Biosynthesis and biotechnological production of ginsenosides

Biosynthesis and biotechnological production of ginsenosides

    Biosynthesis and biotechnological production of ginsenosides Yu-Jin Kim, Dabing Zhang, Deok-Chun Yang PII: DOI: Reference: S0734-975...

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    Biosynthesis and biotechnological production of ginsenosides Yu-Jin Kim, Dabing Zhang, Deok-Chun Yang PII: DOI: Reference:

S0734-9750(15)00051-8 doi: 10.1016/j.biotechadv.2015.03.001 JBA 6910

To appear in:

Biotechnology Advances

Received date: Revised date: Accepted date:

30 October 2014 28 February 2015 1 March 2015

Please cite this article as: Kim Yu-Jin, Zhang Dabing, Yang Deok-Chun, Biosynthesis and biotechnological production of ginsenosides, Biotechnology Advances (2015), doi: 10.1016/j.biotechadv.2015.03.001

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Title:

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Biosynthesis and biotechnological production of ginsenosides

Yu-Jin Kim1,2, Dabing Zhang1,3*, Deok-Chun Yang2* 1

Shanghai Jiao Tong University–University of Adelaide Joint Centre for Agriculture and Health,

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Joint International Research Laboratory of Metabolic & Developmental Sciences, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University, Shanghai, 200240, China; 2

Hee University, Suwon 449-701, Korea; 3

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Department of Oriental Medicinal Materials and Biotechnology, College of Life Science, Kyung

School of Agriculture, Food and Wine, University of Adelaide, Waite Campus, Urrbrae, South

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Australia 5064, Australia.

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* Corresponding authors. Tel.: 0086-21-34205073 (D. Zhang), 0082-31-2012100 (D.C. Yang)

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E-mail address: [email protected]; (D. Zhang), [email protected] (D.C. Yang).

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ACCEPTED MANUSCRIPT Abstract Medicinal plants are essential for improving human health, and around 75% of the population in developing countries rely mainly on herb-based medicines for health care. As the king of herb plants, ginseng has been used for nearly 5,000 years in the oriental and recently in western

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medicines. Among the compounds studied in ginseng plants, ginsenosides have been shown to have multiple medical effects such as anti-oxidative, anti-aging, anti-cancer, adaptogenic and other

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health-improving activities. Ginsenosides belong to a group of triterpene saponins (also called ginseng saponins) that are found almost exclusively in Panax species and accumulated especially in the plant roots. In this review, we update the conserved and diversified pathways/enzymes biosynthesizing ginsenosides have been presented. Particularly, we highlight recent milestone

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works on functional characterization of key genes dedicated to the production of ginsenosides, and

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their application in engineering plants and yeast cells for large-scale production of ginsenosides.

Keywords: Panax, Ginseng, Ginsenoside, Accumultation, Biosynthesis, Physiological role,

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Transgenic plants, Recommbinant yeast cells, Industrial production

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Abbreviations: CAS, Cycloartenol synthase; DDS, Dammarenediol synthase; DMAPP,

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Dimethylallyl diphosphate; DXP: 1-Deoxy-D-xylulose 5-phosphate; FPP, Farnesyl pyrophosphate; FPS, Farnesyldiphosphate synthase; GAP, Glyceraldehyde 3-phosphate; HMGR, 3-Hydroxy-3methylglutaryl coenzyme A reductase; HMG-CoA, 3-Hydroxy-3-methylglutaryl- coenzyme A; IPP,

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Isopentenyl diphosphate; LAS, Lanosterol synthase; MVA, Mevalonic acid; MVD, Mevalonatediphosphate decarboxylase; MEP, Methylerythritol phosphate; MJ, Methyl jasmonate; OAS, Oleanolic acid synthase; OSC, Oxidosqualene cyclase; PPD, Protopanaxadiol; PPDS, Protopanaxadiol synthase; PPT, Protopanaxatriol; CYP, Cytochrome p450; RNAi, RNA interference; SA, Salicylic acid; SS, Squalene synthase; SE, Squalene epoxidase; βAS, β-amyrin synthase; UGT, Uridine diphosphate-dependent glycosyltransferase

ACCEPTED MANUSCRIPT Contents 1. Introduction 2. Conserved and diversified biosynthetic pathway of ginsenosides 2.1. Enzymes of MVA pathway

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2.2. Squalene synthase 2.3. Squalene epoxidase

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2.4. Dammarenediol II synthase 2.5. Cytochrome p450

2.6. Uridine diphosphate (UDP)-dependent glycosyltransferase

2.8. Regulations of ginsenosides synthesis 3. Dynamics of ginsenoside accumulation

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2.7. Cellular and subcellular locations for synthesizing ginsenoside

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4. Biotechnological approaches for production of ginsenosides 4.1. Tissue culture

4.3. Transgenic plants 4.4. Engineered yeast cells

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4.2. Chemical elicitors

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5. Conclusion and future prospectives

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References

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Acknowledgments

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ACCEPTED MANUSCRIPT 1. Introduction Ginsenosides are triterpenoid saponins (or ginseng saponins) that are secondary metabolites almost exclusively produced in Panax species. Panax Linn, belongs to the Order Umbelliferales,

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Family Araliaceae, is commonly called the ginseng genus, since almost every species within this genus has been used as a source of medicine. Among 17 species of Panax genus, P. ginseng (Asian

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or Korean ginseng), P. notoginseng (Chinese ginseng, also called Sanchi ginseng), and P. quinquefolius (American ginseng) have been widely used as medicinal and functional food (Sharma and Pandit, 2009). The name Panax, first used by the Russian botanist, Carl Anton Von Meyer, came from the Greek word meaning “all-healing”. On the other hand, the English name ‘Ginseng’

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was translated from the Chinese pronunciation of Chinese words “ 人 参 ” (Renshen) that

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conventionally refers to the root of P. ginseng because it resembles a human-like shape (Leung, 2013). As a slow-growing perennial herb plant, P. ginseng root grows actively from the third year, increases in diameter during the fourth year growth, then finally reach about 7-10 cm in length and

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3 cm in diameter at the sixth growth year, resembling the ‘human-body’ (Fig. 1). P. ginseng was discovered over 5,000 years ago in China and since then ginseng roots have been used as a highly

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valued medicinal herb in Traditional Chinese Medicine (Hemmerly, 1977). Literally, ginseng means the essence of man and has been known as the king of all herbs. As a best-selling herbal

