protopanaxadiol and dammarenediol

Transcriptome analysis revealed high expression of saponin biosynthetic genes may account for highly accumulated saponins in 3-year-old Panax notoginseng roots and DS and CYP716A47 - like were functionally verified by transgenic tobacco. Panax notoginseng is a well-known traditional medical herb that contains bioactive compounds known as saponins. Three major dammarene-type triterpene saponins including R1, Rb1, and Rg1 were found to be highly accumulated in the roots of 3-year-old plants when compared to those of 1-year-old plants. However, the underlying cellular mechanism is poorly understood. In this study, transcriptome analysis revealed that most genes involved in saponin biosynthesis in P. notoginseng roots augmented during their growth periods. The analysis of the KEGG pathway indicated that the primary metabolism, cell growth, and differentiation were less active in the roots of 3-year-old plant; however, secondary metabolisms were enhanced, thus providing molecular evidence for the harvesting of P. notoginseng roots in the 3rd year of growth. Furthermore, the functional role of DS and CYP716A47-like, two of the candidate genes involved in saponin biosynthesis isolated from P. notoginseng, were verified via overexpression in cultivated tobacco. Approximately, 0.325 µg g

Topics: Chromatography, High Pressure Liquid; Gene Expression Profiling; Genes, Plant; Nicotiana; Panax notoginseng; Plant Roots; Plants, Genetically Modified; Real-Time Polymerase Chain Reaction; Sapogenins; Saponins; Sequence Analysis, RNA; Triterpenes

Topics: Alkyl and Aryl Transferases; Biosynthetic Pathways; Dammaranes; Gene Expression; Ginsenosides; Oryza; Panax; Plants, Genetically Modified; Sapogenins; Saponins; Triterpenes

Ginsenosides, the major bioactive components of Panax ginseng, are regarded as promising high-value pharmaceutical compounds. In ginseng, ginsenosides are produced from their precursor protopanaxadiol. Recently, an artificial biosynthetic pathway of protopanaxadiol was built in Saccharomyces cerevisiae by introducing a P. ginseng dammarenediol-II synthase, a P. ginseng cytochrome P450-type protopanaxadiol synthase (PPDS), and a Arabidopsis thaliana NADPH-cytochrome P450 reductase (ATR1). In this engineered yeast strain, however, the low metabolic flux through PPDS resulted in a low productivity of protopanaxadiol. Moreover, health of the yeast cells was significantly affected by reactive oxygen species released by the pool coupling between PPDS and ATR1. To overcome the obstacles in protopanaxadiol production, PPDS was modified through transmembrane domain truncation and self-sufficient PPDS-ATR1 fusion construction in this study. The fusion enzymes conferred approximately 4.5-fold increase in catalytic activity, and 71.1% increase in protopanaxadiol production compared with PPDS and ATR1 co-expression. Our in vivo experiment indicated that the engineered yeast carrying fusion protein effectively converted 96.8% of dammarenediol-II into protopanaxadiol. Protopanaxadiol production in a 5 L bioreactor in fed-batch fermentation reached 1436.6 mg/L. Our study not only improved protopanaxadiol production in yeast, but also provided a generic method to improve activities of plant cytochrome P450 monooxygenases. This method is promising to be applied to other P450 systems in yeast. Biotechnol. Bioeng. 2016;113: 1787-1795. © 2016 Wiley Periodicals, Inc.

Topics: Cytochrome P-450 Enzyme System; Fermentation; Metabolic Engineering; Saccharomyces cerevisiae; Sapogenins; Saponins; Synthetic Biology; Triterpenes

Protopanaxadiol (PPD) is an aglycone of dammarene-type ginsenoside and has high medicinal values. In this work, we reported the PPD production in transgenic tobacco co-overexpressing PgDDS and CYP716A47. PPD is an aglycone of ginsenosides produced by Panax species and has a wide range of pharmacological activities. PPD is synthesized via the hydroxylation of dammarenediol-II (DD) by CYP716A47 enzyme. Here, we established a PPD production system via cell suspension culture of transgenic tobacco co-overexpressing the genes for PgDDS and CYP716A47. The concentration of PPD in transgenic tobacco leaves was 2.3-5.7 µg/g dry weight (DW), depending on the transgenic line. Leaf segments were cultured on medium with various types of hormones to induce callus. Auxin treatment, particularly 2,4-D, strongly enhanced the production of DD (783.8 µg g(-1) DW) and PPD (125.9 µg g(-1) DW). Treatment with 2,4-D enhanced the transcription of the HMG-Co reductase (HMGR) and squalene epoxidase genes. PPD production reached 166.9 and 980.9 µg g(-1) DW in a 250-ml shake flask culture and in 5-l airlift bioreactor culture, respectively.

Topics: 2,4-Dichlorophenoxyacetic Acid; Alkyl and Aryl Transferases; Bioreactors; Biosynthetic Pathways; Cells, Cultured; Cytochrome P-450 Enzyme System; Gas Chromatography-Mass Spectrometry; Genes, Plant; Ginsenosides; Mevalonic Acid; Nicotiana; Panax; Plant Proteins; Plants, Genetically Modified; Sapogenins; Saponins; Triterpenes

Panax quinquefolius is one of perennial herbs and well known for its outstanding pharmacological activity. Ginsenosides are thought to be the main active ingredients in Panax quinquefolius and exist in many kinds of plant genus Panax (ginseng). Dammarenediol synthase, which is considered as a key enzyme in ginsenoside biosynthesis pathway can convert 2, 3-oxidosqualene into dammarenediol-II. However, the dammarenediol synthase gene in Panax quinquefolius has not been identified. Here, we cloned and identified a dammarenediol synthase gene from Panax quinquefolius (PqDS, GenBank accession No. KC316048) at the first time, and reverse transcription-PCR (RT-PCR) analysis also showed an obvious transcription increase of PqDS in the methyl jasmonate (MeJA)-induced hairy roots. Ectopic expression of PqDS in yeast resulted in the production of dammarenediol-II was confirmed by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC/APCIMS). Moreover, overexpression of PqDS in transgenic hairy roots could increase the transcription of gene PqDS and another P450 gene PqD12H (encoding protopanaxadiol synthase in Panax quinquefolius), the accumulation of ginsenosides also increased at the same time. In addition, both PqDS and PqD12H gene co-expressed in recombinant yeast result in the production of protopanaxadiol was detected by LC/APCIMS; this result also provides a new strategy for the abundant production of protopanaxadiol in vitro.

Topics: Amino Acid Sequence; Cytochrome P-450 Enzyme System; Gene Expression; Molecular Sequence Data; Panax; Phylogeny; Plant Proteins; Plant Roots; Saccharomyces cerevisiae; Sapogenins; Saponins; Triterpenes

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.

Topics: Acetates; Biocatalysis; Biosynthetic Pathways; Chromatography, Liquid; Cyclopentanes; Cytochrome P-450 Enzyme System; DNA, Complementary; Expressed Sequence Tags; Gene Expression Regulation, Enzymologic; Gene Expression Regulation, Plant; Gene Library; Genes, Plant; Ginsenosides; Mass Spectrometry; Oxylipins; Panax; Phylogeny; Plants, Genetically Modified; RNA, Messenger; Saccharomyces cerevisiae; Sapogenins; Saponins; Sequence Analysis, DNA; Transcription, Genetic; Triterpenes