Method for producing beta-amyrin with saccharomyces cerevisiae engineering bacterium

A technology of Saccharomyces cerevisiae and arysinol, applied in the direction of microorganism-based methods, biochemical equipment and methods, fermentation, etc., which can solve the problems of low accumulation of β- arysinol and difficult synthesis by chemical methods, and achieve easy genetic manipulation , Simplify the fermentation process and reduce the cost

Active Publication Date: 2015-04-15
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method for producing β-amyresin by using Saccharomyces cerevisiae engineering bacteria to solve the limitations of low accumulation of β-amyresin in licorice and difficult synthesis by chemical methods

Method used

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  • Method for producing beta-amyrin with saccharomyces cerevisiae engineering bacterium
  • Method for producing beta-amyrin with saccharomyces cerevisiae engineering bacterium
  • Method for producing beta-amyrin with saccharomyces cerevisiae engineering bacterium

Examples

Experimental program
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Effect test

Embodiment 1

[0016] Embodiment 1: Construction of Saccharomyces cerevisiae Genetic Engineering Bacteria

[0017] 1. The 2,3-oxidized squalene monooxygenase SQE gene fragment was cloned from Candida albicans CGMCC2.2086, which was consistent with the Candida albicans 2,3-oxidized squalene monooxygenase gene sequence published by Genbank (Genbank registration sequence number U69674) was compared by BLAST, and the homology analysis showed that the homology of the SQE gene fragment of the clone and the Candida albicans 2,3-oxidosqualene monooxygenase gene announced by the Genbank gene bank was 100% (such as SEQ ID shown in No.1).

[0018] The primer sequences are:

[0019] Primer 1: 5'> GATTAGCATCCATAACCGCATACTCTAATTGACGATAAC ATGAGTTCAGTTAAGTATGATYS1 overlapping sequences.

[0020] Primer 2: 5'> GCCAAGTAGGCAATTTTTAGTACTGTCAGTATTGTTAT CTATCTTACAATCTCGTTCCTYS1 overlapping sequences.

[0021] 2. Obtained the bAS gene fragment of Glycyrrhiza glabra β-amyresinol synthase (Genbank registratio...

Embodiment 2

[0050] Example 2: Verification of Saccharomyces cerevisiae Engineering Bacteria Fermentative Production of β-Arysinol

[0051] Select the Saccharomyces cerevisiae engineered bacterium screened out in Example 1, culture in a shake flask at 30° C. in a medium containing 2% glucose, 2% peptone and 1% yeast powder, centrifuge at 5000 rpm for 5 min after 5 days, and use 10 mL of 20% hydrogen for precipitation Resuspend in potassium oxide solution (containing 50% ethanol), then boil in boiling water for 10 min, extract twice with n-hexane after cooling at room temperature, combine the extracts and concentrate to 1 mL, then add 100 μL of pyrimidine and 50 μL of N,O to the concentrated solution -Bis(trimethylsilyl)trifluoroacetamide, react at 37°C for 2h. Shimadzu gas chromatography-mass spectrometry instrument GCMS-QP2010 equipped with Agilent chromatographic column DB-1 (30m*0.25mm*0.25um) was used to analyze the fermentation production of β-amyresinol by Saccharomyces cerevisiae en...

Embodiment 3

[0052] Example 3: Application of Saccharomyces cerevisiae Engineering Bacteria in the Fermentative Production of β-Arysinol

[0053] Seed liquid culture: Pick the single colony of Saccharomyces cerevisiae engineered bacteria screened in Examples 1 and 2 from the plate, inoculate in 40mL medium containing 2% glucose, 2% peptone and 1% yeast powder, 30°C, 170rpm Shake culture for 36h.

[0054] Fermentation test: Transfer the inoculum solution to a 300mL shake flask filled with 100mL fermentation medium (2% glucose, 2% peptone, 1% yeast powder, pH7.0) according to the inoculum size of the initial absorbance value of 0.10, 30°C, 170rpm Shake culture for 120 hours, sampling once every 24 hours, and measure the content of β-amyresinol in Saccharomyces cerevisiae cells. The results showed that the content of β-amyresinol reached 11.7 mg / L after fermentation of S. cerevisiae engineered bacteria.

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Abstract

The invention provides a method for producing beta-amyrin with a saccharomyces cerevisiae engineering bacterium, and belongs to the field of biochemical engineering. According to the method, a 2,3-oxidized squalene monooxygenase gene is cloned from candida albicans, a glycyrrhiza glabra beta-amyrin synthase gene subjected to codon optimization is chemically synthesized, a gene expression cassette is constructed by utilizing a saccharomyces cerevisiae constitutive promoter, the gene expression cassette and a plasmid after double digestion jointly invert saccharomyces cerevisiae, and assembly of the gene expression cassette and the plasmid is achieved by a homologous recombination function of the saccharomyces cerevisiae, so that 2,3-oxidized squalene monooxygenase is reinforced, and beta-amyrin synthase is led in. Saccharomyces cerevisiae engineering bacterium metabolizable glucose directly synthesizes beta-amyrin, and synthesis of beta-amyrin is coupled with growth of the saccharomyces cerevisiae, so that artificial synthesis of the saccharomyces cerevisiae of a plant secondary metabolism product, namely beta-amyrin is achieved. The method requires no effect agent induction, is simple in technology, and can be used for fermenting and producing beta-amyrin.

Description

technical field [0001] The invention relates to a construction of Saccharomyces cerevisiae engineering bacteria and a method for fermenting and producing β-amyresinol, which belongs to the field of biochemical industry. Background technique [0002] Terpenoids are a class of plant secondary metabolites with special value. They have important functions in resisting biotic and abiotic stresses such as infection, ultraviolet radiation, and drought. Many terpenes have shown pharmaceutical activity against human diseases. Glycyrrhizic acid is the most important terpenoid compound of the traditional Chinese medicinal plant licorice, which has many functions such as anti-infection, anti-inflammation, anti-ulcer, anti-virus and prevention and treatment of tumors. β-Arysinol is an important precursor for the biosynthesis of glycyrrhizic acid. It not only has similar biological functions as glycyrrhizic acid, but also has special effects in antihypertensive and hypolipidemic aspects. ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/63C12P33/00C12R1/865
Inventor 李春张根林周晓宏刘静竹
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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