Progress in understanding of ginsenoside biosynthesis

Ginseng is an economically important medicinal plant. The major bioactive ingredients of ginseng are ginsenosides, which are triterpene saponins. Because of difficulties in ginseng cultivation and the low productivity of ginseng cell and tissue culture, it has become important to improve ginsenoside levels by using metabolic engineering based on the biosynthetic pathway of ginsenosides. During the last decade, substantial advances have been made in biosynthesis of ginsenosides. This review is concerned with recent developments in our understanding of the biosynthesis of ginsenosides.     

Integrative transcriptome analysis identifies new oxidosqualene cyclase genes involved in ginsenoside biosynthesis in Jilin ginseng

BackgroundJilin ginseng, Panax ginseng, is a valuable medicinal herb whose ginsenosides are its major bioactive components. The ginseng oxidosqualene cyclase (PgOSC) gene family is known to play important roles in ginsenoside biosynthesis, but few members of the gene family have been functionally studied.MethodsThe PgOSC gene family has been studied by an integrated analysis of gene expression-ginsenoside content correlation, gene mutation-ginsenoside content association and gene co-expression network, followed by functional analysis through gene regulation.ResultsWe found that five of the genes in the PgOSC gene family, including two published ginsenoside biosynthesis genes and three new genes, were involved in ginsenoside biosynthesis. Not only were the expressions of these genes significantly correlated with ginsenoside contents, but also their nucleotide mutations significantly influenced ginsenoside contents. These results were further verified by regulation analysis of the genes by methyl jasmonate (MeJA) in ginseng hairy roots. Four of these five PgOSC genes were mapped to the ginsenoside biosynthesis pathway. These PgOSC genes expressed differently across tissues, but relatively consistent across developmental stages. These PgOSC genes formed a single co-expression network with those published ginsenoside biosynthesis genes, further confirming their roles in ginsenoside biosynthesis. When the network varied, ginsenoside biosynthesis was significantly influenced, thus revealing the molecular mechanism of ginsenoside biosynthesis.ConclusionAt least five of the PgOSC genes, including the three newly identified and two published PgOSC genes, are involved in ginsenoside biosynthesis. These results provide gene resources and knowledge essential for enhanced research and applications of ginsenoside biosynthesis in ginseng.     

Both the mevalonate and the non-mevalonate pathways are involved in ginsenoside biosynthesis

Key Message

When one of them was inhibited, the two pathways could compensate with each other to guarantee normal growth. Moreover, the sterol biosynthesis inhibitor miconazole could enhance ginsenoside level.

Abstract

Ginsenosides, a kind of triterpenoid saponins derived from isopentenyl pyrophosphate (IPP), represent the main pharmacologically active constituents of ginseng. In plants, two pathways contribute to IPP biosynthesis, namely, the mevalonate pathway in cytosol and the non-mevalonate pathway in plastids. This motivates biologists to clarify the roles of the two pathways in biosynthesis of IPP-derived compounds. Here, we demonstrated that both pathways are involved in ginsenoside biosynthesis, based on the analysis of the effects from suppressing either or both of the pathways on ginsenoside accumulation in Panax ginseng hairy roots with mevinolin and fosmidomycin as specific inhibitors for the mevalonate and the non-mevalonate pathways, respectively. Furthermore, the sterol biosynthesis inhibitor miconazole could enhance ginsenoside levels in the hairy roots. These results shed some light on the way toward better understanding of ginsenoside biosynthesis.     

Ginsenosides: prospective for sustainable biotechnological production

Panax ginseng C.A. Meyer (ginseng) is a well-known medicinal plant that has been traditionally used in the oriental countries for centuries. Wild ginseng is a scarce and rare commodity. Field cultivation of the ginseng plant is a time-consuming and labor-intensive process. Ginsenosides, a group of glycosylated triterpenes, also known as saponins, are the principal bioactive constituents of ginseng. The use of cell and organ culture processes has been sought as a potential alternative for the efficient mass production of ginseng raw material. Various bioprocessing strategies have been developed to date. Cells and adventitious roots have been cultured in large-scale bioreactors and various strategies have been developed accordingly for the enhancement of biomass and ginsenoside accumulation. This review highlights the recent progress in the cultivation of ginseng cell and organ cultures for the production of ginsenosides from bioreactor cultures. In addition, the metabolism and biochemistry of ginsenoside biosynthesis, genomic and proteomic studies in ginseng, metabolic engineering, biosafety, toxicological evaluation, and efficacy assessment of ginseng raw material are also summarized and thoroughly discussed.     

