# Progress on the Studies of the Key Enzymes of Ginsnoside Biosynthesis
## 6 Dammarenediol-II Synthase and \(\beta\)-Amyrin Synthase  
Oxidosqualene cyclases (OSCs) catalyze 2,3-oxidosqualene cyclization, which is a committed step in the biosynthesis of phytosterols and triterpenoids. OSCs belong to a multigene family. The cyclization of oxidosqualene can generate more than 100 kinds of triterpenoids with different skeletons. By far, the most OSC genes have been cloned from many plants. Two kinds of OSC genes involved in ginseng biosynthesis were isolated from _P. ginseng_ and named dammarenediol-II synthase (DS) and \(\beta\)-amyrin synthase (AS). They are the key enzymes for synthesizing dammarane-type and oleanane-type ginsengides, respectively. [PERSON] et al. [28] prepared a microsomal fraction from the hairy roots of _P. ginseng_ and found that it could cyclize 2,3-oxidaseuene to dammarendiol-II in vitro. [PERSON] et al. [29] designed degenerate primers according to the conserved sequence of OSC genes, and then cloned a new DS gene named PNA from hairy root cultures of _P. ginseng_. After introducing PNA into a lanosterol synthase deficient (erg7) yeast strain GIL7/7, dammarendiol-II was detected in the transformant, demonstrating PNA encodes DS. Based on PNA gene sequence, [PERSON] et al. [30] cloned a DS gene named DDS from the _P. ginseng_ flower EST library. Their studies showed that yeast transformed by DDS gene produced dammarendiol-II and hydroxydammarenone. Methyl jasmonate can upregulate the expression of DDS gene in adventitious roots of _P. ginseng_. RNAi of DDS in transgenic _P. ginseng_ resulted in silencing of DDS expression which led to reduction of ginseng production to 84.5% in roots. These results indicate that expression of DDS plays a vital role in the biosynthesis of ginsenosides in _P. ginseng_. Furthermore, the silencing of DDS caused increased expression of other OSCs, implying tight coordination of DDS with other OSCs because of the utilization of the same precursor. [PERSON] et al. [31] induced the DDS gene into tobacco and produced dammarendiol-II, resulting in the increased resistance of tobacco to tobacco mosaic virus. The result also shows that DS has an important effect on ginsenoside biosynthesis. The aglycones of dammarane-type ginsenosides, PPD and PPT, are rarely detected in _P. ginseng_ roots and hairy root cultures, implying that expressions of hydroxyles and glycosyltransferases are higher than that of DS, and thus dammarendiol-II formation can be considered as a rate-limiting step in ginsenoside biosynthesis. This implies that introduction and overexpression of DS in _P. ginseng_ can directly lead to increased production of ginsenosides. Therefore, availability of DS gene can make it possible to engineer ginsenoside biosynthesis to improve _P. ginseng_ quality, since ginseng with high dammarane-type ginsenoside content is rated as high grade. [PERSON] et al. [32] cloned PnDS from _P. notoginseng_ with high identity of 99% with DDS from _P. ginseng_, suggesting that DS is conserved across the _Panax_ species. This protein contains 769 amino acids with six QW-motifs and the substrate binding DCTAE motif. [PERSON] et al. [33] reported that _P. ginseng_ DS and _P. quinquefulus_ DS showed 99.5% identity, _P. quinquefulus_ DS and _P. notoginseng_ DS showed 99.0% identity, _P. ginseng_ DS and _P. notoginseng_ DS showed 98.7% identity. AS, which catalyzes the cyclization of 2,3-oxidaseuene and produces \(\beta\)-amyrin, is a key enzyme for the synthesis of oleanane-type ginsenosides Ro. [PERSON] et al. [34] cloned the cDNA sequence PNY of AS from _P. ginseng_ hairy roots. After introducing PNY into yeast, \(\beta\)-amyrin was produced. **7. Cytochrome P450**  
Cytokrome P450s (CYP450s), a superfamily of monooxygenase, play critical roles in biosynthesis of plant secondary metabolites. The specific CYP450s are involved in the reactions of oxidation and hydroxylation in ginsenoside biosynthesis.