Recent trends in ginseng research

Ginseng (Panax ginseng C.A. Meyer) is one of the most popular medicinal herbs and contains pharmacologically active components, ginsenosides, in its roots. Ginsenosides, a class of tetracyclic triterpene saponins, are thought to be synthesized from dammarenediol-II after hydroxylation by the Cyt P450 (CYP) enzyme and then glycosylation by glycosyltransferase (GT). However, no genes encoding the hydroxylation and glycosylation in ginsenoside biosynthesis have been identified. Here, we identify protopanaxadiol synthase, which is a CYP enzyme (CYP716A47), to be involved in the hydroxylation of dammarenediol-II at the C-12 position to yield protopanaxadiol. Nine putative full CYP sequences were isolated from the expressed sequence tags (ESTs) of methyl jasmonate (MeJA)-treated adventitious ginseng roots. The CYP716A47 gene product was selected as the putative protopanaxadiol synthase because this gene was transcriptionally activated not only by MeJA treatment but also in transgenic ginseng that overexpresses squalene synthase and overproduces ginsenosides. In vitro enzymatic activity assays revealed that CYP716A47 catalyzed the oxidation of dammarenediol-II to produce protopanaxadiol. Ectopic expression of CYP716A47 in recombinant WAT21 yeasts that were fed dammarenediol-II yielded protopanaxadiol. Furthermore, co-expression of the dammarenediol synthase gene (PgDDS) and CYP716A47 in yeast yielded protopanaxadiol without adding dammarenediol-II. The chemical structures of the protopanaxadiol products from dammarenediol-II were confirmed using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC/APCIMS). Thus, CYP716A47 is a dammarenediol 12-hydroxylase that produces protopanaxadiol from dammarenediol-II.

Ginseng (Panax ginseng C.A. Meyer) is one of the most popular medicinal herbs, and the root of this plant contains pharmacologically active components, called ginsenosides. Ginsenosides, a class of tetracyclic triterpene saponins, are synthesized from dammarenediol-II after hydroxylation by cytochrome P450 (CYP) and then glycosylation by a glycosyltransferase. Protopanaxadiol synthase, which is a CYP enzyme (CYP716A47) that catalyzes the hydroxylation of dammarenediol-II at the C-12 position to yield protopanaxadiol, was recently characterized. Here, we isolated two additional CYP716A subfamily genes (CYP716A52v2 and CYP716A53v2) and determined that the gene product of CYP716A53v2 is a protopanaxadiol 6-hydroxylase that catalyzes the formation of protopanaxatriol from protopanaxadiol during ginsenoside biosynthesis in P. ginseng. Both CYP716A47 and CYP716A53v2 mRNAs accumulated ubiquitously in all organs of ginseng plants. In contrast, CYP716A52v2 mRNA accumulated only in the rhizome. Methyl jasmonate (MeJA) treatment resulted in the obvious accumulation of CYP716A47 mRNA in adventitious roots. However, neither CYP716A52v2 nor CYP716A53v2 mRNA was affected by MeJA treatment during the entire culture period. The ectopic expression of CYP716A53v2 in recombinant WAT21 yeast resulted in protopanaxatriol production after protopanaxadiol was added to the culture medium. In vitro enzymatic activity assays revealed that CYP716A53v2 catalyzed the oxidation of protopanaxadiol to produce protopanaxatriol. The chemical structures of the protopanaxatriol products were confirmed using liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC/APCIMS). Our results indicate that the gene product of CYP716A53v2 is a protopanaxadiol 6-hydroxylase that produces protopanaxatriol from protopanaxadiol, which is an important step in the formation of dammarane-type triterpene aglycones in ginseng saponin biosynthesis.

Summary Panax ginseng C. A. Meyer, reputed as the king of medicinal herbs, has slow growth, long generation time, low seed production and complicated genome structure that hamper its study. Here, we unveil the genomic architecture of tetraploid P. ginseng by de novo genome assembly, representing 2.98 Gbp with 59 352 annotated genes. Resequencing data indicated that diploid Panax species diverged in association with global warming in Southern Asia, and two North American species evolved via two intercontinental migrations. Two whole genome duplications (WGD) occurred in the family Araliaceae (including Panax) after divergence with the Apiaceae, the more recent one contributing to the ability of P. ginseng to overwinter, enabling it to spread broadly through the Northern Hemisphere. Functional and evolutionary analyses suggest that production of pharmacologically important dammarane‐type ginsenosides originated in Panax and are produced largely in shoot tissues and transported to roots; that newly evolved P. ginseng fatty acid desaturases increase freezing tolerance; and that unprecedented retention of chlorophyll a/b binding protein genes enables efficient photosynthesis under low light. A genome‐scale metabolic network provides a holistic view of Panax ginsenoside biosynthesis. This study provides valuable resources for improving medicinal values of ginseng either through genomics‐assisted breeding or metabolic engineering.