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Method for improving 7-ACA producing bacterium

A 7-ACA and bacteria-producing technology, applied in the biological field, can solve the problems of hypotoxicity, allergic reaction, yield not reaching industrialized production, and high cost, and achieve the effects of convenient screening, increased yield, and large genetic differences.

Inactive Publication Date: 2013-10-30
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

[0002] There are more than 60 kinds of cephalosporin semi-synthetic antibiotics on the market. They have the advantages of wide antibacterial spectrum, strong antibacterial activity, resistance to penicillinase, high curative effect, low toxicity, and less allergic reactions. 7-Aminocephalosporanic acid (7-ACA ) is an important intermediate in the semi-synthesis of this class of antibiotics. The industry widely uses two-step enzymatic cleavage of cephalosporin C to carry out the industrial production of 7-ACA, but its cost is high, involving two-step reactions, and requires in vitro enzyme production. Preparation and immobilization requirements
In the patent application CN200810205094.0, the genetically engineered Cephalosporium acremonium can be directly fermented to produce 7-ACA, but the output still does not meet the requirements of industrial production

Method used

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  • Method for improving 7-ACA producing bacterium
  • Method for improving 7-ACA producing bacterium
  • Method for improving 7-ACA producing bacterium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 ecs-50 gene and its function

[0048] 1. Error-prone PCR for the starting sequence and construction of product expression plasmid

[0049] 1.1 Perform error-prone PCR on the starting sequence ecs gene

[0050] The starting sequence is obtained by ligation of segmented PCR products, and its gene sequence is shown in pyg232 in FIG. 8 . When using DNA polymerase to amplify the target gene, by adjusting the reaction conditions, increasing the concentration of magnesium ions or adding manganese ions, using low-fidelity DNA polymerase to change the mutation frequency during the amplification process, so as to increase the mutation frequency at a certain frequency. Randomly introduce mutations into the target gene to obtain random mutants of protein molecules. The PCR reaction system used is as follows:

[0051]

[0052] The sequences of primers P1 and P2 are:

[0053] P 1 : 5'-GCTT GTC GAC TCTAGATCAGTGGTG-3' (the underlined part is the Sal I restriction si...

Embodiment 2

[0082] Example 2 Construction and transformation of expression plasmid pYG236 into Cephalosporium acremonium (Acremonium chrysogenum, ATCC 11550)

[0083] 1. Construct the Cephalosporium acremonium expression plasmid pYG236 containing the resistance gene hygB and the exogenous gene ecs, use the Ptrp promoter to activate the ecs gene, and the pcpc promoter to activate the resistance gene hygB. The pYG236 plasmid construction process is as follows: use XbaI+EcoRI to double-digest the plasmid pYG232-50 to obtain the ecs-50 gene, from the pDH25 plasmid (Huang Dafang et al., Plasmid pDH25 carrying the hygromycin resistance gene to transform Botrytis cinerea research, China Bioengineering magazine 1990 05 period) with EcoRI+KpnI digestion to obtain the Ptrp promoter, the obtained ecs-50 gene and Ptrp promoter were connected with the pUC18 plasmid digested with KpnI+XbaI to obtain the plasmid pUC18+ecs-50+Ptrp plasmid. Digested with SalI+HindIII to obtain pcpc+hgh gene, connected wit...

Embodiment 3

[0105] Example 3 PCR verification of transformant Acremonium chrysogenum / pYG236

[0106] Pick Cephalosporium acremonium-resistant transformants from the regenerative transformation plate of Example 1 and inoculate into the basal medium containing 10 μg / ml hygromycin [wort juice 20% (w / v), maltose 4.0% (w / v) , polypeptone 1% (w / v), pH7.0, add purified agar powder 2% (w / v)] culture on the plate for 10-14 days (d), when the spores grow plump, spread it to Continue culturing on the slant of the basic medium for 10 to 14 days, then scrape off the spores from the slant, and extract genomic DNA by liquid nitrogen extraction for PCR verification.

[0107] 1. Liquid nitrogen extraction method to extract the genome of Cephalosporium acremonium:

[0108] (1) Scrape off a small amount of spores from the inclined surface and add an appropriate amount of liquid nitrogen to grind;

[0109] (2) Dissolve with 1ml extract after grinding, extract (Tris-HCl pH7.50.2mol / ml, NaCl 0.5mol / l, EDTA 0...

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Abstract

The invention discloses a method for improving 7-ACA producing bacterium. The method comprises the following steps of: (1) obtaining two parents containing different resistance screening marker genes, wherein the first parent is the Acremonium chrysogenum genetically engineered bacterium of a gene containing an exogenous wild type cephalosporin C acyltransferase, and the second parent is the Acremonium chrysogenum genetically engineered bacterium of a gene containing an exogenous cephalosporin C acyltransferase forward mutant, compared with the wild type, the cephalosporin C acyltransferase forward mutant has the advantages that the enzymatic activity and the tolerance of a cephalosporin C substrate are improved; preparing protoplasts by using the first parent and the second parent respectively; (2) carrying out protoplast fusion; and (3) fermenting fusant by shaking a flask, and selecting the fusant with the 7-ACA content higher than those of the two parents, which is the improved 7-ACA producing bacterium. The method can be used for conveniently, effectively and rapidly obtaining the bacterial strain with the substantially increased 7-ACA yield.

Description

technical field [0001] The invention belongs to the field of biotechnology, in particular to a method for improving 7-ACA producing bacteria. Background technique [0002] There are more than 60 kinds of cephalosporin semi-synthetic antibiotics on the market. They have the advantages of wide antibacterial spectrum, strong antibacterial activity, resistance to penicillinase, high curative effect, low toxicity, and less allergic reactions. 7-Aminocephalosporanic acid (7-ACA ) is an important intermediate in the semi-synthesis of this class of antibiotics. The industry widely uses two-step enzymatic cleavage of cephalosporin C to carry out the industrial production of 7-ACA, but its cost is high, involving two-step reactions, and requires in vitro enzyme production. Preparation and immobilization requirements. In the patent application CN200810205094.0, the genetically engineered Cephalosporium acremonium can be directly fermented to produce 7-ACA, but the yield still does not...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/04C12N1/15C12R1/645
Inventor 胡又佳刘艳谢丽萍刘春磊朱宝泉王颖秋
Owner SHANGHAI INST OF PHARMA IND CO LTD
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