Gene engineering bacterium for high efficiency conversion of echinocandin B and preparation method thereof

A technology for genetically engineered bacteria and echinocandin, which is applied to the field of genetically engineered bacteria for efficient transformation of echinocandin B and its preparation, can solve the problems of low yield of deacylase and low biotransformation efficiency of ECB, and achieves improved efficiency. Transformation efficiency, stable transformation efficiency, and the effect of increasing yield

Inactive Publication Date: 2012-05-09
SHANGHAI INST OF PHARMA IND CO LTD
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Therefore, the technical problem to be solved in the present invention is to provide a kind of ECB deacylase yield for the low yield of ECB deacylase in the existing wild strain of Actinomyces uthazolans, and the low biotransformation efficiency of ECB. Genetically engineered Actinomycetes uthaensis bacteria with higher ECB transformation efficiency and preparation method thereof

Method used

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  • Gene engineering bacterium for high efficiency conversion of echinocandin B and preparation method thereof
  • Gene engineering bacterium for high efficiency conversion of echinocandin B and preparation method thereof
  • Gene engineering bacterium for high efficiency conversion of echinocandin B and preparation method thereof

Examples

Experimental program
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Effect test

Embodiment 2

[0036] Example 2 Construction of specific site integration plasmid, conjugative transfer

[0037] The echinocandin B deacylase gene (EcoRI / XbaI) was ligated into the plasmid pSET152 (EcoRI / XbaI) purchased from Takara Company to obtain the recombinant plasmid pYG-LAJ-2. This recombinant plasmid is the integrated plasmid that multiplies the echinocandin B deacylase gene. Use this recombinant plasmid to transform Escherichia coli DH5α, pick the transformant and culture it in LB, extract the plasmid for enzyme digestion and PCR verification, and finally construct A site-specific integration plasmid pYG-LAJ-2 was formed. The schematic diagram of the above plasmid construction process is shown in figure 1 .

[0038] The slant cultured echinocandin B deacylase-producing bacteria——Actinoplanesutahensis NRRL 12052. Pick an appropriate amount of bacteria from the slope and culture it in 50ml TSB for about 72 hours to reach the logarithmic growth phase. Transfer 1% of the inoculum to ...

Embodiment 3

[0039] Example 3 Screening of Site-specific Recombinant Engineering Bacteria

[0040] The zygotes were picked and cultured in TSB containing abramycin (50 μg / ml), and then the bacterial solution was applied to a slant medium containing abramycin (50 μg / ml) (yeast extract 0.3%, malt leaching 0.3% peptone, 0.5% peptone, 1.0% glucose, agar 2.7, pH7.2), and cultured at 30°C. Because pSET152 contains phage The C31 integration site (attP) can specifically homologously integrate with the attB site in the actinomycete genome, and insert the Am resistance gene and echinocandin B deacylase gene carried by the plasmid pYG-LAJ-2 The zygote can stably express Am resistance gene and echinocandin B deacylase gene by reaching the attB site in the chromosome of Actinomyces Utahii and synchronously amplified with chromosome replication. The site-specific integration method, the schematic diagram is shown in figure 2 . The zygotes were picked and placed in a small test tube of 4ml TSB (con...

Embodiment 4

[0041] Example 4 Production of Echinocandin B Deacylase Transformation Echinocandin B Verification of Engineering Bacteria

[0042] Pick the mutant strain that can amplify the 4000bp fragment in Example 3 and cultivate it in slant medium (0.3% yeast extract, 0.3% malt extract, 0.5% peptone, 1.0% glucose, agar 2.7, pH7.2) at 30°C After culturing for one week, inoculate in the seed culture solution (2.0% sucrose, 2.0% oatmeal, 0.5% malt extract, 0.25% yeast, K 2 HPO 4 0.1%, KCl 0.05%, MgSO 4 ·7H 2 O 0.05%, FeSO 4 ·7H 2 O 0.0002%, pH natural), 30 ℃ for 4 days and then transferred to the fermentation medium (sucrose 2.0%, peanut powder 1.0%, K 2 HPO 4 0.12%, KH 2 PO 4 0.05%, MgSO 4 ·7H 2 O 0.025%, pH natural) (10% inoculum size). After 3 days of cultivation at 30°C and 250 r / m, the bottles were released. Get the fermented liquid and wash it several times with deionized water, after centrifugal precipitation (4000rpm, 20min), suspend with the fresh 0.1mol / L phosphate...

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Abstract

The invention discloses a gene engineering bacterium for the high efficiency conversion of echinocandin B and a preparation method thereof. The echinocandin B is an engineering bacterium of an expression cassette integrated with echinocandin B deacetylase gene in a genome of Actinoplanes utahensis wild strain. The conversion of echinocandin B verifies that the conversion efficiency of the gene engineering bacterium is 1.8 times the conversion efficiency of the wide stain.

Description

technical field [0001] The invention belongs to the field of bioengineering, in particular to a genetic engineering bacterium for efficiently transforming echinocandin B and a preparation method thereof. Background technique [0002] In the past two decades, fungal infections that seriously endanger human life and health have been increasing in terms of mortality and types of infections, especially in immunosuppressed patients. The main drugs currently used to treat clinical deep fungal infections are azole antifungal drugs and amphotericin B. Although these two types of drugs have played an important role in controlling clinical deep fungal infections, due to poor selectivity and drug resistance of these drugs The emergence of fungi and the lack of susceptibility to fungi such as Aspergillus and Candida albicans make the mortality rate of deep fungal infections high. [0003] Echinocandins (Echinocandin) antifungal antibiotics are a group of natural products discovered in ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12N15/63C12N15/55C12P21/04C12R1/045
Inventor 李继安刘爱娟邵雷陈代杰陈继业
Owner SHANGHAI INST OF PHARMA IND CO LTD
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