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medicine used as a tonic and adaptogenic and antiaging agent in more than 35 countries around the world, the global market of ginseng was estimated to be about $2,000 million USD, particularly two ginseng species: P. ginseng and P. quinquefolius, consumed in China, South Korea, Canada, and

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the United States (Baeg and So, 2013). Among the components in ginseng plants, ginsenosides have been shown to be the major pharmacological ingredient in Panax plants. Ginsenosides differ from each other in the number, linkage position and type of sugar moiety. To date, there are more than 150 naturally occurring ginsenosides identified from Panax species (reviewed by Christensen, 2009). According to the skeleton of aglycones, ginsenosides have been classified into two major types: dammarane and oleanane. The dammarane-type ginsenosides contain three groups: protopanaxadiol (PPD), protopanaxatriol (PPT), and ocotillol, each with characteristic genuine aglycone moieties (Fig. 2). PPD-group saponins, such as ginsenosides Ra1, Ra2, Ra3, Rb1, Rb2, Rb3, Rc and Rd; quinquenoside R1; Rs1 to Rs3, and malonylginsenoside Rb1, Rb2, Rc, and Rd are glycosides and each contains an aglycone with a dammarane skeleton, with sugar moieties attached to the β-OH at C-3 and/or C-20. Ginsenosides Re, Rf, Rg1, Rg2, Rh1, F1, and notoginsenoside R1 and R2 belong to the PPT-group saponins which consist of sugar moieties attached to the α-OH at C-6 and/or β-

ACCEPTED MANUSCRIPT OH at C-20 (Fig. 2). Ocotillol-group ginsenosides observed in other Panax species, such as P. quinquefolium, P. japonicus, and P. vietnamensis have a five membered epoxy ring at C-20 (reviewed by Christensen, 2009). The oleanane-group only has one identified ginsenoside Ro with minor amounts in P. ginseng and it contains a pentacyclic structure with aglycone oleanolic acid

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(Fuzzati, 2004). In addition, some other types of ginsenosides, such as the panaxatriol-type and dammarenediol-type ginsenosides, have been also identified from Panax species. As the

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technologies for isolation and analysis advance, more chemicals are expected to be identified from Panax plants. Interestingly, the anticancer activity of ginseng saponins negatively correlates with the number of sugar molecules, number and position of hydroxyl groups, and stereoselectivity (reviewed by Nag et al., 2012).

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Previously, several excellent reviews have summarized advances on the knowledge of the pharmaceutical effects, chemical structures, methodological analyses, biosynthesis and production

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of ginsenosides (Baque et al., 2012; Choi, 2008; Christensen, 2009; Leung, 2013; Liang and Zhao, 2008; Liu, 2012; Murthy et al., 2014; Nag et al., 2012; Peng et al., 2012; Thanh et al., 2014; Wang et al., 2012). In this review, we update the knowledge of conserved and diversified biosynthetic

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pathways of ginsenosides, as well as improvements to increase the yield of ginsenosides using

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tissue culture, transgenic plants and engineered yeast cells.

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2. Conserved and diversified biosynthetic pathway of ginsenosides

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Even though the significant pharmacological importance of ginsenosides is well established, their biosynthetic enzymes and the mode of regulation remains largely to be elucidated (Augustin et al 2011; Haralampidis et al., 2002; Jenner et al., 2005; Liang and Zhao; 2008; Osbourn et al., 2011; Sawai and Saito 201; Thimmappa et al., 2014). Generally, formation of triterpenoid saponins is considered to occur from a linear C30 molecule, squalene, which is produced by the head-to-head assembly of two farnesyl diphosphate (FPP) molecules, and each FPP is derived from dimethylallyl diphosphate (DMAPP) together with two molecules of isopentenyl diphosphate (IPP) (Lee et al., 2004). Squalene can be converted into (S)-2,3-oxidosqualene (Han et al., 2010), with subsequent cyclization via formation of a dammarenyl-cation catalyzed by oxidosqualene cyclases (OSCs) (Phillips et al., 2006). Subsequently, various kinds of ginsenosides are formed by multiple oxidations (e.g. mediated by cytochrome P450-dependent monooxygenases) (Han et al., 2011;2012;2013) and glycosylation (Haralampidis et al., 2002; Jenner et al., 2005; Thimmappa et al., 2014) and these steps remain to be identified. 5

ACCEPTED MANUSCRIPT The triterpene ginsenosides are mainly biosynthesized utilizing the precursor IPP through the mevalonic acid (MVA) pathway in the cytosol and the methylerythritol phosphate (MEP) pathway in the plastid (Fig. 3; Table 1). The MVA pathway is likely to be an ancestral metabolic route in all organisms, and eukarytes have conserved enzymes from MVA to IPP synthesis except partial

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divergence for the Archaea pathway (Hemmerlin et al., 2012; Lombard and Moreira, 2011; Tholl and Lee, 2011). The MEP pathway initiates from the formation of 1-deoxy-D-xylulose 5-phosphate

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(DXP) by the condensation of glyceraldehyde 3-phosphate (GAP) and a C2-unit from pyruvate. Subsequently, DXP can be rearranged and reduced to 2-C-methyl-D-erythritol 4-phosphate (MEP), which forms (E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP). Finally, IPP and DMAPP are generated from HMBPP (Eisenreich et al., 2004; Kuzuyama and Seto 2012; Zhao et al., 2014).

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Inhibition assays indicated that both the MVA and MEP pathways can compensate for each other in producing metabolites in P. ginseng hairy roots to guarantee ginseng growth (Zhao et al., 2014). 13

CO2 pulse-chase experiments using 6-year-old P.

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However, Schramek et al. (2014) performed a

ginseng in the field, and concluded that ginsenosides are mainly produced in P. ginseng via the MVA route. These studies suggest the ginsenoside biosynthesis is mainly via the MVA pathway,

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but could be compensated by MEP pathway under limited supply of products by MVA pathway.

