Escherichia coli mutant strain with S-equol resistance and application

A technology of Escherichia coli and equol is applied in the field of screening and application of mutant engineering bacteria, achieving the effects of wide application, stable system and convenient use

Active Publication Date: 2018-01-30
ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to solve the deficiency that the fermentation product S-equol in the current microbial fermentation production process can inhibit the growth of engineering bacteria, and obtain a strain with S-equol resistance by using the method of screening the marine transposon mutation library. Escherichia coli mutant G2

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  • Escherichia coli mutant strain with S-equol resistance and application
  • Escherichia coli mutant strain with S-equol resistance and application
  • Escherichia coli mutant strain with S-equol resistance and application

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Embodiment 1

[0025] Example 1: Escherichia coli mutant strain screening with anti-S-equol ability

[0026] The present invention obtains an Escherichia coli mutant strain with anti-S-equol function by using the screening method of the marine transposon mutation library, which mainly includes the following steps:

[0027] 1. Construction of transposon library

[0028] (1) E.coli DH5α(pRK2013) (purchased from Beijing Lebo Biotech Co., Ltd.), the preparation method of the culture solution is: take the E.coli DH5α(pRK2013) bacterial solution stored in the refrigerator at -80°C, and pipette it after slightly dissolving at room temperature. Inoculate 50 μl of bacterial liquid into a test tube containing 5 ml of LB medium, add 5 μl of 15 mg / ml kanamycin aqueous solution at the same time, shake at 200 rpm at 37°C overnight, and obtain E.coli DH5α (pRK2013) culture medium.

[0029] The preparation method of E. coli WM3064 (PFAC) culture medium is as follows: extract plasmid PFAC from E. coli DH5α ...

Embodiment 2

[0039] Application of embodiment 2 anti-S-equol mutant bacteria

[0040] 1. Construction of Escherichia coli mutant strains with S-equol resistance

[0041] Plasmids PETDuet-1-L-DDRC-DZNR (constructed with patent application 2014102431488 Example 1) and PCDFDuet-1-L-DHDR–THDR (constructed with patent application 2014102431488 Example 1) were simultaneously transformed into E .coli BL21(D3)-PFAC-G2. Then clones resistant to both antibiotics were screened on LB plates containing carbenicillin (50 μg / ml) and streptomycin (50 μg / ml) to obtain S-equol-resistant E. coli mutants DDDT-G2. The final concentration of the LB plate consists of: tryptone 10g / L, yeast extract 5.0g / L, sodium chloride 10g / L, agar powder 12g / L, the solvent is distilled water, and the pH value is 7.0.

[0042] 2. Validation of Escherichia coli mutant strains with S-equol resistance

[0043] The present invention carries out functional verification to the ability of engineering bacteria to produce S-equol by...

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Abstract

The invention discloses an Escherichia coli mutant strain with S-equol resistance and application. The Escherichia coli mutant strain is obtained by cloning L-DDRC, L-DZNR, L-DHDR and L-THDR genes coming from Lactococcus sp. 20-92 to Escherichia coli BL21-G2 for converting. The Escherichia coli mutant strain is simple in fermentation condition, convenient to use, stable in system and wide in application range, has S-equol resistance and has ability of converting daidzein in LB culture media into S-equol. Equol yield of engineering bacterium DDDT-G2 is 1.25-2 times that of engineering bacteriumDDDT-BL21 in the patent application 2014102431488.

Description

(1) Technical field [0001] The invention relates to the screening and application of a mutant engineering bacterium, in particular to the screening of an Escherichia coli mutant strain with an anti-S-equol function and its application in the production of S-equol by transformation of daidzein. (2) Background technology [0002] S-equol is a phytoestrogen, which is a metabolite produced by soy food after being transformed by specific microorganisms in the intestine, and has higher biological activity than its corresponding precursor soybean isoflavones. Compared with estrogen, S-equol binds to the estrogen-β receptor more strongly, so S-equol can prevent various estrogen-dependent cancers. A large number of clinical studies have shown that the prevention of various cardiovascular diseases, women's climacteric syndrome, gynecological cancer, and skin whitening and skin care effects of soy foods are all due to the biological activity of its metabolite S-equol, so whether the hu...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/21C12P17/06C12R1/19
Inventor 尹业师李海亮陈华海王燕玲王欣
Owner ZHEJIANG ACADEMY OF AGRICULTURE SCIENCES
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