A kind of Saccharomyces cerevisiae engineered bacteria producing glycyrrhetinic acid or its precursor and its construction method

A technology of Saccharomyces cerevisiae and glycyrrhetinic acid, applied in the direction of microorganism-based methods, biochemical equipment and methods, botany equipment and methods, etc., can solve the problems of low efficiency, high cost, difficult chemical synthesis under molecular structure synthesis conditions, etc. , to achieve the effect of broad application prospects and convenient production process

Active Publication Date: 2019-11-08
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Plant extraction has disadvantages such as long cycle, low efficiency, high cost, and great environmental damage.
The complex molecular structure and harsh synthesis conditions of glycyrrhetinic acid also make it difficult to carry out chemical synthesis

Method used

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  • A kind of Saccharomyces cerevisiae engineered bacteria producing glycyrrhetinic acid or its precursor and its construction method
  • A kind of Saccharomyces cerevisiae engineered bacteria producing glycyrrhetinic acid or its precursor and its construction method
  • A kind of Saccharomyces cerevisiae engineered bacteria producing glycyrrhetinic acid or its precursor and its construction method

Examples

Experimental program
Comparison scheme
Effect test

experiment example 1

[0053] Experimental example 1: Acquisition of related genes in the glycyrrhetinic acid synthesis pathway

[0054] A. Acquisition of β-amyresinol synthase gene

[0055] According to the sequence of the β-amyresinol synthase gene (Genbank registration sequence number is AB037203), through codon optimization, the codons of the β-amyresinol synthase gene have yeast preference, and the optimized gene sequence generated is SEQ ID No. 1.

[0056] B. Acquisition of the CYP450 oxidase gene that oxidizes the carbon 11 of β-amyresinol

[0057] According to the sequence of the CYP450 oxidase CYP88D6 gene (Genbank registration number is AB433179), the codon of the CYP88D6 gene has yeast preference through codon optimization, and the optimized gene sequence generated is SEQ ID No.2.

[0058] Primers were designed according to the sequence of CYP450 oxidase CYP88D6 gene (Genbank registration sequence number is AB433179), SEQ ID No.3: 5'-ATGGAAGTACATTGGGTTTGC-3' and SEQ ID No.4: 5'-CTAAGCAC...

experiment example 2

[0068] Experimental Example 2: Construction of Expression Cassette

[0069] Construction of A, β-amyresinol synthase expression cassette

[0070] The left homology arm HOL, yeast promoter P FBA1 , β-amyresinol synthase gene, yeast terminator T CYC1 The method of overlapping extension PCR is used to connect in this way to obtain the expression cassette P of β-amyresinol synthase FBA1 -bAS-T CYC1

[0071] B. Construction of the CYP450 oxidase expression cassette at the carbon 30 position of oxidizing 11-oxygen-β-amyresinol

[0072] Yeast terminator T CYC1 , yeast promoter P GPD , CYP450 oxidase gene CYP72A154 or CYP72A63, yeast terminator T ADH1 The method of overlapping extension PCR is used for this connection, and the CYP450 oxidase T at the carbon 30 position of oxidizing 11-oxo-β-amyresinol is obtained CYC1 -P GPD –CYP72A154-T ADH1 or T CYC1 -PGPD -CYP72A63-T ADH1

[0073] C, construction of the CYP450 oxidase expression cassette at the carbon 11 position of ox...

experiment example 3

[0079] Experimental Example 3: Acquisition of Saccharomyces cerevisiae Engineering Bacteria Producing Glycyrrhetinic Acid

[0080] Yeast Transformation of Fragments Using the Lithium Acetate Method

[0081] Using the homologous recombination function of the yeast itself, all the expression cassettes constructed above were co-transferred into the HO site of the Saccharomyces cerevisiae INVSc1 genome. After transformation, use the YPD solid plate supplemented with Geneticin (G418) for screening, and the obtained transformants are transferred to the YPD liquid medium supplemented with Geneticin (G418) for 1-2 days, the genome is extracted, and positive clones are identified by PCR , plate streak or glycerol bacteria preservation.

[0082] On the basis of the existence of the MVA pathway in the yeast itself, the glycyrrhetinic acid synthetase is introduced exogenously to obtain the engineering strain of Saccharomyces cerevisiae with the function of producing glycyrrhetinic acid. ...

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Abstract

The invention discloses a saccharomyces cerevisiae engineered strain used for producing glycyrrhetinic acid and a precursor of glycyrrhetinic acid, namely, 11-hydroxy-beta-amyrin, or 11-oxo-beta-amyrin, or 30-hydroxy-beta-amyrin, or 11,30-hydroxy-beta-amyrin, or 30-hydroxy-11-oxo-beta-amyrin, or 30-aldehydo-11-oxo-beta-amyrin and a construction method of the saccharomyces cerevisiae engineered strain. The construction method comprises the following steps: constructing a beta-amyrin synthase expression box, an oxidase expression box of the 11th site of carbon of beta-amyrin, an oxidase expression box of the 30th site of carbon of 11-oxo-beta-amyrin, and an oxidordeuctase expression box of cytochrome P450, and importing the expression boxes to a saccharomyces cerevisiae genome. According to the saccharomyces cerevisiae engineered strain and the construction method, the synthesis of glycyrrhetinic acid is coupled with the growth of the yeast, so that the yeast synthesis of glycyrrhetinic acid or the precursor of glycyrrhetinic acid is realized, for the method, the inducing of an effector is not needed, the process is simple, and the method can be used for fermentation production of glycyrrhetinic acid and the key precursor of glycyrrhetinic acid.

Description

technical field [0001] The invention relates to the construction and fermentation of a Saccharomyces cerevisiae engineering bacteria to produce glycyrrhetinic acid or its precursor: 11-hydroxy-β-amyrin (11-hydroxy-β-amyrin), or 11-oxo-β-amyrin Resin alcohol (11-oxo-β-amyrin), or 30-hydroxy-β-amyrin alcohol (30-hydroxy-β-amyrin), or 11,30-hydroxy-β-amyrin alcohol (11,30-hydroxy -β-amyrin), or 30-hydroxy-11-oxo-β-amyrin (30-hydroxy-11-oxo-β-amyrin), or 30-formyl-11-oxo-β-amyrin ( Glycyrrhetaldehyde) belongs to the field of bioengineering. Background technique [0002] Terpenoids are a kind of plant secondary metabolites, which have various biological activities and pharmacological values. Pharmacology has proved that triterpenoids such as glycyrrhizin (GL) have very important effects in liver protection, anti-inflammation, anti-tumor, anti-virus, etc., and can also be used as sweeteners or food additives. Glycyrrhetinic acid (Glycyrrhetinic acid, GA) is the precursor substa...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/53C12N15/81C12N1/19C12P33/00C12R1/865
CPCC12N9/0081C12N9/90C12P33/00C12Y114/15006C12Y504/99039
Inventor 李春朱明王彩霞孙文涛周安琪
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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