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2.1. Enzymes of MVA pathway

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Although most of MVA-pathway genes in ginseng plants have not been functionally characterized, their corresponding sequence homologs have been identified in P. ginseng and other

of

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Panax species. In animals and plants, the MVA pathway begins with the condensation of three units acetyl-CoA

to

form

3-hydroxy-3-methylglutaryl-CoA

(HMG-CoA)

by

acetyl-CoA

acetyltransferase and HMG-CoA synthase (HMGS), respectively. Subsequently, S-HMG-CoA is converted into R-MVA by two reduction steps, and then further reduced to MVA by the key enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR) (Stermer et al., 1994). It has been shown that HMGR acts as the first rate-limiting enzyme of the MVA pathway in plants (Bach and Lichtenthaler, 1982) and animals (Goldstein and Brown, 1990). Unlike the single HMGR member in animals, plants have multiple HMGR members, and different isoforms display varied spatial and temporal expression patterns. P. ginseng contains two copies of HMGRs (called PgHMGR1 and PgHMGR2) (Kim et al., 2014b), similar to the model dicot Arabidopsis thaliana. High levels of PgHMGR1 transcription occurs in petioles of 2-week-old seedlings, whereas relatively weak expression of PgHMGR2 was observed in this tissue. In 3-year-old and 6-year-old ginseng, PgHMGR1 is abundantly expressed in roots, while PgHMGR2 expression gradually increases as the ginseng plant develops. Promoter

ACCEPTED MANUSCRIPT activity analysis revealed that both PgHMGR1 and PgHMGR2 are active in root vasculature. Furthermore, continuous dark exposure can promote the expression of PgHMGR1 and increase the total ginsenosides content in 3-year-old ginseng. These findings suggest that PgHMGR1 may play a general role in secondary metabolite production, whereas PgHMGR2 may be responsible for age-

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dependent ginsenosides accumulation in roots (Kim et al., 2014b). Additionally, competitive inhibition assays of HMGR enzyme activity, using the specific inhibitor mevinolin, significantly

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reduced the total content of ginsenosides in adventitious roots; by contrast, overexpression of PgHMGR1 triggers the production of triterpenes in Arabidopsis and ginseng. Moreover, PgHMGRs have been shown to be localized to the endoplasmic reticulum, peroxisomes and plastids (Kim et al., 2014b), which is consistent with the location of the enzymes involved in the plant MVA pathway,

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in that they are mainly localized in the endoplasmic reticulum/cytosolic compartments, and peroxisomes (Clastre et al., 2011; Sapir-Mir et al., 2008).

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Furthermore, the evolutionary importance of HMGR was evidenced by the genetic complementation of the mutant of Arabidopsis counterpart of HMGR by overexpressing PgHMGR1 (Kim et al., 2014b). In Arabidopsis, there are two HMGRs (HMG1 and HMG2), silencing of HMG1

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showed early senescence, sterility and a dwarf phenotype (Suzuki et al., 2009). Even though Arabidopsis hmg2 mutant showed no visible phenotype, hmg1 hmg2 double mutants were lethal

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(Suzuki et al., 2009), indicating that both HMG1 and HMG2 genes play a major role in the biosynthesis of triterpene metabolites required for development (Ohyama et al., 2007). In contrast,

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PgHMGR2 failed to complement the mutant of AtHMGR1 (Kim et al., 2014b), suggesting the evolutionary divergence of HMGR isozymes in spite of same catalytic activity. Overall, these

ginsenosides.

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findings demonstrate that ginseng HMGRs play a crucial role in biosynthesis of the triterpene

MVA can be converted to IPP by two sequential ATP-dependent phosphorylation steps, catalyzed by mevalonate kinase and phosphomevalonate kinase, respectively, and then decarboxylation by mevalonatediphosphate decarboxylase (MVD) (Sandmann and Albrecht, 1994; Throll and Lee, 2011). Although how these three enzymes are involved in biosynthesizing triterpene in plants remains unclear, overexpression of P. ginseng MVD results in an increased amount of campesterol, stigmasterol and β-sitosterol, with the up-regulated expression of cycloartenol cyclase (CAS), but no significant increase of ginsenosides and decreased β-amyrin in transgenic ginseng hairy roots (Kim et al., 2014c). This result suggests that MVD, the last enzyme of the mevalonate pathway for production of IPP, plays a key role in phytosterol biosynthesis in P. ginseng, rather than ginsenoside biosynthesis (Kim et al., 2014c).

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ACCEPTED MANUSCRIPT Isopentenyl diphosphate isomerase is capable of catalyzing the reversible conversion of fivecarbon IPP and its allylic isomer DMAPP. Furthermore, nucleophile IPP and electrophilic DMAPP can be readily condensed to form C10-geranylpyrophosphate which can be further combined with another IPP to yield FPP by farnesyldiphosphate synthase (FPS), whereas geranyl diphosphate

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synthase produces C10 for monoterpene synthesis in the chloroplast. FPS has been shown to contribute to the biosynthesis of ginsenosides and phytosterols in Centella asiatica (Kim et al.,

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2010a) and P. ginseng; overexpression of PgFPS caused an approximate 2.4-fold increase in ginsenoside content in transgenic ginseng hairy roots (Kim et al., 2014c). Furthermore, PgFPS overexpression caused the upregulation of ginsenoside synthesizing genes, indicating the important

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role of PgFPS in ginsenosides synthesis in P. ginseng (Kim et al., 2014c).

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2.2. Squalene synthase

Squalene synthase (SS; farnesyl diphosphate:farnesyl diphosphate farnesyl transferase) is a membrane-bound enzyme that condenses two FPP (C15) molecules to yield C30-squalene, the

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common biosynthetic precursor for synthesizing triterpenes and phytosterols (Haralampidis et al., 2002). Yeast (Jennings et al., 1991) and humans (Robinson et al., 1993) have one single copy of SS,

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whereas plants contain one to three copies. Arabidopsis has two annotated SS genes, but only SS1 is functional and SS2 has no SS activity (Busquets et al. 2008). P. ginseng has three SS genes and all

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have biochemical function of SS (Kim et al., 2011). The expression of PgSS1 is ubiquitously observed in ginseng tissues, but mRNAs of PgSS2 and PgSS3 were only detected in specific organs

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(Kim et al., 2011). In situ hybridization analysis revealed that both PgSS1 and PgSS3 are preferentially expressed in vascular bundle tissues and resin ducts in petioles. The biochemical function of these three PgSSs was confirmed by complementation of the yeast erg9 mutant strain 2C1 lacking a functional endogenous SS and the recombinant strains, respectively. Individual expression of PgSS1, PgSS2 and PgSS3 could recover the production of squalene, squalene epoxide and ergosterol in the yeast mutant (Kim et al., 2011). Overexpression of PgSS1 caused the upregulation of downstream ginsenoside-synthetic genes, including squalene epoxidase (SE), βamyrin synthase (βAS), and CAS, eventually causing a dramatic increase in the levels of phytosterols and ginsenosides in adventitious roots of transgenic P. ginseng (Lee et al., 2004). These observations suggest the conserved and diversified role of SSs in ginseng and that their biochemical activities are essential for the production of triterpenes and phytosterols in ginseng.

