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A kind of genetically engineered bacteria producing protopanaxadiol and its method

A technology of protopanaxadiol and genetically engineered bacteria, which is applied in the field of genetic engineering, can solve problems such as unfavorable industrial production, slow growth of yeast, complex medium, etc., to achieve clear technology and related knowledge, simple medium, and short fermentation time Effect

Active Publication Date: 2021-11-05
SHANGHAI INST OF PHARMA IND CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The technical problem to be solved by the present invention is to provide a protopanaxadiol capable of producing protopanaxadiol to overcome defects such as slow growth rate of yeast, long fermentation time, complicated medium, and unfavorable industrial production in the prior art. Escherichia coli engineering bacteria for panaxadiol and corresponding enzyme gene, recombinant vector and method for preparing protopanaxadiol

Method used

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  • A kind of genetically engineered bacteria producing protopanaxadiol and its method
  • A kind of genetically engineered bacteria producing protopanaxadiol and its method
  • A kind of genetically engineered bacteria producing protopanaxadiol and its method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] Example 1 Construction of plasmid pCDFDuet-1SSSE

[0061] (1) Cloning of squalene synthase (SS) gene from Saccharomyces cerevisiae

[0062] Extract the genome of Saccharomyces cerevisiae ATCC204508 (purchased from ATCC) by the fungal genome DNA extraction kit (Beijing Soleibao Technology Co., Ltd.), such as electrophoresis Figure 4 ; Taking the extracted Saccharomyces cerevisiae genomic DNA as a template, using "SS up" and "SS down" as primers to amplify the SS gene (GenBank accession number: EU675328), the sequence is shown in SEQ ID NO.1.

[0063] ①Amplification primer sequence:

[0064] On SS: CGGGATCCGATGGGAAAGCTATTACAATTG (see SEQ ID NO.11 for details);

[0065] Under SS: CCAAGCTTTCACGCTCTGTGTAAAGTGTATATATAATAAAAC (see SEQ ID NO. 12 for details).

[0066] ②Amplification system:

[0067] Max DNA polymerase: 25 μL;

[0068] Primers (10 μM): 2 μL each;

[0069] Saccharomyces cerevisiae S288C genomic DNA (template): 2 μL;

[0070] Add distilled water to 50 μL...

Embodiment 2

[0092] Example 2 Construction of plasmid pACYCDuet-1D612C46P72C46

[0093] (1) D612C46 gene cloning

[0094] A. Using the dammar diene synthase gene (DDS, accession number: AB265170) as a template, through codon optimization in Escherichia coli, chemically synthesize the D gene (sequence shown in SEQ ID NO.8), using the D gene as a template, After hydrophobicity analysis, the amino acid sequence after the 612th amino acid sequence was removed, and the D612 gene was amplified with primers "D up" and "D down". The sequence of the D612 gene is shown in SEQ ID NO.3.

[0095] ①Amplification primer sequence:

[0096] Upper D: CGCGGATCCGATGTGGAAGCAGAAGGGCGCACAG (see SEQ ID NO.15 for details);

[0097] Bottom D: GTGGTCTTCTTCCATGTCGACCCAGAGCCATCCGGCATCTGGTTGC (see SEQ ID NO. 16 for details).

[0098] ②Amplification system:

[0099] Max DNA polymerase: 25μL;

[0100] Primers (10 μM): 2 μL each;

[0101] D gene (template): 2 μL;

[0102] Add distilled water to 50 μL.

[0103] ③...

Embodiment 3

[0169] Example 3 Construction of recombinant strain pCDFDuet-1SSSE pACYCDuet-1D612C46P72C46BL21(DE3)

[0170] The plasmid pCDFDuet-1SSSE and the plasmid pACYDuet-1D612C46P72C46 were simultaneously transformed into Escherichia coli BL21 (DE3) competent cells, spread on LB containing 100 μg / mL streptomycin and 30 μg / mL chloramphenicol (peptone 10%, yeast extract 5%, NaCl 10%, agarose 12%) on a solid plate, and positive clones were screened with 2×Taq Master Mix (Dye Plus), thereby obtaining the recombinant strain pCDFDuet-1SSSEpACYD612C46P72C46BL21(DE3).

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Abstract

The invention discloses a genetically engineered bacterium for producing protopanaxadiol, which expresses squalene synthase gene, 2,3-oxide squalene synthase gene, dammarene synthase gene and dammarene synthase gene in Escherichia coli. Enzyme gene, protopanaxadiol synthase gene and nicotinamide adenine dinucleotide phosphate-cytochrome P450 reductase gene engineering bacteria. The biosynthesis of protopanaxadiol by using the genetically engineered bacteria has the advantages of short fermentation time, simple culture medium, meeting the requirements of modern industry, being beneficial to popularization and application, and the like.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a biosynthesis method of protopanaxadiol and a production strain thereof. Background technique [0002] Protopanaxadiol (PPD) belongs to tetracyclic triterpenoids and is aglycon of ginseng saponins Ra1, Ra2, Rb1, Rb2, Rc, Rd, Rg3 and Rh2, etc. And other Araliaceae plants and Cucurbitaceae plants such as Gynostemma pentaphyllum. It has the effects of anti-tumor, anti-depression, and improving human immunity. At present, it mainly comes from the extraction of ginseng plants, which is costly and inefficient, and its source is limited by the natural environment, such as the amount of land input and climate. [0003] Synthetic biology is an emerging engineering discipline born in the early 21st century. It is a new research field that is based on engineering and biology and is oriented by innovation. It hopes to solve the problems of biochemical engineering, ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N1/21C12N15/70C12N15/60C12N15/53C12P33/00C12R1/19
CPCC12N9/0042C12N9/0073C12N9/0083C12N9/1085C12N9/88C12N15/70C12P33/00C12Y106/02004C12Y114/99007C12Y205/01021
Inventor 谢丽萍胡又佳刘海元龚桂花张伟韩姝潘洁
Owner SHANGHAI INST OF PHARMA IND CO LTD
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