Regulation and differential expression of protopanaxadiol synthase in Asian and American ginseng ginsenoside biosynthesis by RNA interferences

Asian ginseng (Panax ginseng) and American ginseng (Panax quinquefolium), are thought to be representative plant of Panax species, have important commercial value and are used in worldwide. Panax species produces triterpene saponins called ginsenosides, which are classified into two groups by the skeleton of aglycones, namely dammarane-type and oleanane-type. Dammarane-type ginsenosides dominate over oleanane-type not only in amount but also in structural varieties. Researches shows that the saponins content in American ginseng is higher than that in Asian ginseng, the higher part of ginsenosides is from dammarane-type biosynthesis. It has been proposed that protopanaxadiol derived from dammarenediol-II, is a key hydroxylation by cytochrome P450 for the biosynthesis of ginsenosides, and the gene number of protopanaxadiol synthase has been published independent in Asian ginseng (PgCYP716A47). However, little is known about genes involved in hydroxylation and glycosylation in American ginseng ginsenoside biosynthesis. Here, we first cloned and identified a P450 gene named PqD12H encoding enzymes catalyzed dammarenediol-II to protopanaxadiol by RT-PCR using degenerate primers designed based on sequence homology. In vitro, the ectopic expression of PqD12H in recombinant WAT21 yeast resulted in protopanaxadiol production after dammarenediol-II was added to the culture medium. In vivo, we established both PgCYP716A47 and PqD12H RNAi transgenic. The RT-PCR and HPLC analysis of the final products of protopanaxadiol and protopanaxatriol showed a result that declined level of protopanaxadiol-type and protopanaxatriol-type ginsenosides. It suggested that the P450 synthase content or expression in American ginseng exceed than in Asian ginseng. The result elucidated the evolution relationship of P450s and the reason of different saponins content among Panax species.     

Expression and RNA interference-induced silencing of the dammarenediol synthase gene in Panax ginseng

Panax ginseng is one of the most highly valued herbal medicines in the Orient, where it has gained an almost magical reputation for being able to maintain the quality of life. The root of ginseng contains noble tetracyclic triterpenenoid saponins, which are thought to be the major effective ingredients in P. ginseng. The first committed step in ginsenoside synthesis is the cyclization of 2,3-oxidosqualene to dammarenediol II by oxidosqualene cyclase, dammarenediol synthase (DDS). The gene encoding DDS has been characterized. Here, we investigated the expression of the DDS gene together with the genes involved in ginsenoside biosynthesis (SS, SE, PNX, PNY, PNY2 and PNZ). Expression of DDS mRNA was higher in flower buds compared with root, leaf and petiole of ginseng plants. Elicitor (methyl jasmonate) treatment up-regulated the expression of DDS mRNA. Ectopic expression of DDS in a yeast mutant (erg7) lacking lanosterol synthase resulted in the production of dammarenediol and hydroxydammarenone which were confirmed by liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC/APCIMS). RNA interference (RNAi) of DDS in transgenic P. ginseng resulted in silencing of DDS expression which leads to a reduction of ginsenoside production to 84.5% in roots. These results indicate that expression of DDS played a vital role in the biosynthesis of ginsenosides in P. ginseng.     

Ginsenoside Rb1 in asymmetric somatic hybrid calli of Daucus carota with Panax quinquefolius

American ginseng (Panax quinquefolius L.) is one of the most valuable herbs in the world. Its major active components are ginsenosides. In order to produce ginsenoside heterogeneously, somatic hybridization, a novel approach for genetic introgression, was employed in this study. Protoplasts derived from respective calli of carrot (Daucus carota var. sativus Hoffm.) and American ginseng (P. quinquefolius L.) were used as the fusion partners. Hybrid calli derived from single cell lines containing chromatin of American ginseng were confirmed by the analyses of isozyme, Random amplified polymorphic DNA (RAPD) and genomic in situ hybridization (GISH). High performance liquid chromatography (HPLC) results showed that the ginseng monomer Rb₁ was synthesized in seven of the hybrid calli identified as well as in the parent American ginseng calli but not in the parent carrot calli. Results indicated that hybrid introgression lines could produce ginsenoside Rb₁ and the ginsenoside Rb₁ biosynthesis pathway has been introgressed into carrot cells via somatic hybridization. From the point of biosafety view concerning the consumer acceptance, the potential predominance to produce ginsenosides with somatic hybridization other than with genetic transformation is discussed. Lu Han and Chuanen Zhou contributed equally to this work.     