2.3. Squalene epoxidase

ACCEPTED MANUSCRIPT SE catalyzes the oxygenation of the double bond of squalene to produce 2,3-oxidosqualene, representing one rate-limiting step in the phytosterol and triterpenoid saponin biosynthetic pathway (Vincken et al., 2007). Yeast and mouse only have a single copy of SE, by contrast, plants so far

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examined contain two or more copies of SE genes. In Arabidopsis, there are 6 copies of SEs, among them, SE1, SE2 and SE3 are functional and only SE1 is essential for normal plant development

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(Rasbery et al., 2007), especially for root sterol biosynthesis (Posé et al., 2009). P. ginseng was found to have two copies of SE genes; two sequences have low similarity at the N-terminal regions, transcripts of PgSE1 abundantly exist in all ginseng organs, and PgSE2 is only weakly expressed except in petioles and flower buds. In situ hybridization assay showed that the expression of both

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PgSE1 and PgSE2 occurs preferentially in vascular bundle tissue and resin ducts of petioles. The phloem is associated with long-distance transport of assimilates from the source leaves to non-

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photosynthesizing sink organs, such as roots, flowers and developing seeds (Lucas et al., 2013). The accumulation of PgSE1 and PgSE2 transcripts in phloem and resin ducts suggests that PgSE1 and PgSE2 may participate in the synthesis and long-distance transport of ginsenosides. Treatment of

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MJ enhanced expression of PgSE1 in roots, but suppressed expression of PgSE2 (Han et al., 2010), and two homologues from P. notoginseng (PnSE1 and PnSE2) also showed similar expression

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pattern by MJ treatment (Niu et al., 2013). Knock-down of PgSE1 using RNA interference (RNAi) caused a reduction of ginsenosides in transgenic plants. Interestingly, PgSE1 RNAi plants had

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upregulated expression of PgSE2 and CAS (also called PNX), leading to enhanced phytosterol accumulation (Han et al., 2010). These results indicate that PgSE1 and PgSE2 have divergent roles

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during evolution, with PgSE1 mainly responsible for ginsenoside biosynthesis in P. ginseng, but not for phytosterols.

The presence of multiple genes and isoforms of biosynthetic enzymes may be associated with the diverse regulatory control on triterpene biosynthesis. The different isoforms exhibit varied expression patterns in tissues and developmental stages, which might be linked with developmental and/or environmental signals. The organ-specific transcription of PgHMGR, PgSS, and PgSE could contribute to the organ-specific accumulation of phytosterols and triterpenes in P. ginseng plants. Future functional characterization of these genes and their regulators will provide insight into the regulation for producing ginsenosides and phytosterols.

2.4. Dammarenediol II synthase

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ACCEPTED MANUSCRIPT Ginsenoside biosynthesis is mainly achieved through three reaction steps; i.e., cyclization of 2,3oxidosqualene catalyzed by OSCs, and subsequent hydroxylation and glycosylation (Wang et al., 2012; Xu et al., 2004). Cyclization of 2,3-oxidosqualene in fungi, animals, and plants for the production of sterol or triterpene products includes the most complex enzymatic reactions

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(reviewed by Thimmappa et al., 2014). Unlike one single copy of OSC, lanosterol synthase (LAS), for sterol biosynthesis identified in the each species of animals and fungi, so far more than 80 OSCs

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have been identified and their functional characterization were mostly conducted through their heterologous expression in appropriate yeast strains (reviewed by Thimmappa et al., 2014). OSCs control the branch metabolic flow toward specific secondary metabolites in plants. In P. ginseng, LAS and CAS are responsible for phytosterol production similar to that in other plants (Ohyama et

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al., 2009; Suzuki et al., 2006). In addition, β-amyrin synthase (β-AS) and dammarenediol synthase (DDS) have been identified for triterpene biosynthesis in P. ginseng (Suzuki et al., 2006). Most

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triterpenes identified from plants are pentacyclic triterpenes, derived from β-amyrin, α-amyrin, or lupeol. Whereas, in P. ginseng, a DDS encoding gene (also called PNA) has been characterized to be the first committed step in ginsenoside synthesis in cyclizing 2,3-oxidosqualene into tetracyclic

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dammarenediol by the ectopic expression in a LAS deficient Saccharomyces cerevisiae strain GIL77 (erg7). The recombinant yeast strain containing DDS has detectable dammarenediol-II and

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hydroxydammarenone (Han et al., 2006; Tansakul et al., 2006). RNAi of DDS caused a reduction of ginsenoside production to 84.5% in transgenic P. ginseng adventitious roots. DDS expression was

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high in floral buds, and is promoted by MJ (Han et al., 2006). Furthermore, the importance of DDS in synthesizing ginsenosides was shown by experiments using hairy root of transgenic plant

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suppressing other branch CAS; these roots showed increased DDS enzyme activity and contained 50–100% higher ginsenoside levels compared with control roots (Liang et al., 2009). Heterooverexpression of DDS successfully produced dammarenediol-II in tobacco cells (Lee et al., 2012; Han et al., 2014) providing a new way to produce ginsenosides with other downstream genes in yeast cell system (Dai et al., 2014; Jung et al., 2014; Yan et al., 2014). DDS is conserved across the Panax species (Niu et al., 2014), and it contains 769 amino acids with six QW-motifs and the substrate binding DCTAE motif. DDS shares similarity to P. ginseng PNY2 (β-AS), PNY (β-AS), PNX (CAS), and PNZ (LAS) (Suzuki et al., 2006). DDS shares relatively low identities with other OSC proteins in P. ginseng, and has higher sequence similarity with lupeol synthase compared with β-ASs or CASs from other higher plants, suggesting that P. ginseng evolved a specialized triterpene synthase, DDS (Han et al., 2006). Structurally, 2,3-oxidosqualene is folded into a chair-boat-chair-boat conformation for the formation of the transient tetracyclic protosteryl cation intermediate by CAS and LAS (Lee et al., 2004; Tansakul et al., 2006). In contrast, most pentacyclic triterpene skeletons are produced from

ACCEPTED MANUSCRIPT the dammarenyl cation by D-ring expansion to form lupeol or further E-ring expansion to form βamyrin based on the chair-chair-chair-boat configuration (Xu et al., 2004). DDS reacts on the transient dammarenyl cation with water generating epimeric C-20 dammarenediol, precursors of the dammarene-type triterpene saponins (Dewick, 2004; Haralampidis et al., 2001; Tansakul et al., 13

CO2-labeled Rg1 and Rb1 characterized by specific NMR coupling

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2006). Furthermore,

confirmed the chair-chair-chair-boat conformation of the (S)-2,3-oxidosqualene precursor for the

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cyclization of the dammarenyl intermediate (Schramek et al., 2014).

