# Progress on the Studies of the Key Enzymes of Ginsnoside Biosynthesis
## 6 Dammarenediol-II Synthase and \(\beta\)-Amyrin Synthase
Several candidate CYP450s that might be involved in ginsenoside biosynthesis have been discovered through the combination of the next generation sequencing technology and bioinformatical analysis [35]. [PERSON] et al. [36] isolated nine putative full CYP450 sequences from the EST library of _P. ginseng_ adventitious roots induced by methyl jasmonate. Among these genes, the CYP716A47 gene product was selected as the putative PPD synthase because this gene was transcriptionally activated not only by methyl jasmonate treatment but also in transgenic ginseng that overexpressed squalene synthase and overproduced ginsenosides. In vitro enzymatic activity assays revealed that CYP716A47 catalyzed the oxidation of dammarendiol-II to produce PPD. Ectopic expression of CYP716A47 in recombinant WAT21 yeast yielded PPD when fed with dammarendiol-II. Furthermore, co-expression of DS and CYP716A47 genes in yeast yielded PPD without supplementing dammarendiol-II. Thus, CYP716A47 is a dammarendiol 12-hydroxylase that catalyzes the production of PPD from dammarendiol-II. This was the first report on the functional verification of CYP450 gene involved in ginsenoside biosynthesis. [PERSON] et al. [37] analyzed the transcriptomes of _P. ginseng_ and identified 133 CYP450 genes by 454 sequencing technology. Their study laid an important foundation for the further screening of CYP450 involved in ginsenoside biosynthesis. [PERSON] et al. [38] obtained 150 CYP450s in the 454 cDNA library of _P. quinquefulus_ roots,some of which encode enzymes responsible for the conversion of the ginseng backbone into the various ginsengides. Finally, transcript contig00248 was selected as the CYP450 candidate most likely to be involved in ginseng biosynthesis through a combination of a methyl jasmonate inducibility experiment and tissue-specific expression pattern analysis based on a real-time PCR assay. [PERSON] et al. [PERSON] et al. (2018) discovered the candidate CYP450 genes in _P. notoginseng_. Pn02132 and Pn00158 were most likely to be involved in hydroxylation of aglycones for gensinoside biosynthesis from 174 cytochrome P450s by phylogenetic analysis. The transcript Pn00158 was very similar to the contig00248 of _P. quinquefolius_ candidate CYP450. The deduced amino acid sequence of Pn00158 had a high identity 97.95% with that of _P. ginseng_ CYP716A47 which has been functionally verified. Thus Pn00158 may be CYP450 involved in ginseng synthesis of _P. notoginseng_. [PERSON] et al. [PERSON] et al. (2018) isolated CYP716A53v2 which belongs to CYP716A subfamily genes from _P. ginseng_ adventitious root cDNA library. The ectopic expression of CYP716A53v2 in recombinant WAT21 yeast resulted in PPT production when PPD was added to the culture medium. In vitro enzymatic activity assays revealed that the recombinant CYP716A53v2 catalyzed the oxidation of PPD to produce PPT. The results suggested that the gene product of CYP716A53v2 was a PPD 6-hydroxylase which catalyzed the production of PPT from PPD, an important step in the formation of dammarane-type triterpene aglycones in ginseng biosynthesis. [PERSON] et al. [PERSON] et al. (2018) engineered the synthetic pathway of PPT by introducing DS, PPD synthase, PPT synthase and NADPH-cytochrome P450 reductase from different plants in _Saccharomyces cerevisiae_. The engineered yeast strains in this work can serve as the basis for producing PPT in yeast rather than extraction from plant sources. These studies promote research on the biosynthesis pathway of ginsenosides and provide an alternative way for ginseng production by synthetic biology.