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Recombinant saccharomyces cerevisiae capable of producing glycyrrhetinic acid, and construction method and applications thereof

A glycyrrhetic acid and yeast technology, applied in the field of glycyrrhetic acid production, can solve the problems of ecological environment destruction, failure to meet pharmacopoeia, complex structure of glycyrrhetic acid and the like

Active Publication Date: 2017-05-10
INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current licorice resources are mainly planted licorice, but the main problem in planting licorice is that the content of glycyrrhizic acid, its functional component, is less than 2%, which cannot meet the requirements of the Pharmacopoeia
[0005] Glycyrrhetinic acid has a complex structure and cannot be chemically synthesized at present
Extraction and separation from licorice is limited by limited licorice resources and its low content. At the same time, large-scale licorice excavation will bring damage to the ecological environment and cause desertification.

Method used

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  • Recombinant saccharomyces cerevisiae capable of producing glycyrrhetinic acid, and construction method and applications thereof
  • Recombinant saccharomyces cerevisiae capable of producing glycyrrhetinic acid, and construction method and applications thereof
  • Recombinant saccharomyces cerevisiae capable of producing glycyrrhetinic acid, and construction method and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1. Acquisition of genes required for glycyrrhetinic acid synthesis and yeast's own gene elements in modular regulation

[0034] Using fresh licorice as a material, RNA was extracted by methods known in the art, and cDNA was obtained by reverse transcription. According to conventional methods in the field, primers for β-AS, CYP88D6, CYP72A154, and Cyb5 genes were designed to amplify the above-mentioned fragments; ERG10, ERG12, ERG13, ERG19, ERG20, Primers for ERG9, ERG1, ERG8, IDI1, and tHMG gene fragments to amplify the above gene fragments in yeast.

Embodiment 2

[0035] Example 2. Acquisition of genes required for glycyrrhetinic acid synthesis and expression clusters composed of yeast's own gene elements in modular regulation

[0036]Specifically, an expression cluster refers to a promoter+gene fragment sequence+terminator. The construction of further expression clusters is divided into two steps: first, the constitutive promoter and terminator sequences commonly used in yeast are amplified, and the amplified promoters here include P-TEF1, P-TEF2, P-TDH3, and P-ADH1 and P-PGK1, the terminator sequences include: T-PGK1, T-CYC1, T-TDH3 and T-ADH1, the primers used to amplify the above promoter and terminator sequences are shown in Table 1. The amplified promoter sequence and terminator sequence were connected to the cloning vector puc19L in the order of promoter-restriction site-terminator to construct puc19L-Ptef1-Tpgk1, puc19L-Ptdh3-Tcyc1, puc19L-Ppgk1-Tadh1, For the puc19L-Ppgk1-Tpgk1, puc19L-Ptef2-Tcyc1, and puc19L-Ptdh3-Ttdh3 vecto...

Embodiment 3

[0040] Example 3. Creation of a synthetic pathway for glycyrrhetinic acid in yeast

[0041] The gene expression clusters of Ppgk1-β-AS-Tadh1, Ptdh3-CYP88D6-Tcyc1, Padh1-CYP72A154-adh1 and Ptdh3-CPR1-Tcyc1 were in the form of homologous recombination ( Figure 7 shown) was integrated into the rDNA site of the yeast chromosome, positive clones were detected by PCR, and the ability of the positive clone strains to produce glycyrrhetinic acid was detected, and the yeast strain with the highest glycyrrhetinic acid production was obtained and named GA-1. Among them, the sequence of integration of exogenous gene expression cluster fragments is as follows: Figure 1A shown.

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Abstract

The invention relates to a recombinant saccharomyces cerevisiae capable of producing glycyrrhetinic acid, and a construction method and applications thereof, and more specifically relates to a recombinant saccharomyces cerevisiae which is capable of producing glycyrrhetinic acid and is numbered as CGMCC13126, a construction method thereof, and applications of the recombinant saccharomyces cerevisiae in producing glycyrrhetinic acid.

Description

technical field [0001] The invention relates to the technical field of glycyrrhetinic acid production. Specifically, the present invention relates to glycyrrhetinic acid-producing recombinant Saccharomyces cerevisiae, its construction method and use. Background technique [0002] Licorice (Glycyrrhiza uralensis Fisch) is a perennial herb of the leguminous licorice genus. Its roots and stems are used as medicine. Chinese medicinal materials. At present, more than 200 compounds have been isolated from licorice, mainly divided into triterpenoids, flavonoids and polysaccharides. Among them, glycyrrhizic acid (glycyrrhizic acid), also known as glycyrrhizin (glycyrrhizin), is an oleanane-type pentacyclic triterpenoid saponin and is the main active ingredient of licorice, accounting for 2-8% of the dry weight of licorice. Modern pharmacological research has confirmed that glycyrrhizic acid has various pharmacological activities, such as antibacterial and anti-inflammatory, regul...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12N15/81C12P33/00C12R1/865
CPCC12N9/0042C12N9/90C12N15/81C12P33/00C12Y106/02004C12Y504/99039
Inventor 陈士林王彩霞孙伟苏新尧张梦婷孙梦楚
Owner INST OF CHINESE MATERIA MEDICA CHINA ACAD OF CHINESE MEDICAL SCI
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