Beside the major ginsenoside dammarenediol skeleton catalyzed by DDS, the oleanane-type ginsenoside R0, a minor ginsenoside with a pentacyclic skeleton in P. ginseng and P. quinqefolius, is derived from β-amyrin (also called PNY), catalyzed by β-AS which shows 65% identity with

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DDS in P. ginseng (Suzuki et al., 2006). PNY1 ubiquitously expresses in all tissues, whereas PNY2 only expresses in flower bud and roots (Han et al., 2006). Furthermore, the silencing of DDS caused

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the increased expression of PNY2 and PNZ (Han et al., 2006), implying the tight coordination of DDS with other OSCs because of the utilization of the same precursor. Phylogenetic analysis suggests that DDS and β-AS originated from the ancestral LAS in higher plants mainly through

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tandem duplication as well as subsequent positive selection and diversifying evolution (Xue et al., 2012).

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Intriguingly, the DDS gene was also characterized from C. asiatica by expression in yeast, which shows 78% identity with PgDDS (Kim et al., 2005). In addition, the occurrence of dammarane-type

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triterpene in Gymnostemma pentaphyllum (Cui et al., 1999) implies the possible existence of DDS

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in this species beside Panax species.

2.5. Cytochrome p450 After the production of a basic triterpene skeleton by OSCs, the triterpene skeletons are converted to various ginsenosides via hydroxylation by cytochrome p450 (CYP) and glycosylation by uridine diphosphate (UDP)-dependent glycosyltransferase (UGT). In contrast to the limited gene number and common substrate of OSC genes within plants, UGTs and CYPs belong to large gene families with significant functional diversity (Augustin et al., 2011; Nelson et al., 2008). In ginseng plants, among reported ginseng CYP genes (Balusamy et al., 2013; Devi et al., 2012), three CYP genes are involved in ginsenoside biosynthesis, CYP716A47 acts as protopanaxadiol synthase (PPDS) hydroxylating dammarenediol-II at the C-12 position, yielding PPD (Han et al., 2011); two additional CYP716A subfamily members: CYP716A53v2 as the protopanaxadiol 6hydroxylase (or called protopanaxatriol synthase, PPTS), catalyzing the generation of PPT from 11

ACCEPTED MANUSCRIPT PPD for the synthesis of dammarene-type ginsenosides in P. ginseng (Han et al., 2012), CYP716A52v2 (oleanolic acid synthase, OAS) acting as a β-amyrin 28-oxidase modifying βamyrin into oleanolic acid for the synthesis of an oleanane-type saponin in P. ginseng (Han et al., 2013). PPDS and PPTS express ubiquitously in all ginseng organs, and the expression of PPDS is

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promoted by MJ in adventitious roots, but neither for PPTS and OAS (Han et al., 2012). Furthermore, the counterpart of PPDS in P. quinquefolium, PqD12H, was shown to have conserved

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role in production of PPD by the analyses of in vitro yeast expression and RNAi-transgenic plants (Sun et al., 2013). The RNAi lines of PgPPDS and PqD12H in transgenic hairy root of P. ginseng and P. quinquefolius, respectively, caused reduction of growth (Sun et al., 2013), implying its positive correlation between plant growth and the synthesis of ginsenosides.

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Even though P. notoginseng contains varied ginsenosides from those of P. ginseng and P. quinquefolius, it has almost identical sequences of PgPPDS, PgPPTS and PgOAS (Luo et al., 2011),

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suggesting a close evolutionary relationship among P. ginseng, P. quinquefolius, and P. notoginseng at the DNA sequence level. However, PPDS is expressed at higher levels in P. quinquefolius than P. ginseng, suggesting the emergence of different transcriptional control

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mechanisms between P. quinquefolius and P. ginseng (Sun et al., 2013).

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2.6. Uridine diphosphate (UDP)-dependent glycosyltransferase

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Glycosylation of triterpenes frequently increases their water solubility and modifies their biological activity. UGTs are widely distributed in organisms capable of recognizing a wide range

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of natural products as acceptor molecules. UGTs belong to a multiple family and are involved in the diversification of natural products in plants. In ginseng plants, UGTs are assumed to play an important role in producing different ginsenosides by adding monosaccharides to triterpene aglycones mainly at C-3 and/or C-20 for PPD-type ginsenosides and at C-6 and/or C-20 for PPTtype ginsenosides. Even though several UGT enzymes have been identified in glycosylating triterpene aglycones (Jung et al., 2014; Khorolragchaa et al., 2014; Luo et al., 2011; Xiang et al., 2012; Yan et al., 2014; Yue and Zhong, 2005), the biochemical functions of UGTs in ginseng plants remain largely unknown. Two UGTs, UGRdGT from P. notoginseng and UGRh2GT from P. ginseng, were purified, and were shown to be responsible for the synthesis of ginsenoside Rb1 from Rd, and Rg3 from Rh2, respectively (He and Yue, 2010; Yue and Zhong, 2005). Recently Yan et al. (2014) identified UGTPg1 in glycosylating several intermediates, including dammarenediol, PPD, Rh2, and Rg3 at C-20 positions for the production of new compound 20S-O-β-(D-glucosyl)dammarenediol II (DMG), compound K, a kind of ginsenoside not detectable in ginseng plants, but in human blood, F2, and Rd, respectively, providing insight into ginsenoside biosynthesis. This

ACCEPTED MANUSCRIPT finding suggests that P. ginseng may have the ability to synthesize compound K, but it cannot be accumulated due to its instability in plants. Jung et al. (2014) characterized that PgUGT74AE2 catalyzes the transfer of a glucose moiety from UDP-glucose to the C3 hydroxyl groups of PPD and compound K, yielding Rh2 and F2, respectively, PgUGT94Q2 can transfer a glucose moiety from

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UDP-glucose to Rh2 and F2 to generate Rg3 and Rd, respectively. Khorolragchaa et al. (2014) showed 12 putative UGTs from ginseng plants, future biochemical characterization of the UGTs

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may reveal the mechanism controlling ginsenoside biosynthesis, and provide new approaches for modifying ginsenoside production.

