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A kind of Saccharomyces cerevisiae engineering bacteria for producing betulinic acid and construction method thereof

A technology of Saccharomyces cerevisiae and betulinic acid, applied in the field of genetic modification of microorganisms, can solve problems such as waste of resources

Active Publication Date: 2019-07-19
湖北济安堂药业股份有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the main source of betulinic acid is directly extracted and purified from the bark of white birch, and there is a serious waste of resources.

Method used

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  • A kind of Saccharomyces cerevisiae engineering bacteria for producing betulinic acid and construction method thereof
  • A kind of Saccharomyces cerevisiae engineering bacteria for producing betulinic acid and construction method thereof
  • A kind of Saccharomyces cerevisiae engineering bacteria for producing betulinic acid and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] Example 1 Construction of Saccharomyces cerevisiae strain WAT11-LUP-BPLO producing betulinic acid

[0035] 1. Construction of yeast integration vector YIPlac204-LUP-BPLO

[0036] The LUP gene of Arabidopsis thaliana and the BPLO gene from white birch (Betulaplatyphylla) were respectively connected to the GAL1 and GAL10 promoters of the same PESC-Trp vector to obtain the plasmid PESC-TRP-LUP-BPLO( figure 1). Due to the requirement of the enzyme cleavage site, a base of the EcoRV enzyme cleavage site in the LUP gene was mutated (cytosine in GATATC was mutated into thymine). The specific method is as follows: the plasmid PESC-TRP-LUP-BPLO is used as a template, and two pairs of primers P1 / P4 and P2 / P3 are respectively used for the first round of PCR. Then, the products of the two groups of PCRs in the first round were mixed in equal amounts as templates, and the second round of PCR was performed with the primer pair P3 / P4. The second-round product was subjected to agaro...

Embodiment 2

[0043] Example 2 Construction of Saccharomyces cerevisiae strain W80 with high betulinic acid production

[0044] The Saccharomyces cerevisiae strain WAT11-LUP-BPLO constructed in Example 1 has been able to produce betulinic acid. On this basis, the inventors continued to study and unexpectedly found that when the regulatory factor GAL 80 gene was mutated, the content of the product betulinic acid be greatly improved. According to this finding, the inventors further improved the Saccharomyces cerevisiae strain WAT11-LUP-BPLO by knocking out the GAL 80 gene in the genome of the Saccharomyces cerevisiae strain WAT11-LUP-BPLO by means of homologous recombination. The specific method is as follows:

[0045] 1. Construction of homologous knockout fragments

[0046] Using the pUG6 plasmid (Euroscarf) as a template, use primers P5 / P6 to amplify the loxP-KanMX-loxP selection marker fragment (1724bp), using Saccharomyces cerevisiae WAT11 genomic DNA as a template, use primers P7 / P8 t...

Embodiment 3

[0059] Example 3 Production and extraction of betulinic acid using the Saccharomyces cerevisiae strain of the present invention

[0060] 1. Production and extraction of betulinic acid

[0061] Inoculate the Saccharomyces cerevisiae strain WAT11-LUP-BPLO or W80 obtained in Example 1 or 2 into SD-Trp-liquid medium with 2% glucose as carbon source, place at 30°C, and cultivate at 250rpm / min for 16 hours , and then collected the cells and washed with sterile water and resuspended in SD-Trp-medium with 2% galactose as carbon source to the initial OD 600 The value is 0.8, placed at 30° C., 250 rpm / min for induction culture for 7 days. The yeast culture solution obtained above was acidified (adjusted pH to 2.0 with 2M HCl), and extracted three times with an equal volume of ethyl acetate. The three ethyl acetate phases were combined and dried. After drying, reconstitute with a small amount of ethyl acetate, transfer to a 2ml centrifuge tube, and dry. betulinic acid

[0062] 2. Id...

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Abstract

The invention relates to an engineered strain of saccharomyces cerevisiae for producing a betulinic acid. The engineered strain of the saccharomyces cerevisiae is characterized in that an LUP gene expression cassette and a BPLO gene expression cassette are integrated in a genome on the basis of a saccharomyces cerevisiae strain WAT11; and a GAL80 gene is knocked out with priority from the engineered strain of the saccharomyces cerevisiae. The invention further relates to a method for building the engineered strain of the saccharomyces cerevisiae and a method for producing the betulinic acid employing the engineered strain of the saccharomyces cerevisiae. Through the engineered strain of the saccharomyces cerevisiae, the betulinic acid can be produced employing the saccharomyces cerevisiae in a microbial fermentation manner; the operation is simple; the yield is high; the production cycle is short; and the floor area is small.

Description

technical field [0001] The invention relates to the genetic modification of microorganisms, more particularly to a Saccharomyces cerevisiae engineering bacterium producing betulinic acid and a construction method thereof. Background technique [0002] Betulinic acid (BA), also known as betulinic acid, is a pentacyclic triterpenoid compound. Pentacyclic triterpenoids are a very important class of plant secondary metabolites, which usually have a wide range of pharmacological effects and biological activities. This type of compound exists widely in the plant kingdom, and contains a large number of types. The main pentacyclic triterpenes are oleanane type, ursane type, lupinane type and suberane type. Betulinic acid is a typical representative of lupine-type pentacyclic triterpenoids. [0003] A large number of studies in recent years have shown that betulinic acid has significant pharmacological effects and biological activities, mainly in anti-tumor, anti-HIV, anti-inflamma...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/19C12N15/81C12P33/00C12R1/865
CPCC07K14/395C07K14/415C12N9/90C12N15/81C12N2800/102C12P33/00C12Y504/99041
Inventor 陈国平陈斌章焰生周晨
Owner 湖北济安堂药业股份有限公司
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