人参皂苷生物合成和次生代谢工程

人参皂苷属于植物三萜皂苷类化合物,是传统名贵药材人参和西洋参的主要活性成分,具有抗炎、抗氧化作用,还有广泛的抗肿瘤作用。人参皂苷与植物甾醇共享前期代谢途径,通过2, 3-氧化鲨烯环化步骤进入三萜代谢分支途径,在三萜碳环骨架复杂修饰的基础上形成人参皂苷。综述了近年人参皂苷生物合成途径及关键酶基因研究的最新进展,揭示了人参皂苷生物合成的基本途径,对途径中关键酶的基因进行了综述,并结合次生代谢工程技术, 探讨了该技术在人参皂苷生物合成中的应用前景。     

Studies on the biochemical action of ginseng saponin. I. Purification from ginseng extract of the active component stimulating serum protein biosynthesis.

Systematic isolation and purification of the biologically active component of ginseng extract were followed by observing the incorporation of labeled leucine into serum protein at 6 hr after a single intraperitoneal injection in a mouse. Ginseng saponin mixture (fraction 5) exhibited high activity for such incorporation. Seven saponins were isolated from fraction 5 by means of preparative TLC, and assayed. Administration of all these saponins (ginsenoside-Rb2, Rc, Rc2, Rd, Re, and Rg1)except for ginsenoside-Rb1, caused an increase of leucine incorporation over that in control animals. The incorporation rate was directly proportional to the dose in the case of ginsenoside-Rd, which had the highest activity. The increase specific radio-activity of serum protein was not due to a decrease in the pool size of free amino acids in the liver. It was conclusively shown that the active component stimulating serum protein biosynthesis is saponin.     

Inhibitory effect of ginsenosides on NMDA receptor-mediated signals in rat hippocampal neurons

Alternative medicines such as herbal products are increasingly being used for preventive and therapeutic purposes. Ginseng is the best known and most popular herbal medicine used worldwide. In spite of some beneficial effects of ginseng on the CNS, little scientific evidence shows at the cellular level. In the present study, we have examined the direct modulation of ginseng on the activation of glutamate, especially NMDA, receptors in cultured hippocampal neurons. Using fura-2-based digital imaging techniques, we found ginseng total saponins inhibited NMDA-induced but less effectively glutamate-induced increase in [Ca2+]i. Ginseng total saponins also modulated Ca2+ transients evoked by depolarization with 50mM KCl along with its own effects on [Ca2+]i. Furthermore, we demonstrated that ginsenoside Rg3 is an active component for ginseng actions on NMDA receptors. The data obtained suggest that the inhibition of NMDA receptors by ginseng, in particular by ginsenoside Rg3, could be one of the mechanisms for ginseng-mediated neuroprotective actions.     

Discovery of genes for ginsenoside biosynthesis by analysis of ginseng expressed sequence tags

Expressed sequence tags (ESTs) provide a valuable tool that can be used to identify genes in secondary metabolite biosynthesis. Ginseng (Panax ginseng C.A Meyer) is a medicinal plant that accumulates ginsenosides in roots. We sequenced 11,636 ESTs from five ginseng libraries in order to create a gene resource for biosynthesis of ginsenosides, which are thought to be the major active component in roots. Only 59% of the ginseng ESTs exhibited significant homology to previously known polypeptide sequences. Stress- and pathogen-response proteins were most abundant in 4-year-old ginseng roots. ESTs involved in ginsenoside biosynthesis were identified by a keyword search of BLASTX results and a domain search of ginseng ESTs. We identified 4 oxidosqualene cyclase candidates involved in the cyclization reaction of 2,3-oxidosqualene, 9 nine cytochrome P450 and 12 glycosyltransferse candidates, which may be involved in modification of the triterpene backbone.Abbreviations cDNA Complementary DNA - ESTs Expressed sequence tagsCommunicated by I.S. Chung     

Biotransformation of Panax notoginseng saponins into ginsenoside compound K production by Paecilomyces bainier sp. 229