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2.7. Cellular and subcellular locations for synthesizing ginsenosides Saponins are frequently accumulated in specific tissues. Chemical analysis and immunological

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staining demonstrated that the root epidermis contains a high level of ginsenosides (Fukuda et al., 2006; Taira et al., 2010; Yokota et al., 2011). Furthermore, histochemical staining revealed the distribution of ginsenosides mainly in the oil canals in the periderm and outer cortex regions of the

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root, but not in the xylem or pith, the two zones which comprise most of the root (Christensen et al., 2006; Tani et al., 1981) (Fig. 4). The ginsenoside distribution in root differs from the tissues in

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which ginsenoside biosynthetic genes are expressed, namely the vasculature and especially in resin ducts and phloem (Han et al., 2010; Kim et al., 2014b). This suggests that phloem and resin ducts

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are both metabolically active sites for sterol and ginsenoside biosynthesis and that the ginsenosides can then be reallocated into other tissues, such as the epidermis (Fukuda et al., 2006; Li et al.,

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2013).

Furthermore, in leaves, organelles such as plastids, peroxisome, and vacuoles have been shown to be involved in synthesizing ginsenosides (Poustka et al., 2007; Yokota et al., 2011) (Fig. 4). The synthesized ginsenosides in leaves can be transported into the roots through the phloem for storage or to play a role in defense. The transport of saponin glycosides was assumed to be mediated by an ATP binding cassette transporter (Jasin´ski et al., 2001) or multidrug and toxic compound extrusion transporters (Goodman et al., 2004; Yazaki, 2005). Further studies on the mechanism of ginsenoside transport will provide additional insight into ginsenoside transport, and this could be a target to improve the accumulation of ginsenosides. Although some genes have been identified to be associated with ginsenoside synthesis, the enzymes responsible for ginsenoside modification and biosynthesis largely remain to be characterized. Moreover, the enzymes related to the biosynthesis of ocotillol-type ginsenosides identified from P. notoginseng and P. japonicas, at the sequence level, have not been investigated. 13

ACCEPTED MANUSCRIPT Other dammarenediol-type ginsenosides, such as notoginsenoside I isolated from P. notoginseng, P. vietnamensis, and P. japonicas (reviewed by Christensen, 2009) appear to be directly biosynthesized from dammarenediol followed by glycosylation. Beside the efforts on understanding the enzymes involved in uncharacterized biosynthetic steps, understanding the regulatory

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will be helpful for further improvement of ginsenosides.

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mechanisms of biosynthetic genes/enzymes and the dynamics of ginsenosides production in plants

2.8. Regulations of ginsenosides synthesis

To date, little is known about the regulatory mechanism controlling genes/enzymes relative to

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plant triterpene biosynthesis. In animals, HMGR is known as the rate-limiting enzyme in cholesterol synthesis, thus HMGR is transcriptionally and post-transcriptionally regulated by

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multiple feedback mediated by sterol and nonsterol end-products of MVA pathway (Goldstein and Brown, 1990). In plants, HMGR activity is also regulated at the transcriptional and posttranslational level, including the inhibition by reversible kinase-mediated phosphorylation by SNF1

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(sucrose nonfermenting)-related protein kinase 1 (SnRK1), mediated by Pleiotropic Regulatory Locus1 (PRL1) (reviewed by Throll and Lee, 2011), light-regulated proteolytic degradation (Korth

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et al., 2000), and negative regulation by protein phosphatase 2A (PP2A) (Leivar et al., 2011). Since PP2A is involved in abscisic acid and auxin signaling and PRL1 functions as a regulator of sugar,

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stress, and hormone response, it is possible that the HMGR activity is regulated by phytohormone and stress response. Furthermore, both MVA and MEP pathways have been shown to be

2010).

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coordinated by sugar and hormone molecules via the global regulator PRL1 (Flores-Perez et al.,

In animal, cholesterol synthesis is mainly regulated by the transcriptional control mediated by the bHLH transcription factors called sterol regulatory element binding proteins (SREBPs) (Yokoyama et al., 1993). Nearly all of genes encoding cholesterol synthesizing enzymes involved in MVA pathway, OSCs and P450s are the targets of SREBPs (Sharpe and Brown, 2013). By contrast, transcription factors controlling the expression of plant MVA pathway genes remain unknown so far. More recently, Kemen et al, (2014) observed that sterol/lipid-binding class IV homeodomain leucine zipper (HD-ZIP IV) transcription factors may act as potential regulator of βAS by the promoter analysis in oat (Avena strigosa). In plants, gene duplication followed by functional divergence is particularly important for the diversification of biochemical metabolites (Liu et al., 2012). Gene duplication is considered to be a major mechanism in the generation of evolutionary novelty and adaptation. Different from the presence of a single copy of HMGR, SS, SE in yeast and animals, plants contain one more genes of

ACCEPTED MANUSCRIPT HMGR, SS, SE. The presence of multiple genes and isoforms of biosynthetic enzymes may contribute to flexible regulatory control over triterpene biosynthesis. The organ-specific transcription of PgHMGR, PgSS, and PgSE could contribute to the organ-specific accumulation of phytosterols and triterpenes in P. ginseng plants. Due to the unclear function of these duplicated

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members in P. ginseng plants, future functional analysis and identification of these members and their regulators will help in clarifying the biochemical mechanism for synthesizing ginsenosides

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and phytosterols.

Metabolic gene clusters provide another strategy of transcriptional co-regulation. An operon-like gene cluster required for the synthesis of the triterpene avenacin has been discovered in diploid oat (Qi et al., 2004; Chu et al., 2011). In Arabidopsis, two gene clusters for the synthesis of novel

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triterpenes, thalianol and marneral, were identified (Field and Osbourn, 2008; Field et al., 2011). Gene cluster for the biosynthesis of a novel triterpene, dihydroluepol was also recently discovered

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in the model legume plant Lotus japonicas (Krokida et al., 2013). Most clusters have shown that the pair of terpene synthase and CYP are clustered and coordinately expressed across monocots and eudicots (Boutanaev et al., 2015). It will be interesting to investigate whether ginseng genomes also

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contain operon-like gene clusters for ginsenoside biosynthesis.

