The Cyt P450 Enzyme CYP716A47 Catalyzes the Formation of Protopanaxadiol from Dammarenediol-II During Ginsenoside Biosynthesis in Panax ginseng

Abstract

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.

OSC2 and CYP716A14v2 Catalyze the Biosynthesis of Triterpenoids for the Cuticle of Aerial Organs of Artemisia annua

“…CYP716A12 from Medicago truncatula (Carelli et al, 2011), CYP716A15 and CYP716A17 from Vitis vinifera (Fukushima et al, 2011), CYP716AL1 from C. roseus (Huang et al, 2012), CYP716A52v2 from P. ginseng , and CYP716A75 from Maesa lanceolata (Moses et al, 2015) are C-28 oxidases of a-amyrin, b-amyrin, and lupeol. CYP716Y1 from Bupleurum falcatum is a C-16a oxidase of a-amyrin and b-amyrin (Moses et al, 2014), and CYP716A47 and CYP716A53v2 from P. ginseng are C-12 and C-6 oxidases of dammaranes, respectively (Han et al, 2011(Han et al, , 2012. The CYP716A12 protein was used to screen for candidate CYP716 family P450s in the A. annua transcriptome data set.…”

Combinatorial biosynthesis of sapogenins and saponins in Saccharomyces cerevisiae using a C-16α hydroxylase from Bupleurum falcatum

“…Hydroxylases from Panax ginseng that oxidize the dammarenediol-II backbone on C-6, C-12, or C-28 (19)(20)(21), and a C-20 hydroxylase from Lotus japonicus (22) that modifies lupeol, have also been identified. To characterize these P450s, they have been ectopically expressed in yeast strains either producing β-amyrin or externally supplied with candidate substrates.…”

Production of bioactive ginsenoside compound K in metabolically engineered yeast

“…As briefly illustrated in Supplementary information, Figure S1B, the biosynthesis of PPD (1a) from dammarenediol II (DM) (1b) was achieved recently via coexpressing cDNAs encoding a cytochrome P450 (CY-P716A47) identified from P. ginseng and an NADPHcytochrome P450 reductase ATR2-1 from Arabidopsis thaliana [7]. Catalyzed by a dammarenediol synthase isolated from P. ginseng (PgDDS), DM (1b) could be synthesized from 2,3-(S)-oxidosqualene (1c) [8], which, together with squalene (1d) and other intermediates, could be traced back to the isoprenoid precursors produced by the well studied mevalonate (MEV) pathway in yeast.…”

“…Chassis vectors expressing ORFs of the 16 UGTPgs (pHCD-UGTPg1 to pHCD-UGTPg16), were transformed into a chassis yeast, Saccharomyces cerevisiae strain BA21 individually [7] ( Figure 1A) and a perspective CK compound (ca. 240 μg/L) was detected in AK1 harboring the UGTPg1 gene, but not in any of the other recombinant strains ( Figure 1A).…”