Jung-Yeon Han

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

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.

Cytochrome P450 CYP716A53v2 Catalyzes the Formation of Protopanaxatriol from Protopanaxadiol During Ginsenoside Biosynthesis in Panax Ginseng

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.

Regulation of ginsenoside and phytosterol biosynthesis by RNA interferences of squalene epoxidase gene in Panax ginseng.

Squalene epoxidase catalyzes the first oxygenation step in phytosterol and triterpenoid saponin biosynthesis and is suggested to represent one of the rate-limiting enzymes in this pathway. Here, we investigated the roles of two squalene epoxidase genes (PgSQE1 and PgSQE2) in triterpene and phytosterol biosynthesis in Panax ginseng. PgSQE1 and PgSQE2 encoded deduced proteins of 537 and 545 amino acids, respectively. Amino acid sequences deduced from PgSQE1 and PgSQE2 share 83% homology, but the N-terminal regions (first 60 amino acids) are highly different. PgSQE1 mRNA abundantly accumulated in all organs. PgSQE2 was only weakly expressed and preferentially in petioles and flower buds. Methyl jasmonate (MeJA) treatment enhanced the accumulation of PgSQE1 mRNA in roots, but rather suppressed expression of PgSQE2. Precursor (squalene) treatment coordinately upregulated the expression of both PgSQE1 and PgSQE2. In situ hybridization analysis established that both PgSQE1 and PgSQE2 mRNAs accumulated preferentially in vascular bundle tissue and resin ducts of petioles. RNA interference of PgSQE1 in transgenic P. ginseng completely suppressed PgSQE1 transcription. Concomitantly, the interference of PgSQE1 resulted in reduction of ginsenoside production. Interestingly, silencing of PgSQE1 in RNAi roots strongly upregulated PgSQE2 and PNX (cycloartenol synthase) and resulted in enhanced phytosterol accumulation. These results indicate that expression of PgSQE1 and PgSQE2 were regulated in a different manner, and that PgSQE1 will regulate ginsenoside biosynthesis, but not that of phytosterols in P. ginseng.

Expression and functional characterization of three squalene synthase genes associated with saponin biosynthesis in Panax ginseng.

Squalene synthase (SQS) catalyzes the biosynthesis of squalene by condensing two molecules of farnesyl pyrophosphate (FPP), a key precursor in sterol and triterpene biosynthesis. Previously, we reported that PgSS1 overexpression results in the enhanced biosynthesis of both phytosterols and triterpene saponins in Panax ginseng. Here, cDNAs encoding two new SQS homologs (PgSS2 and PgSS3) from a P. ginseng expressed sequence tag (EST) library are described. Functional complementation analysis revealed that ectopic expression of PgSS1, PgSS2 and PgSS3 in the yeast erg9 mutant strain 2C1 lacking SQS activity restored ergosterol prototrophy. The recombinant mutant yeast produced squalene, squalene epoxide and ergosterol. PgSS1 (mRNA) was highly transcribed in all organs, whereas PgSS2 and PgSS3 (mRNAs) were only transcribed in specific organs. All three genes were activated positively by an elicitor (methyl jasmonate), but their transcriptional patterns were different. In situ hybridization analysis revealed that both PgSS1 and PgSS3 transcripts were preferentially accumulated near conducting tissue in the petiole. The PgSS1 and PgSS3 promoters were isolated, and the tissue- and organ-specific regulation of PgSS genes was examined. Transgenic ginseng was constructed by introducing PgSS1 and PgSS3 promoters fused to the β-glucuronidase (GUS) gene. GUS expression driven by the PgSS1 promoter was found in both roots and shoots, but PgSS3-driven GUS was only found in shoots. These results suggest that the three SQS genes are differently expressed and that all three SQS enzymes are involved in squalene production in P. ginseng.

Gene regulation patterns in triterpene biosynthetic pathway driven by overexpression of squalene synthase and methyl jasmonate elicitation in Bupleurum falcatum

The root of Bupleurum falcatum L. (Apiaceae) has long been one of the most important traditional herbal medicines in Asian countries. A group of triterpene saponins (saikosaponins) are the major constituents of this plant. Squalene synthase (SS) may play a regulatory role in directing triterpene intermediates and sterol pathways. Here, we investigated the regulatory role of the squalene synthase (BfSS1) gene in the biosynthesis of phytosterol and triterpene in B. falcatum. BfSS1 mRNA accumulated ubiquitously in plant organs and markedly increased in roots after treatment with methyl jasmonate (MeJA), ABA and ethephon. Transgenic B. falcatum constructs overexpressing BfSS1 in the sense and antisense orientations were assembled using the Agrobacterium-mediated method. Transgenic roots overexpressing BfSS1 in the sense orientation resulted in enhanced production of both phytosterol and saikosaponins. Overexpression of the BfSS1 gene in the sense orientation increased the mRNA accumulation of downstream genes such as squalene epoxidase and cycloartenol synthase but unexpectedly decreased the mRNA levels of β-amyrin synthase (β-AS), a triterpene synthase mRNA. MeJA treatment of wild-type roots strongly stimulated β-AS mRNA accumulation and saikosaponin production but suppressed phytosterol production. MeJA treatment of transgenic roots overexpressing BfSS1 in the sense orientation failed to stimulate β-AS mRNA accumulation but still enhanced saikosaponin and phytosterol production. These results indicate that overexpression of BfSS1 in B. falcatum regulates more powerfully the downstream genes than elicitor (MeJA) treatment in triterpene and phytosterol biosynthesis.