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3. Dynamics of ginsenoside accumulation and the physilogical function

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The quantity and quality of ginsenosides in plants are affected by biological and environmental factors such as the species, age, different plant tissues, harvest season, differences in cultivation

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methods, and preservation methods (reviewed by Christensen, 2009). It has been demonstrated that most of the ginseng plant organs, such as roots, stems, leaves, flowers, and fruits can synthesize ginsenosides even though different organs have varied amount and types of ginsenosides (Attele et al., 1999) (Fig.1). Interestingly, during both P. ginseng and P. quinquefolius grow from one to six years, roots or root hairs of older ginseng accumulate a higher content of ginsenosides, such as Rb1, whereas the leaves accumulate ginsenoside maximally during early growth stages (mainly during the first year and the second year) (Li et al., 2012; Shi et al., 2007; Wang et al., 2006) (Fig.1). In addition, during the foliation of leaves from previous year roots, ginsenoside content of leaves become markedly increased, conversely, those in the roots decrease (Kim et al., 2014a), suggesting the dynamic movement or synthesis during each year of growth. Even though investigations have been performed to understand the mechanism of ginsenoside synthesis and distribution, the longdistance allocation of ginsenosides remains largely unknown. More recently,

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C-labeling assays

indicated that precursors for ginsenoside biosynthesis can be transported from ginseng leaves into 15

ACCEPTED MANUSCRIPT roots. Schramek et al. (2014) proposed that the products of photosynthetic metabolites, such as glucose and fructose, contribute to the synthesis or movement of ginsenosides into roots. In addition, different genotypes of ginseng plants influence the ginsenoside content. Although P. quinquefolium, P. ginseng, and P. notoginseng are morphologically and phylogenetically similar,

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each ginseng species contains characteristic types and/or levels of ginsenosides (Chan et al., 2000; Fuzzati, 2004; Li et al., 2000; Wan et al., 2007; reviewed by Kim, 2012), which also make different

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contents and composition for ginsenosides in tissue culture (Table 2). These differences among various ginseng species not only reflect the genetic diversity in synthesis and accumulation of ginsenosides in different ginseng species, but also can be used for discrimination of ginseng species and product quality control. In the future, it will be important to investigate the genetic and

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biochemical mechanisms controlling the biosynthesis of ginsenosides among Panax species. Although triterpenoid saponins have been considered as defense compounds against pathogenic

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microbes and herbivores (Osbourn, 1996; reviewed by Augustin et al., 2011), little is known about the physiological roles of ginsenosides in plants. Currently, the most important physiological role of ginsenosides has been shown as the phytoanticipins to protect plants against pathogens. For

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instance, the major secreted ginsenosides in the rhizosphere have allelopathic effects on the soil fungal community. The possible mechanism in blocking pathogen attacks is that ginsenosides

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interact with fungal membrane sterols, leading to damage in membrane integrity (Bernards et al., 2006; Morrissey and Osbourn, 1999; Nicol et al., 2002; Sung and Lee, 2008). On the other hand,

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the fungitoxic properties of ginsenosides can be attenuated through enzymatic degradation by pathogen-formed glycosidases (Bernards et al., 2006), and the degraded ginsenosides may

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subsequently serve as allelopathic growth stimulators of more virulent root pathogens, such as Fusarium spp., and Cylindrocarpon destructans (Nicol et al., 2002; Yousef and Bernards, 2006), implying the evolutionary interaction between ginsenosides rhizosphere and soil fungi has decided for defense or susceptibility. Moreover, ginsenosides were also shown to have potential anti-insect activity (Mallvadhani et al., 2003), possibly via interfering with the receptor of insect hormone ecdysteroid, and influencing the life cycle of herbivorous insects (Harada et al., 2009). In addition, dammarenediol-II, the intermediate structure of ginsenosides, was demonstrated to have antiviral activity in transgenic tobacco (Lee et al., 2012). However, how ginsenosides are recognized and sensed by pathogens remains to be addressed. Although the major role of ginsenosides is plant defense, triterpenes also function in plant growth and development. Lupeol, a triterpene saponin, is synthesized in the nodules of the Lotus japonicas and regulates nodule development (Delis et al., 2011). Interestingly, the unusual triterpenes thalianol and marneral affect plant growth, including root length and embryogenesis, respectively, in Arabidopsis thaliana (Field and Osbourn, 2008). Surprisingly, recently β-amyrin

ACCEPTED MANUSCRIPT was shown to change the epidermal cell patterning in Avena strigosa (Kemen et al., 2014). In ginseng, the allelopathy effect of ginsenosides on ginseng plant growth has been reported (Zhang et al., 2011). PPD-group ginsenosides inhibit the seedling growth of P. quinquefolius at higher concentrations, whereas it has stimulatory effects at lower concentrations (Zhang et al., 2011). By

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contrast, the PPT-group of ginsenosides has stimulatory effects on P. quinquefolius seedling growth at various concentrations. The different effect of PPD and PPT-group ginsenosides on plant growth

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reflects varied signaling responses of plants to ginsenosides, which remains to be elucidated.

4. Biotechnological approaches for production of ginsenosides

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4.1. Tissue culture

Field cultivation of ginseng generally involves 4 to 6 years, and requires extensive effort on quality

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control as ginseng growth is susceptible to environmental factors, such as soil, climate, shade, pathogens and pests. To solve these problems, cell and tissue culture methods have been extensively explored for more rapid and massive production of ginsenosides in P. ginseng

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(reviewed by Baque et al., 2012; Murthy et al., 2014; Paek et al., 2009; Wu and Zhong, 1999; Thanh et al., 2014) (Fig. 5). Among the studies of Panax major species for ginsenoside production

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by cell lines and chemicals, more focus is on the culture of suspension cells of P. notoginseng and adventitious roots of P. ginseng (Table 2).

