Study On The Heterologous Coexpression Of Key Enzymes Genes DS And D12H In The Biosynthesis Of Ginsenoside

Panax ginseng is a perennial herb belonging to Araliaceae, it is a precious medicinal herb.The main effective components of ginseng are ginsenosides, which are secondary metabolites ofginseng. With the development of society and improve-ment of people’s living standard, thedemand for ginseng are increasing. So in recent years, the study on ginsenosides biosynthesis,including tissue culture, regulation of metabolic engineering, genetic engine-ering and so on,has become a hot spot. The main purpose of this study is to construct the biosynthetic pathway ofginsenoside precursor, protopanaxadiol in vitro. Because the biosyn-thetic pathway ofdammarenediol precursor,2,3-oxidosqualene is found in Saccharomyces cerevisiae.2,3-oxidos-qualene generates dammarenediol in the catalytic conversion of DS, dammarenediolgenerates protopanaxadiol in the catalytic conversion of D12H, protopana-xadiol furtherglycosylated to form a variety of ginsenosides.In this study, we cloned two key genes, DS andD12H in ginseng hairy roots by PCR, connected the two genes with expression vector,transfected into Saccharomyces cerevisiae, detected target protein by SDS-PAGE to verify thegene expression, detected protopanaxadiol content by HPLC to verify biological activity of thetwo gene expression products, meanwhile,optimized conditions of yeast engineering bacteria forproducing protopanaxadiol through experiments. The main results of study are as follows:1. Ginseng total RNA were extracted from ginseng hairy roots conserved by our labrotary,cDNA were gained through reverse transcription. Primers were designed according to the knownkey enzyme genes, DS and D12H in ginseng saponin biosyn-thesis. Using cDNA as a template,DS gene and D12H gene were successfully cloned and connected to PMD-18T vector. Thecloning vector was sent to sequencing. The result showed that DS gene similarity is99.68%andD12H genes is99.66%, in line with the test requirements.2. The restriction sites at both ends of the gene were designed according to sequencingresults, DS gene and D12H gene were expression vectors by using T4DNA ligase. The twoexpression vectors were constructed and named DS-pAUR123and D12H-pAUR123respe-ctively. The two expression vectors were transfected into Saccharo-myces cerevisiae.Saccharomyces cerevisiae engineering strain was successfully constructed by identification.3. Proteins in Saccharomyces cerevisiae were extracted and analysed by SDS-PAGE. Theobtained results were two genes were normally expressed in Saccharomyces cerevisiae andproduced dammarenediol synthase and protopanaxadiol synthase.4. Protopanaxadiol was detected in yeast extract by HPLC, showed that the two genes expressed protein are biologically active and had catalytic function. The experiment successfullyconstructed protopanaxadiol synthesis pathway.5. Through optimized culture conditions of Saccharomyces cerevisiae and tested content ofits secondary metabolites of protopanaxadiol, the optimum temperature of yeast engine-eringbacteria for producing protopanaxadiol is33℃, the optimal incubation time is36hours, theoptimum rotation is160rpm/min.Protopanaxidiol content per milliliter of bacterial liquid testedis3.01×10-4mg under such culture conditions.