The Involvement of β-Amyrin 28-Oxidase (CYP716A52v2) in Oleanane-Type Ginsenoside Biosynthesis in Panax ginseng

Panax species are the most popular medicinal herbs. The root of these plants contains pharmacologically active triterpene saponins, also known as ginsenosides, compounds that are divided into dammarane- and oleanane-type triterpenes. Two CYP716A subfamily genes (CYP716A47 and CYP716A53v2) were recently characterized, encoding an enzyme catalyzing the hydroxylation of dammarane-type triterpenes in Panax ginseng. Herein, we report that one CYP716A subfamily gene (CYP716A52v2) isolated from P. ginseng encodes a β-amyrin 28-oxidase, which is suggested to modify β-amyrin into oleanolic acid, a precursor of an oleanane-type saponin (mainly ginsenoside Ro) in P. ginseng. The ectopic expression of both PNY1 and CYP716A52v2 in recombinant yeast resulted in erythrodiol and oleanolic acid production, respectively. In vitro enzymatic activity assays biochemically confirmed that CYP716A52v2 catalyzed the oxidation of β-amyrin to produce oleanolic acid, and the chemical structure of the oleanolic acid product was confirmed using gas chromatography-mass spectrometry (GC/MS). Transgenic P. ginseng plants were generated via Agrobacterium tumefaciens-mediated transformation: the overexpression of CYP716A52v2 greatly increased the content of oleanane-type ginsenoside (ginsenoside Ro), whereas RNA interference against CYP716A52v2 markedly reduced it. Furthermore, the levels of other dammarene-type ginsenosides were not affected in these transgenic lines. These results indicate that CYP716A52v2 is a β-amyrin 28-oxidase that plays a key role in the biosynthesis of oleanane-type triterpenes in P. ginseng.

Induction of adventitious roots and analysis of ginsenoside content and the genes involved in triterpene biosynthesis inPanax ginseng

Adventitious roots were produced directly from root segments ofPanax ginseng seedlings when cultured on an MS solid medium containing 3.0 mg L-1 IBA. Omitting NH4NO3 from this medium greatly enhanced both the frequency of adventitious root formation and the number of roots per expiants. This frequency declined markedly with the age of the root, but could be increased through repeated sub-culturing events. A two-step procedure that included NH4NO3 free media for the first two weeks of culture, followed by transfer onto media containing NH4NO3 for another four weeks, greatly improved total fresh weights of these adventitious roots compared with a method of continuous culture over six weeks in media that always contained NH4NO3. Expression of the genes involved in triterpene biosynthesis was analyzed by RT-PCR. Ginsenoside contents were enhanced by the omission of NH4NO3 and were also greatly increased by treatment with methyl jasmonate.

Dammarenediol-II Production Confers TMV Tolerance in Transgenic Tobacco Expressing Panax ginseng Dammarenediol-II Synthase

Panax ginseng is one of the famous medicinal plants. Ginsenosides, a class of tetracyclic triterpene saponins, are mainly responsible for its pharmacological activity. Most ginsenosides are composed of dammarenediol-II aglycone with various sugar moieties. Dammarenediol-II synthase is the first enzyme in the biosynthesis of ginsenosides. Here, we report that transgenic tobacco expressing the P. ginseng dammarenediol-II synthase gene (PgDDS) produced dammarenediol-II, and conferred resistance to Tobacco mosaic virus (TMV). Upon infection with TMV, lesions developed more rapidly in transgenic tobacco plants, and their size was smaller than those of wild-type plants. Transgenic tobacco plants showed a low level of both the viral titer and mRNA accumulation of TMV coat protein (CP) compared with the wild type. The production of dammarenediol-II in transgenic tobacco stimulated the expression of tobacco pathogenesis-related genes (PR1 and PR2) under both virus-untreated and -treated conditions. When the leaves of wild-type plants were inoculated with a mixture of TMV and dammarenediol-II, the leaves exhibited a reduced viral concentration and TMV-CP expression than those receiving TMV treatment alone. When the leaves of P. ginseng were infected with TMV, transcription of PgDDS was significantly increased. Transgenic P. ginseng plants harboring a β-glucuronidase (GUS) gene driven by the PgDDS promoter were constructed. The GUS expression was activated when the transgenic ginseng plants were treated with TMV. These results indicate that the medicinally important dammarenediol-II can be ectopically produced in tobacco, and the production of dammarenediol-II in tobacco plants allows them to adopt a viral defense system.

Metabolic engineering for production of ginsenosides in Panax ginseng

【Panax ginseng roots produce triterpene saponins called ginsenosides, which are high value secondary metabolites and has been used as drugs, detergents, sweeteners, and cosmetics. In the recent years plant cell, tissue and organ cultures have developed as important alternative sources for the saponin production in Panax ginseng. Adventitious roots and hairy roots have been successfully induced and cultured for the improvement of saponin contents. Genetic and metabolic engineering to regulate saponin biosynthesis in P. ginseng might be important way to improve the medicinal values of P. ginseng. Here we introduced the protocol of genetic transformation and recent progress of functional characterization of genes involved in saponin biosynthesis in P. ginseng.】