Aims: Development and optimization of an efficient and inexpensive biotransformation process for ginsenoside compound K production by Paecilomyces bainier sp. 229. Methods and Results: We have determined the optimum culture conditions required for the efficient production of ginsenoside compound K by P. bainier sp. 229 via biotransformation of ginseng saponin substrate. The optimal medium constituents were determined to be: 30 g sucrose, 30 g soybean steep powder, 1 g wheat bran powder, 1 g (NH₄)₂SO₄, 2 g MgSO₄·7H₂O and 1 g CaCl₂ in 1 l of distilled water. An inoculum size of 5–7·5% with an optimal pH range of 4·5–5·5 was essential for high yield. Conclusions: The Mol conversion quotient of ginseng saponins increased from 21·2% to 72·7% by optimization of the cultural conditions. Scale‐up in a 10 l fermentor, under conditions of controlled pH and continuous air supply in the optimal medium, resulted in an 82·6% yield of ginsenoside compound K. Significant and Impact of the Study: This is the first report on the optimization of culture conditions for the production of ginsenoside compound K by fungal biotransformation. The degree of conversion is significantly higher than previous reports. Our method describes an inexpensive, rapid and efficient biotransformation system for the production of ginsenoside compound K.     

Antimelanogenic Activity of Ocotillol‐Type Saponins from Panax vietnamensis

The ocotillol (OCT)‐type saponins have been known as a tetracyclic triterpenoid, possessing five‐ or six‐membered epoxy ring in the side chain. Interestingly, this type saponin was mostly found in Panax vietnamensis Ha et Grushv ., Araliaceae (VG), hence making VG unique from the other Panax spp. Five OCT‐type saponins, majonoside R2, vina‐ginsenoside R2, majonoside R1, pseudoginsenoside RT4, vina‐ginsenoside R11, together with three protopanaxadiol (PPD)‐type saponins and four protopanaxatriol (PPT)‐type saponins from VG were evaluated for their antimelanogenic activity. All of isolates were found to be active. More importantly, the five OCT‐type saponins inhibited melanin production in B16‐F10 mouse melanoma cells, without showing any cytotoxicity. Besides ginsenoside Rd and ginsenoside Rg3 in PPD and notoginsenoside R1 in PPT‐type saponins, majonoside R2 was the most potent melanogenesis inhibitory activity in OCT‐type saponins. In this article, we highlighted antimelanogenic activity of OCT‐type saponins and potential structure–activity relationship (SAR) of ginsenosides. Our results suggested that OCT‐type saponins could be used as a depigmentation agent.     

Enzymatic preparation of genuine prosapogenin, 20(S)-ginsenoside Rh1, from ginsenosides Re and Rg1

It was found that a lactase preparation from Penicillium sp. nearly quantitatively hydrolyzed ginsenosides Re and Rg1, which are major saponins in roots of Panax ginseng, to a minor saponin, 20(S)-ginsenoside Rh1 [6-O-beta-D-glucopyranosyl-20(S)-protopanaxatriol]. This is the first report on the enzymatic preparation of ginsenoside Rh1 with a high efficiency. This enzyme also readily hydrolyzed ginsenoside Rg2 to ginsenoside Rh1.     

Proteomic analysis of Korean ginseng (Panax ginseng C.A. Meyer)

Although many reports have been published regarding the pharmacological effects of ginseng, little is known about the biochemical pathways operant in ginsenoside biosynthesis, or the genes involved therein. Proteomics analysis is an approach to elucidate the physiological characteristics and biosynthetic pathways of ginsenosides, main components of ginseng. In this review, we introduced the recent progress in proteomics studies of ginseng (Panax ginseng C.A. Meyer). We briefly reference the genomic analyses of P. ginseng, without which proteomics approaches would have been impossible. Functional genomics studies regarding secondary metabolism in P. ginseng are also introduced here, in order to introduce possible future prospects for further study.     

Stimulation of Rg3 ginsenoside biosynthesis in ginseng hairy roots elicited by methyl jasmonate

It has been recognized that ginsenoside Rg3 is not naturally produced in ginseng although this ginsenoside can accumulate in red ginseng as the result of a thermal process. In order to determine whether or not Rg3 is synthesized in ginseng, hairy roots were treated with methyl jasmonate (MJ). From HPLC analysis, no peak for Rg3 was observed in the controls. However, Rg3 did accumulate in hairy roots that were MJ-treated for 7?days. Rg3 content was 0.42?mg/g (dry weight). To gain more insight into the effects of MJ on UDP-glucosyltransferase (UGT) activity, we attempted to evaluate ginsenoside Rg3 biosynthesis by UGT. A new peak for putative Rg3 was observed, which was confirmed by LC-MS/MS analysis. Our findings indicate that the proteins extracted from our hairy root lines can catalyze Rg3 from Rh2. This suggests that our ginseng hairy root lines possess Rg3 biosynthesis capacity.