Recombinant microorganism for preparing dharma diene and protopanoxadiol and construction method thereof

A technology encoding protopanaxadiol and dammar diene synthase, which is applied in the biological field and can solve problems such as species degradation, ginsenoside compound production that cannot meet social needs, and growth environment damage

Active Publication Date: 2013-02-13
TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the main source of ginsenosides is directly extracted from ginseng, a traditional Chinese medicine. However, with the reclamation of wasteland and deforestation, the growth environment of wild ginseng resources has been severely damaged, and ginseng resources are extremely tight; factors such as degradation, large land and human costs
The output of ginsenoside compounds has been far from meeting the needs of the society, which has seriously affected the clinical application of ginseng and the development and application of ginsenoside pharmaceutical raw material intermediates. It is urgent to provide new resources.

Method used

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  • Recombinant microorganism for preparing dharma diene and protopanoxadiol and construction method thereof
  • Recombinant microorganism for preparing dharma diene and protopanoxadiol and construction method thereof
  • Recombinant microorganism for preparing dharma diene and protopanoxadiol and construction method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0087] Embodiment 1, gene element cloning and plasmid construction containing corresponding gene element

[0088] 1. The cloning of genetic elements is divided into the following three steps:

[0089] (1) Yeast genomic DNA extraction

[0090] Pick Saccharomyces cerevisiae BY4742 (Saccharomyces cerevisiae BY4742, recorded in Carrie baker brachmann et al., 1998, YEAST, 14:115-132, the public can obtain from Tianjin Institute of Industrial Biotechnology and Institute of Traditional Chinese Medicine, China Academy of Chinese Medical Sciences.) Plaques in YPD liquid medium (recipe: 1% Yeast Extract (yeast extract), 2% Peptone (peptone), 2% Dextrose (glucose)), 30°C, 200rpm, cultured for 24h. 10000g, 5 minutes to collect the bacteria in a 1.5ml centrifuge tube, wash twice with water, resuspend the bacteria in the yeast wall breaking solution (25ul yeast wall breaking enzyme, 470ul sorbitol buffer, 5ul β-ME), incubate at 30°C Centrifuge after 1h; resuspend the bacteria in 500ul TEN...

Embodiment 2

[0180] Construction of Saccharomyces cerevisiae Genetic Engineering Strain ZD-PPD-000

[0181] 1) rDNA-LEU2-up, P PGK1 -PgDDS-T ADH1t , P TDH3 -AtCPR1-T TPI1 , P TEF1 -PgPPDS-T CYC1 and rDNA-down preparation

[0182]Use the PCR templates and primers described in Table 14 to perform PCR to obtain functional modules: M1 (rDNA-LEU2-up), M2 (P PGK1 -PgDDS-T ADH1t ), M3 (P TDH3 -AtCPR1-T TPI1 ), M4 (P TEF1 -PgPPDS-T CYC1 ), M5 (rDNA-down) and other functional modules. The amplification system is: NewEngland Biolabs Phusion 5Xbuffer 10ul, dNTP (10mMeach dNTP) 1ul, DNA template 20ng, primers (10uM) 1ul, PhusionHigh-Fidelity DNAPolymerase (2.5U / ul) 0.5ul, add distilled water to a total volume of 50ul . The amplification conditions are 98°C pre-denaturation for 1.5 minutes (1 cycle); 98°C denaturation for 10 seconds, annealing for 10 seconds (annealing temperature 58°C), 72°C extension for 2 minutes (32 cycles); 72°C extension for 8 minutes (1 cycle), the product is recov...

Embodiment 3

[0192] Embodiment 3, the construction of Saccharomyces cerevisiae genetically engineered bacteria ZD-PPD-010

[0193] The plasmid p δ-tHMG1 was single-digested with Xho1, and the target fragment δ-tHMG (containing the expression cassette P PGK1 -tHMG1-T ADH1t ).

[0194] The same method as in Example 2 was used to prepare and transform ZD-PPD-000 competent cells, transfer into δ-tHMG1, and culture in screening culture to obtain transformants. The medium for screening culture is: 0.8% yeast selection medium SD-Ura-Trp-Leu-HIS3, 2% glucose, 0.005% HIS3., 0.01% Trp.; the conditions for screening culture are: 30 degrees, culture for more than 36 hours.

[0195] Using Sac11-pGK1 and Sac11-Pme-ADHt primers for PCR identification, the fragment of about 2539bp was the correct positive clone, named strain ZD-PPD-010.

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Abstract

The invention discloses a recombinant microorganism for preparing dharma diene and protopanoxadiol and a construction method of the recombinant microorganism. The construction method of the recombinant bacteria comprises a step of adding dharma diene synthase, protopanoxadiol synthase and nicotinamide adenine dinucleotide phosphate - cytochrome P450 reductase encoding gene into saccharomyces cerevisiae to obtain recombinant bacteria I. According to the recombinant microorganism for preparing the dharma diene and the protopanoxadiol and the construction method of the recombinant microorganism, by means of homologous recombination, the dharma diene synthase, the protopanoxadiol synthase and the nicotinamide adenine dinucleotide phosphate - the cytochrome P450 reductase encoding gene are all added into the saccharomyces cerevisiae to obtain the initial recombinant bacteria, and the effect that the initial recombinant bacteria can produce trace amount of the dharma diene and trace amount of the protopanoxadiol is discovered; tHMG1 activity of the initial recombinant bacteria is further improved, and therefore intermediate recombinant bacteria are obtained, and by means of the intermediate recombinant bacteria, the yield of the dharma diene and the yield of the protopanoxadiol are significantly increased; the activity of one or two or three of ERG1, ERG9 and ERG20 are improved on the basis of the intermediate recombinant bacteria, and the effect that the recombinant bacteria which can be used to increase the yield of the dharma diene and the yield of the protopanoxadiol are constructed is also discovered. By means of the recombinant microorganism for preparing the dharma diene and the protopanoxadiol and the construction method of the recombinant microorganism, the foundation is laid for artificially synthesizing the dammar diene and the protopanoxadiol.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a recombinant microorganism producing dammardiene and protopanaxadiol and a construction method thereof. Background technique [0002] Protopanaxadiol is a triterpene saponin extracted from the medicinal plant ginseng, and Dammarenediol-II is its biosynthetic precursor compound. Protopanaxadiol has various pharmacological activities such as anticancer, antidepressant, activating chloride ion channels and inhibiting the depolarization of sodium ion channels, and inhibiting the growth of human embryonic kidney HEK-293 cells and Helicobacter pylori. Glycosylation products of protopanaxadiol: Panaxadiol-type ginsenoside compounds, such as ginsenoside Rh2, ginsenoside Rg3, etc., also have good pharmacological activities in cardiovascular and cerebrovascular, anti-tumor and other aspects. Related products of such compounds It has been widely used clinically. At present, the main source o...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12P33/00C12R1/865
Inventor 张学礼黄璐琦戴住波刘怡张夏楠施明雨马延和
Owner TIANJIN INST OF IND BIOTECH CHINESE ACADEMY OF SCI
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