278%, MgCl 2 0.203%) Place at 20°C for 10 minutes. After centrifugation at 5000 rpm for 5 minutes, the supernatant was removed. The precipitate was washed once with P buffer solution. The supernatant was removed after centrifugation at 5000 rpm for 5 minutes. The precipitate was dissolved in an appropriate volume of P buffer solution and added to 7% soft agar preheated at 60℃. Pour immediately Regenerate the upper layer of the plate (tryptone 3.0%, soy peptone 0.8%, NaCl 0....

Embodiment 3

[0074] Example 3 Fermentation and cultivation of Cephalosporium acremonium

[0075] Using PEG-CaCl 2 Mediated protoplast transformation method transforms the constructed plasmid into Cephalosporium acremonium. The specific steps are as follows: Take 7μl of 2μg / μl high-purity plasmid pYG280 and add 100μl with a concentration greater than 10 8 Protoplasts per ml, ice bath for 20 minutes, add P buffer containing 30% PEG6000 (the formula of P buffer is: KCl4.47%, CaCl 2 0.278%, MgCl 2 0.203%) Place at 25°C for 30 minutes. After centrifugation at 5000 rpm for 5 min, the supernatant was removed. The pellet was washed once with P buffer solution. The supernatant was removed after centrifugation at 5000 rpm for 5 min. The pellet was dissolved in an appropriate volume of P buffer solution and added to 7% soft agar preheated at 70°C. Pour immediately Regenerate the upper layer of the plate (tryptone 3.0%, soy peptone 0.8%, NaCl 0.8%, sucrose 20.6%, pH 7.0). After the upper layer is solidifi...

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Abstract

The invention discloses a preparation method of cephalosporin C and a genetically engineered bacterium used therein, and further discloses a preparation method of the genetically engineered bacterium. The preparation method of cephalosporin C includes the following steps: (1), enabling a recombinant expression vector carrying nucleic acid shown as SEQ ID NO:1 in a sequence table to be transfected with Acremonium chrysogenum to obtain a recombinant expression transformant; (2), performing fermentation culture on the recombinant expression transformant, and separating cephalosporin C from culture. AcveA genes are additionally introduced into a cephalosporin CPC high-yield strain, so that transcription level of CPC synthesized genes is improved, regulatory genes AcveA are synthesized by guiding in CPC, and yield of CPC is increased.

Description

Technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a preparation method of cephalosporin C and a genetically engineered bacteria used therein. Background technique [0002] Cephalosporin C (CPC) is the main raw material for producing 7-aminocephalosporanic acid (7-ACA), an important intermediate of cephalosporin antibiotics. Cephalosporium acremonium is an important industrial microorganism that produces CPC by fermentation. After years of research, the biosynthetic pathway of CPC in Cephalosporium acremonium has been explained more clearly. All the synthetic genes involved have been cloned, and most of the enzymes encoded by these genes have been purified (see the literature MartinJF.Alpha -aminoadipyl-cysteinyl-valinesynthetasesinbeta-lactamproducingorganisms.FromAbraham'sdiscoveriestonovelconceptsofnon-ribosomalpeptidesynthesis[J].JAntibiot(Tokyo),2000,53(10):1008-1021). The study found that the genes in the CPC biosynthet...

Claims

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Application Information

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IPC IPC(8): C12P35/06C12N1/15C12R1/645
Inventor胡又佳龚桂花张伟刘艳谢丽萍朱宝泉
OwnerSHANGHAI INST OF PHARMA IND