Building method of saccharomyces cerevisiae engineered strains capable of synthesizing glycyrrhetinic acid

A technology of Saccharomyces cerevisiae and glycyrrhetinic acid, which is applied in the field of construction of Saccharomyces cerevisiae engineering bacteria, can solve problems such as difficulties in production and chemical synthesis methods, and achieve the effects of saving fermentation costs, low raw material costs, and stable expression genetics

Inactive Publication Date: 2017-07-28
SHIHEZI UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the limitations and difficulties of glycyrrhetinic acid hydrolysis production and chemical synthesis

Method used

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  • Building method of saccharomyces cerevisiae engineered strains capable of synthesizing glycyrrhetinic acid
  • Building method of saccharomyces cerevisiae engineered strains capable of synthesizing glycyrrhetinic acid

Examples

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Embodiment 1

[0026] A method for constructing an engineered Saccharomyces cerevisiae capable of synthesizing glycyrrhetinic acid, firstly using the β-aromatic alcohol synthase GgbAS gene derived from Glycyrrhiza glabra (Genbank registration number is AB037203) and the cytochrome P450 derived from Glycyrrhiza uralensis The oxidase CYP88D6 gene (Genbank registration number is AB433179) and the cytochrome P450 oxidase CYP72A154 gene (Genbank registration number is AB558146), and the cytochrome reductase CPR1 gene from Arabidopsis thaliana (Genbank registration number is AB433179) GgbAS gene fragment, CYP88D6 gene fragment, CYP72A154 gene fragment, CPR1 gene fragment and CPR2 gene fragment were prepared with cytochrome reductase CPR2 gene (Genbank registration number AB558146). The above gene fragments, yeast promoter and yeast terminator were passed through Two-step overlap extension PCR ligation to obtain gene expression cassettes FBA1p-GgbAS-FBA1t, PGK1p-CYP88D6-GMP1t, ALA1p-CPR1-ALA1t, ENO2p...

Embodiment 2

[0063] Verification of fermentation production of glycyrrhetinic acid by engineering strain of Saccharomyces cerevisiae

[0064] Select the Saccharomyces cerevisiae engineering strains selected in Example 1, and culture them in a 30°C shake flask in a medium containing 2% glucose, 2% peptone and 1% yeast powder. After 7 days, take 50ml of the cultured Saccharomyces cerevisiae liquid and centrifuge at 8000rpm After 10 min, remove the culture medium, wash with 30 ml sterile water, centrifuge at 8000 rpm for 10 min, discard the supernatant, and resuspend the cells with 2 ml saturated sodium chloride solution. Use a cell column breaker to break the cells 5 times for 5 minutes, add an equal volume of ethyl acetate to the centrifuge tube, vortex for 3 minutes, and fully extract the yeast extract. Centrifuge at 8000rpm for 10min, draw the organic phase into a chicken heart bottle, evaporate ethyl acetate in a rotary evaporator, add 1ml methanol to fully dissolve it, pass through a 0.22μ...

Embodiment 3

[0066] Fermentation of Saccharomyces cerevisiae to produce glycyrrhetinic acid

[0067] The single colony of Saccharomyces cerevisiae engineering bacteria selected in Example 1 was inoculated into a 30 mL medium containing 2% glucose, 2% peptone and 1% yeast powder, and cultured with shaking at 30°C and 150 rpm. The culture solution after 36 hours was used as the seed solution . Inoculate the seed liquid according to 10% of the inoculum into a 500 mL shake flask containing 150 mL of culture medium, culture with shaking at 30°C and 170 rpm, add glucose to a concentration of 0.5 to 1.0% every 12 hours after 24 hours, and measure after 5 days of fermentation The content of glycyrrhetinic acid in Saccharomyces cerevisiae cells was found to synthesize 35.5μg / L of glycyrrhetinic acid by Saccharomyces cerevisiae engineering bacteria.

[0068] After 24 hours of fermentation, ethanol was used as a carbon source to feed every 12 hours. It was found that when the supplement concentration was...

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Abstract

The invention belongs to the field of biochemical engineering and particularly relates to a building method of saccharomyces cerevisiae engineered strains capable of synthesizing glycyrrhetinic acid. The building method includes: beta-amyrin synthase gene GgbAS from Glycyrrhiza glabra, cytochrome P450 oxidases CYP88D6 and CYP72A154, cytochrome P450 oxidoreductases CPR1 and CPR2 from Arabidopsis thaliana are used to build gene expression cassettes, the gene expression cassettes are co-transformed in saccharomyces cerevisiae CEN.PK2-1C, and the saccharomyces cerevisiae engineered strains with complete glycyrrhetinic acid biosynthetic pathways are assembled and formed by the homologous recombination ability of the saccharomyces cerevisiae. By the method, the complete glycyrrhetinic acid biosynthetic pathways are introduced in the saccharomyces cerevisiae engineered strains, the glycyrrhetinic acid can be produced through fermentation, and artificial synthesizing of the glycyrrhetinic acid in the saccharomyces cerevisiae is achieved.

Description

Technical field [0001] The invention belongs to the field of biochemical industry, and specifically relates to a construction method of Saccharomyces cerevisiae engineering bacteria capable of synthesizing glycyrrhetinic acid, including a construction method of Saccharomyces cerevisiae engineering bacteria containing glycyrrhetinic acid biosynthesis pathway. Background technique [0002] Terpenoids are representative biologically active molecules in nature. They are essential for plant growth and development. They play an important role in the interaction between plants and the environment. They are also an industry for products such as spices, drugs, pesticides, and sweeteners. Raw materials. Glycyrrhetinic acid is a pentacyclic triterpene compound. Since glycyrrhizic acid, the main active component of licorice, has two molecules less gluconic acid, it is easier to penetrate cell membranes, and it is easier to exert anti-inflammatory, anti-viral, lowering blood lipid and prevent...

Claims

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Application Information

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IPC IPC(8): C12N1/19C12P33/00C12R1/865
CPCC12N9/90C12N9/0042C12N9/0079C12P33/00C12Y106/02004C12Y114/15004C12Y504/99039
Inventor 张根林李宏彪李春王婷
Owner SHIHEZI UNIVERSITY
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