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Eggeria, s-equol producing engineering bacteria and its construction method and application

A technology of eggeria and equol, applied in the field of microbiology

Active Publication Date: 2019-11-12
HEBEI AGRICULTURAL UNIV.
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

A strict anaerobic strain that can convert the substrate genistein to L-5-OH-equol was isolated from fresh chicken feces in our laboratory Slackia sp. AUH-JLC159 (ZL201310043107.X), but the transfer and culture 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. The substrate daidzein is converted to S -equol

Method used

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  • Eggeria, s-equol producing engineering bacteria and its construction method and application
  • Eggeria, s-equol producing engineering bacteria and its construction method and application
  • Eggeria, s-equol producing engineering bacteria and its construction method and application

Examples

Experimental program
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Effect test

Embodiment 1

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

[0056] 1. Strain HAU-JLC44 is a gram-positive strict anaerobic bacterial strain isolated from fresh rooster feces. The anaerobic bacteria has the original transformation effect on the substrate daidzein. The isolation and screening process of strain HAU-JLC44 is :

[0057] 1) Pick up fresh rooster feces with sterilized cotton swabs, put them in 1 mL of fresh BHI liquid medium, and place them in a 37°C anaerobic workstation, as the microbial flora for screening specific functional microbial strains;

[0058] 2) Perform a gradient dilution of 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 set the 100 μl concentration to 10 -5 , 10 -6 , 10 -7 , 10 -8 Spread the diluted micro...

Embodiment 2

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

[0070] The present invention utilizes the wild-type strain Eigeria ( Eggerthella sp.) Genomic DNA cloning of HAU-JLC44 S -Equol biosynthesis genes, linking different functional genes on the same plasmid, constructing a S -Equol production engineering bacteria, the construction of the engineering bacteria mainly includes the following steps:

[0071] 1. Acquisition of equol biosynthesis genes

[0072] Genomic DNA cloning method is used to obtain the target gene. Egger's strain HAU-JLC44 is a strict anaerobic bacterial strain. It is cultivated on Concept 400 anaerobic workstation (Ruskinn, UK) using BHI medium (Bacto, USA) at 37°C, and 1 mL Bacteria, use the genome extraction kit (Beijing Quanshijin Biotechnology Co., Ltd.) to extract the genomic DNA of HAU-JLC44, design primers with restriction sites and carry a repeating sequence of the vector, and amplify by PCR E - dgr Gene and E - tdhd...

Embodiment 3

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

[0120] 1, S -Preparation of seed liquid of engineered bacteria producing equol;

[0121] will S -Equol-producing engineering bacteria were picked from the LB solid plate into 2 mL of LB liquid medium containing ampicillin (100 μg / mL), the rotation of the shaker was 120 rpm, and the seed solution was obtained after 12 hours at 37°C. , Used for vaccination.

[0122] 2. Transformation medium and transformation conditions

[0123] (1) Transformation medium 1: Take 2.5 g of LB solid medium powder, dissolve it with 100 mL of PBS buffer with pH value of 6.5, divide into 2 mL / tube, and autoclave at 121°C for 15 min for later use.

[0124] Transformation medium 2: Add 1.5 g of ground soybean powder to 100 mL of distilled water, and place it in a water bath at 80 ℃ for 1 h. Let stand overnight in the refrigerator. Take out the supernatant to obtain the soybean powder infusion. Add phosphate b...

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Abstract

The invention discloses a kind of Aggeria, S ‑Equol-producing engineering bacteria and its construction method and application relate to the field of microbial technology. Eggeria HAU‑JLC44, the preservation number is CGMCC No.12354. S The construction method of equol-producing engineering bacteria is as follows: three invertase genes involved in equol synthesis are cloned from Eggeria HAU-JLC44: E‑dgr , E‑dhdr and E‑thdr ;Will E‑ dhdr , E‑thdr and the 145 bp non-coding region sequence between the two genes were cloned as one gene, called E‑tdhdr ;Will E‑ dgr and E‑tdhdr Cloned to pETDuet‑1, expressed in BL21 (DE3), to obtain engineering bacteria. The present invention uses Escherichia coli engineering bacteria co-expressing three genes to convert the substrate daidzein or daidzein into S ‑Equol, with stable conversion performance and simple method.

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 by soybeans and other legumes during their growth. Genistein and Daidzein are the main free aglycons of soy isoflavones. . Soy isoflavones have many physiological functions such as anti-oxidation, anti-cancer, reducing osteoporosis, and reducing the incidence of cardiovascular and cerebrovascular diseases. In vivo and in vitro test results show that the soy isoflavones ingested by the human body or other animals will be metabolized by the microbial flora inhabiting the gastrointestinal tract into various metabolites. A large number of research results confirmed that the biological activities of equol, such as estrogen, anti-oxidation, prevention of osteoporosis, and anti-cancer (such as breast cancer and prostate cancer), are significantly higher than soy isoflavones themselves or oth...

Claims

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Application Information

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Patent Type & Authority Patents(China)
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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