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Genetic engineering bacterium and method for producing protopanaxadiol

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

Active Publication Date: 2018-10-23
SHANGHAI INST OF PHARMA IND +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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  • Genetic engineering bacterium and method for producing protopanaxadiol
  • Genetic engineering bacterium and method for producing protopanaxadiol
  • Genetic engineering bacterium and method for producing protopanaxadiol

Examples

Experimental program
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Effect test

Embodiment 1

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

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

[0062] Extract the genome of Saccharomyces cerevisiae ATCC204508 (purchased from ATCC) by fungal genomic DNA extraction kit (Beijing Soleibao Technology Co., Ltd.), such as electrophoresis Figure 4 ; Using 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] 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: 25μL;

[0068] Primer (10μM): 2μL each;

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

[0070] Add distilled water to 50μL.

[0071] ③Amplification conditions:

[...

Embodiment 2

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

[0093] (1) D612C46 gene cloning

[0094] A. Using the damardadiene synthase gene (DDS, accession number: AB265170) as a template, the D gene (sequence shown in SEQ ID NO. 8) was chemically synthesized by E. coli codon optimization, and the D gene was used as a template, After hydrophobic analysis, the amino acid sequence after removing the 612th amino acid sequence was used to amplify the D612 gene with "D upper" and "D lower" as primers. The sequence of D612 gene is shown in SEQ ID NO.3.

[0095] ①Amplification primer sequence:

[0096] Top D: CCGCGGATCCGATGTGGAAGCAGAAGGGCGCACAG (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] Primer (10μM): 2μL each;

[0101] D gene (template): 2μL;

[0102] Add distilled water to 50μL.

[0103] ③Amplification conditions:

[0104] 1)...

Embodiment 3

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

[0170] The plasmid pCDFDuet-1SSSE and plasmid pACYDuet-1D612C46P72C46 were simultaneously transformed into E. coli BL21 (DE3) competent cells, and spread on LB containing 100μg / mL streptomycin and 30μg / mL chloramphenicol (peptone 10%, yeast extract 5%, NaCl 10%, agarose 12%) on solid plates, and 2×Taq Master Mix (Dye Plus) was used to screen positive clones to obtain recombinant strain pCDFDuet-1SSSEpACYD612C46P72C46BL21 (DE3).

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Abstract

The invention discloses a genetic engineering bacterium for producing protopanaxadiol. The genetic engineering bacterium for producing protopanaxadiol is an engineering bacterium for expressing a squalene synthase gene, a 2,3-oxidosqualene synthase gene, a dammarenediol synthase gene, a protopanaxadiol synthase gene and a nicotinamide adenine dinucleoside phosphate-cytochrome P450 reductase gene in Escherichia coli. The protopanaxadiol is biologically synthesized by using the genetic engineering bacterium, so the genetic engineering bacterium and the method have the advantages of short fermentation time, simple medium, meeting the requirements of modern industry, and facilitation of promotion and application.

Description

Technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a biosynthesis method of protopanaxadiol and a production strain thereof. Background technique [0002] Propanaxadiol (PPD) belongs to tetracyclic triterpenoids, and is aglycone of ginsengdiol type saponins Ra1, Ra2, Rb1, Rb2, Rc, Rd, Rg3 and Rh2, mainly derived from ginseng, panax notoginseng and American ginseng 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 plant, which has high cost and low efficiency, 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 at the beginning of the 21st century. It is a new research field that is based on engineering and biology and is ra...

Claims

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Application Information

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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
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