Biosynthesis mehtod of dammarenediol and producing strain thereof

A technology of dammarenediol and synthetase, which is applied in the field of genetic engineering, can solve problems such as inability to synthesize and complex chemical structure, and achieve the effects of increasing production and reducing production costs

Inactive Publication Date: 2013-06-26
QINGDAO VLAND BIOTECH GRP
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, due to the complex chemical structure of these compounds and multiple chiral structures on the skeleton, they have not been able to be synthesized by chemical methods so far.

Method used

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  • Biosynthesis mehtod of dammarenediol and producing strain thereof
  • Biosynthesis mehtod of dammarenediol and producing strain thereof
  • Biosynthesis mehtod of dammarenediol and producing strain thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Example 1 Construction of Dammarenediol Synthase Expression Vector

[0041] The dammarenediol synthase gene GenBank: AB265170.1 in the ginseng genome was codon-optimized according to the codon preference of Pichia pastoris GS115, and the optimized dammarenediol synthase gene sequence was SEQ ID NO: 1. The gene Synthesized by Shanghai Qinglan Biotechnology Co., Ltd.

[0042] Design restriction restriction sites AsuII and XbaI at both ends of the gene fragment. Using primer F respectively:

[0043] 5'-TACTTCGAAATGGCCTGGAAGCAAAAAAGGTGCTC-3' and R:

[0044] 5'-GCCTCTAGATTAAATTTTCAACTGCTGATGTTAG-3' was amplified by PCR. PCR amplification conditions: 94°C, 5min; 94°C, 30S, 58°C, 30S, 30 cycles; 72°C, 2.5min; 72°C, 10min.

[0045] The PCR amplified product and the Pichia pastoris expression plasmid pPicZa (purchased from Invitrogen) were digested with AsuII and XbaI, and digested at 37°C overnight, and the digested product was recovered and purified on agarose gel. Design ...

Embodiment 2

[0046] Example 2 Construction of Engineering Bacteria for Recombinant Expression of Dammarenediol Synthase

[0047]The positive transformants were inoculated in LB medium, cultured at 37°C and 200rpm for 8h, the bacteria were collected, and the recombinant plasmid DS-pPICZa was extracted by conventional methods. SacI single-digestion treatment, overnight at 37°C, PCR purification and recovery, collected in 15ul of water, and stored at 4°C until use.

[0048] Prepare Pichia pastoris GS115 competent cells, add SacI-digested DS-pPICZa plasmid, mix well, place in ice bath for 5min, transfer to electroporation cup, 1.98kv, 5.8ms, electric shock transformation. Add 1ml of sorbitol that was pre-cooled in advance, and incubate at 30°C for 30min. Collect the bacteria by centrifugation, add 1ml of YPD medium, rejuvenate at 200rpm at 30°C for 1 hour, collect the bacteria, smear on 100ug / ml bleomycin resistance screening YPD plate, culture at 30°C for 48h-72h. 800ug / ml bleomycin resista...

Embodiment 3

[0049] Embodiment 3 fermentation culture and the production of dammarenediol

[0050] GS115-DS was fermented in BMMY fermentation medium at 30°C, 200rpm, and induced by adding 0.5% methanol every 24h. After 96 hours of fermentation, the cells were collected by centrifugation, dissolved in 400ul ultrapure water, ultrasonically crushed, and analyzed by SDS-PAGE to detect the expression of intracellular proteins in GS115-DS. The experimental results are as follows figure 2 shown. It is known that the protein molecular weight of dammarenediol synthase is 88kda, figure 2 Compared with the negative control lane 1, where the intracellular protein of GS115-DS is located, there is an extra protein band at about 90kda, which is the position indicated by the arrow, indicating that dammarenediol synthase is expressed in GS115-DS cells .

[0051] Take 2mL of fermentation broth, collect the bacteria by centrifugation, add 400ul 20% NaOH ethanol solution, crush for 30min, mix with 400ul...

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Abstract

The invention relates to a biosynthesis method of dammarenediol and provides a producing strain of the dammarenediol. A pichia pastoris engineering strain, which can recombine and express a dammarenediol synthetic enzyme with high efficiency, is constructed through a genetic engineering technical means, a precursor for synthesizing protopanaxadiol, namely the dammarenediol is further synthesized, and a foundation is finally laid for biosynthesis and large-scale industrial production of the protopanaxadiol.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a biosynthesis method of dammarenediol and a production strain thereof. technical background [0002] For centuries, natural products, especially metabolites of higher plants, have been the main source of human beings to obtain drugs and their active substances with pharmacologically active functions. 80% of the world's population mainly relies on plants and plant extracts to maintain life and health. The role of natural medicines and natural products with pharmacologically active functions in protecting human health has attracted more and more attention from scientists. [0003] Protopanaxadiol belongs to plant triterpene saponins and is the main active ingredient of traditional precious medicinal materials ginseng and American ginseng. It has anti-inflammatory, anti-oxidation and extensive anti-tumor effects. However, as the main source plants of protop...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N1/19C12N15/81C12P33/00C12R1/84
Inventor 王华明黄涛原静黄亦钧
Owner QINGDAO VLAND BIOTECH GRP
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