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Method for synthesizing 3-O-glucose-based oleanolic acid and cellobiose oleanolic acid by using saccharomyces cerevisiae

A kind of Saccharomyces cerevisiae and gene technology, applied in the field of bioengineering, can solve the problems such as the difficult realization of chemical synthesis of molecular structure, and achieve the effects of increasing production, optimizing fermentation process, and reducing costs

Active Publication Date: 2017-01-11
BEIJING INSTITUTE OF TECHNOLOGYGY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, 3-O-glucosyl oleanolic acid and cellobiose oleanolic acid are also difficult to synthesize due to the complexity of their molecular structures.

Method used

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  • Method for synthesizing 3-O-glucose-based oleanolic acid and cellobiose oleanolic acid by using saccharomyces cerevisiae
  • Method for synthesizing 3-O-glucose-based oleanolic acid and cellobiose oleanolic acid by using saccharomyces cerevisiae
  • Method for synthesizing 3-O-glucose-based oleanolic acid and cellobiose oleanolic acid by using saccharomyces cerevisiae

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Synthetic Genes CYP716A12, MtCPR and UGT73C10

[0022] The P450 cytochrome monooxygenase encoded by the gene CYP716A12 and the cytochrome reductase encoded by the gene MtCPR can work together to oxidize β-amyresinol to oleanolic acid (see figure 1 ); the UDP-glucotransferase encoded by the gene UGT73C10 can glycosylate the hydroxyl group at the C-3 position of oleanolic acid to generate 3-O-glucosyl oleanolic acid and cellobiose oleanolic acid (see figure 2 ). The P450 cytochrome monooxygenase gene CYP716A12 from Medicago truncatula (Medicago truncatula), the cytochrome reductase gene MtCPR from Medicago truncatula (Medicago truncatula) and the P450 cytochrome reductase gene MtCPR from European mountain mustard (Barbarea vulgaris) were retrieved from the NCBI database. For the sequence of UDP-glucose transferase gene UGT73C10, the codon optimization software JCAT was used to optimize the codons of the three genes, so that the amino acid coding sequence confo...

Embodiment 2

[0023] Example 2 Construction of expression vectors for genes CYP716A12, MtCPR and UGT73C10

[0024] Using the codon-optimized pUC57-CYP716A12 synthesized by the company as a template, primers CYP716A12-F-BamHI and CYP716A12-R-SalI were used to amplify the P450 cytochrome monooxygenase gene CYP716A12 of Medicago truncatula, A CYP716A12 gene fragment with a length of 1455bp was obtained. The gene fragment and the vector pESC-URA were digested simultaneously by restriction endonucleases BamHI and SalI, the gene fragment obtained above was connected to the expression vector pESC-URA, transformed into Escherichia coli TOP10 strain, and the plasmid pESC-URA-P was extracted GAL1 -CYP716A12-T CYC1 , Sequencing verification of the CYP716A12 gene on the plasmid, its sequence is shown in SEQ ID No.1, and the results show that it is correct.

[0025] Using the codon-optimized pUC57-CPR synthesized by the company as a template, the primers MtCPR-F-ClaI and MtCPR-R-NotI were used to ampl...

Embodiment 3

[0027] Example 3: Construction of engineered Saccharomyces cerevisiae producing oleanolic acid, 3-O-glucosyl oleanolic acid and cellobiose oleanolic acid

[0028] The expression vector pESC-URA-P constructed in Example 2 was transformed by lithium acetate chemical conversion method GAL1 -CYP716A12-T CYC1 -P GAL10 -MtCPR-T ADH1 and pESC-TRP-P GAL1 -UGT73C10-T CYC1 , co-transformed into S. cerevisiae strain Sgib capable of producing β-amyresinol. Spread the transformation product on a screening plate containing 2% glucose basal medium (SD-URA-TRP) lacking uracil and tryptophan, and culture at 30°C for 2-5 days. Randomly select 6-10 clones from the screening plate, and extract their plasmids respectively. Using the extracted plasmid as a template, the primer pair CYP716A12-F-BamHI and CYP716A12-R-SalI were used to amplify the P450 cytochrome monooxygenase gene CYP716A12 of Medicago truncatula; the primer pair MtCPR-F-ClaI and MtCPR-R-NotI amplifies the cytochrome reductase...

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Abstract

The invention provides a method for synthesizing 3-O-glucose-based oleanolic acid and cellobiose oleanolic acid by using saccharomyces cerevisiae engineering bacteria, and belongs to the field of bioengineering. The method comprises the following steps: synthesizing a codon optimized P450 cytochrome monooxygenase gene, a cytochrome reductase gene and a UDP-glucosyltransferase gene by a chemical method; constructing corresponding gene expression boxes by combining a saccharomyces cerevisiae promoter with a terminator; constructing gene expression vectors by a DNA (Deoxyribonucleic Acid) klenow fragment assembling method, and importing the gene expression vectors into the saccharomyces cerevisiae capable of producing beta-amyrin. Direct synthesis of the 3-O-glucose-based oleanolic acid and the cellobiose oleanolic acid serving as plant secondary metabolites in the saccharomyces cerevisiae is realized for the first time; in addition, two synthesized compounds can span cytomembrane of the saccharomyces cerevisiae engineering bacteria, and a downstream separation and extraction process is simplified, so that a new idea is provided for producing pentacyclic triterpene compounds with low water solubility and difficulty in spanning membranes by using the saccharomyces cerevisiae. The method is simple in process and can be used for producing the 3-O-glucose-based oleanolic acid and the cellobiose oleanolic acid by fermenting.

Description

technical field [0001] The invention belongs to the field of biological engineering, and in particular relates to a construction method for producing 3-O-glucosyl oleanolic acid and cellobiose oleanolic acid by using engineered Saccharomyces cerevisiae. Background technique [0002] 3-O-glucosyl oleanolic acid and cellobiose oleanolic acid are important oleanane-type pentacyclic triterpenoids. This type of compound belongs to the plant secondary metabolites with special value, and has many important functions in resisting biotic and abiotic stress, such as anti-infection, anti-radiation, and anti-drought, and many substances have pharmaceutical activity against human diseases. 3-O-glucosyl oleanolic acid and cellobiose oleanolic acid are widely distributed in ginseng (Panax ginseng), licorice (Glycyrrhiza uralensis), Chinese snow gall (Hemsleya chinensis cogn), privet fruit (Glossyprivet fruit), In the leaves, fruits and rhizomes of various plants such as olive (Olea europa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N1/19C12P33/00C12P19/56C12R1/865
CPCC12N9/0042C12N9/0077C12N9/1051C12P19/56C12P33/00C12Y106/02004
Inventor 李春赵雨佳刘啸尘蔚欢樊婧婧
Owner BEIJING INSTITUTE OF TECHNOLOGYGY
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