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Eggerthella sp., engineering bacteria generated by S-equol and construction method and application of engineering bacteria

A technology of Eggeria and equol, applied in the field of microorganisms, to achieve the effects of convenient use, stable system and simple fermentation

Active Publication Date: 2016-08-17
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A strict anaerobic strain, Slackia sp. AUH-JLC159 (ZL201310043107.X), which can convert the substrate genistein to L-5-OH-equol, was isolated from fresh chicken feces samples in our laboratory, but The transfer and cultivation of this strain must be completed under strict anaerobic conditions
In 2007, Japanese scholar Uchiyama et al. reported a strain of facultative anaerobic lactic acid bacteria 20-92. The strain 20-92 can grow both under anaerobic conditions and under aerobic conditions, but it can only grow under anaerobic conditions. Conversion of substrate daidzein to S-equol

Method used

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  • Eggerthella sp., engineering bacteria generated by S-equol and construction method and application of engineering bacteria
  • Eggerthella sp., engineering bacteria generated by S-equol and construction method and application of engineering bacteria
  • Eggerthella sp., engineering bacteria generated by S-equol and construction method and application of engineering bacteria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] Example 1: Isolation and screening of Eggerthella sp. HAU-JLC44, species identification and structural identification of the product produced by transformation of substrate daidzein by strain HAU-JLC44

[0056] 1. Bacterial strain HAU-JLC44 is a Gram-positive strict anaerobic bacterial strain isolated from fresh feces of roosters. The anaerobic bacterium has an original transformation effect on the substrate daidzein. The isolation and screening process of bacterial strain HAU-JLC44 is as follows: :

[0057] 1) Use a sterilized cotton swab to pick up fresh rooster feces, put it into 1 mL of fresh BHI liquid medium, and place it in an anaerobic workstation at 37°C, as a microbial flora for screening specific functional microbial strains;

[0058] 2) Gradually dilute the microbial flora in the BHI liquid medium to a concentration of 10 -1 、10 -2 、10 -3 、10 -4 、10 -5 、10 -6 、10 -7 、10 -8 , and then respectively 100 μl concentration was 10 -5 、10 -6 、10 -7 、10 -...

Embodiment 2

[0069] Example 2: Construction and verification of S-equol-producing engineering bacteria

[0070] The present invention clones the S-equol biosynthetic gene from the genomic DNA of the wild-type strain Eggerthella sp. HAU-JLC44 isolated in our laboratory, connects different functional genes to the same plasmid, and constructs a A kind of S-equol produces engineering bacteria, and the construction of this engineering bacteria mainly includes the following steps:

[0071] 1. Acquisition of equol biosynthesis genes

[0072] Genomic DNA cloning method was used to obtain the target gene. Eggeria strain HAU-JLC44 is a strict anaerobic bacterial strain. It was cultivated in Concept 400 anaerobic workstation (Ruskinn Company, UK) at 37°C using BHI medium (Bacto Company, United States), and 1 mL was taken. Bacterial liquid, use the genome extraction kit (Beijing Quanshijin Biotechnology Co., Ltd.) to extract the genomic DNA of HAU-JLC44, design primers with restriction sites and carr...

Embodiment 3

[0119] Example 3: Application of S-equol-producing engineering bacteria in the synthesis of S-equol

[0120] 1. Preparation of seed liquid of engineering bacteria producing S-equol;

[0121] Pick the S-equol-producing engineered bacteria from the LB solid plate into 2 mL LB liquid medium containing ampicillin (100 μg / mL), shake the table at 120 rpm, and incubate at 37°C for 12 h to obtain Seed solution for inoculation.

[0122] 2. Transformation medium and transformation conditions

[0123] (1) Transformation medium 1: Take 2.5 g of LB solid medium powder, dissolve it in 100 mL of PBS buffer with a pH value of 6.5, aliquot into 2 mL / tube, and sterilize under high temperature and high pressure at 121°C for 15 minutes before use.

[0124] Transformation medium 2: Add 1.5 g of crushed soybean powder to 100 mL of distilled water, bathe in water at 80 °C for 1 h, let it stand in the refrigerator overnight, take out the supernatant to obtain the soybean powder infusion, add phosph...

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Abstract

The invention discloses Eggerthella sp., engineering bacteria generated by S-equol and a construction method and application of the engineering bacteria, and relates to the technical field of microbial technology. The preservation number of Eggerthella sp. HAU-JLC44 is CGMCC No.12354. The construction method of the engineering bacteria generated by S-equol comprises the steps that three invertase genes, namely, E-dgr, E-dhdr and E-thdr, involved in equol synthesis are cloned from the Eggerthella sp. HAU-JLC44; the sequence of a 145 bp noncoding region between the E-dhdr gene and the E-thdr gene is cloned as one gene and named as E-tdhdr; E-dgr and E-tdhdr are cloned to pETDuet-1 and expressed in BL21(DE3), and the engineering bacteria are obtained. According to the Eggerthella sp., the engineering bacteria generated by S-equol and the construction method and application of the engineering bacteria, Escherichia coli engineering bacteria with three coexpression genes are used for converting the substrate daidzein or soybean agglutinin into the S-equol, the conversion performance is stable, and the method is simple.

Description

technical field [0001] The invention relates to the technical field of microorganisms. Background technique [0002] Soy isoflavones (Soy isoflavones) are a class of secondary metabolites produced during the growth of soybeans and other legumes, of which Genistein and Daidzein are the main free aglycones of soybean isoflavones . Soybean isoflavones have various physiological functions such as anti-oxidation, anti-cancer, reducing osteoporosis, and reducing the incidence of cardiovascular and cerebrovascular diseases. The results of in vivo and in vitro experiments show that soybean isoflavones ingested by humans or other animals will be metabolized into various metabolites by the microbial flora living in the gastrointestinal tract. A large number of research results have confirmed that the biological activities of equol, such as estrogen, anti-oxidation, prevention of osteoporosis, and anti-cancer (such as breast and prostate cancer), are significantly higher than those o...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12P17/06C12N15/70C12N1/21C12R1/01C12R1/19
CPCC12N9/0004C12P17/06C12N1/205C12R2001/01
Inventor 王秀伶高雅宁于秀梅张红蕾
Owner HEBEI AGRICULTURAL UNIV.
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