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Suspension culture of ginseng cells is an approach for production of ginseng saponin, and investigations show that medium components such as sugar molecules have dramatic effects of on

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ginsenoside yield in cultured plant cells. For example, the feeding of sucrose from 20 to 40 g/L in P. notoginseng suspension culture promoted 2.3-fold-production of total ginsenosides, which might be due to the high osmotic pressure and reduced nutrient uptake (Zhang et al., 1996a). In case of P. ginseng, sucrose feeding of 60 g/L increased 3.5-fold production of total ginsenosides compared with the control containing 30 g/L sucrose inoculum in media (Akalezi et al., 1999; Wu and Zhong, 1999) or feeding sorbitol combing with casein hydrolysate also increased 3.5-fold of total ginsenosides yield compared with the control containing 30 g/L sucrose (Wu et al., 2005). Furthermore, the increased phosphate concentration in medium from 0 to 1.25 mM, was reported to enhance the 7-fold productivity of ginsenosides in suspension cultures of P. notoginseng (Zhong and Zhu, 1995), and similar accumulation in P. quinquefolium cell culture was observed (Liu and Zhong, 1998). Meanwhile, P. ginseng cell culture supplemented with 0.42 mM phosphate displayed an optimized growth (Liu and Zhong, 1998), implying less demand of phosphate of P. ginseng than other species. When 6.0 μM copper was initially added in the medium, the total production of 17

ACCEPTED MANUSCRIPT ginsenosides could be increased up to 4-fold (Zhong and Wang, 1996). Furthermore, a higher ratio of nitrate to ammonium can also improve the yield of ginsenosides in the suspension cultures of three major Panax species (Zhang et al., 1996b; Zhong and Wang, 1998; Paek et al., 2009), indicating the importance of nitrate ratio for the cell growth and productivity of ginsenosides.

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Additionally, the MJ treatment at a concentration of 200 μM increased the yield of ginsenosides particularly PPD groups in cell suspension cultures in both P. ginseng and P. notoginseng (Thanh et

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al., 2005; Wang et al., 2005; Hu and Zhong, 2008). The addition of N,N'-dicyclohexylcarbodiimide in suspension cultures of P. ginseng cells at a concentration of 10 μM caused 3-fold increase of total ginsenosides compared with that of untreated control, particularly, higher yield of Rg1 and Re than Rb1 ginsenosides due to the increased activities of PPT-biosynthetic enzymes by modulating

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nitric oxide signaling in P. ginseng cells (Huang et al., 2013).

In addition, the centrifugal impeller bioreactor is useful for high-density suspension cultivation

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of P. notoginseng cells compared with the conventional turbine reactor or the shake flask system (Zhong et al., 1999; Zhang and Zhong, 2004). One interesting observation during ginseng production by cell culture is that the synthetic rate of ginsenosides is not proportional to the growth

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rate although the synthesis and the growth of cell culture simultaneously from the initial culture (Kochan and Chmiel, 2011). During the rapid cell growth, after which biomass reached 90% of

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maximum yield, only half ginsenosides was produced, and the final half production occurred during the slow growth phase (Kochan and Chmiel, 2011), suggesting that the accumulation of

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ginsenosides is tightly controlled with growth status, which remains further investigation on its underlying mechanism. Recently, Kochkin et al. (2013) monitored ginsenosides profile over a 4-

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year from a suspension cell culture of P. japonicus, and observed that ginsenoside Rg1, R0, and malonyl-Rb1 accounted for more than 75% of the total pool of ginsenosides in the suspension culture, but the amounts and compositions of ginsenosides varied according to growth stage and subculture cycles, suggesting that the production condition of ginsenosides using suspension cells should be well controlled for high yield (Kochkin et al., 2013). Notably, the maximum content of ginsenosides in the suspension culture is comparable to that of the 3-year-old field grown ginseng plants (Kochan and Chmiel, 2011), suggesting the potential of large-scale of ginsenoside production using cell cultures even though the system of suspension cultivation of P. notoginseng cells is currently being hampered by low yield and productivity (Wu and Zhong, 1999). Alternatively, culture of adventitious roots is regarded as an improvement of ginsenoside production using tissue culture approach because of its higher production of biomass and ginsenosides as well as a high stability against physical and chemical conditions during large-scale cultures (i.e. upto 10-ton scale bioreactor) (Paek et al., 2009). Adventitious roots, induced from leafstalks, lateral roots shoots, young floral bud callus, have been used for ginsenoside production

ACCEPTED MANUSCRIPT for 15 years for P. ginseng and P. notoginseng (Gao et al., 2005; reviewed by Baque et al., 2012; Murthy et al., 2014; Thanh et al., 2014) and established recently for P. quinquefolius (Szymańska et al., 2013). The accumulation of ginsenosides in the adventitious root cultures of ginseng can be improved by medium replenishment strategy for the large-scale production (Sivakumar et al., 2005;

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Jeong et al., 2008). MJ and salicylic acid (SA) can also enhance the accumulation of ginsenosides in P. ginseng roots, and the possibly because MJ and SA can induce an oxidative stress in the

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suspension-cultured P. ginseng roots, resulting in an increase of the synthesis of ginsenosides (Ali et al., 2006a). Furthermore, the combination of phytohormone, indole-3-butyric acid and MJ can synergetically trigger root growth with 7-fold increase of ginsenoside accumulation in adventitious root cultures of P. ginseng compared with the treatment of MJ alone, which showed an inhibitory

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effect of adventitious growth (Kim et al., 2004; 2007). Moreover, the ginsenoside productivity in P. ginseng adventitious root cultures can be increased by the combination of ethephon and MJ (Bae et

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al., 2006). Recently, the bioreactor culture of P. quinquefolius and P. notoginseng hairy roots has been established (Mathur et al., 2010; Kochan et al., 2012; 2013; Zhang et al., 2010). Notably, different cell lines may have 4 to 5 times variation for producing total ginsenosides (Woo et al.,

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2004). Further, permeabilizer such as Tween 80 can enhance the ginsenoside secretion, leading to about 3-fold increase of total ginsenosides yield during the culture of hairy roots (Liang et al., 2014).

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In another way, P. ginseng hairy root cultures have been established by infecting ginseng roots with Agrobacterium rhizogenes, for higher production of ginsenosides because of their the quick

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growth, stability and flexibility for large-scale production using bioreactor (Jeong et al., 2005; Kim et al., 2009; Mallol et al., 2001; Palazón et al., 2003; Woo et al., 2004; Yu et al., 2005a).

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Transformed P. ginseng roots infected with A. rhizogenes displaying traits of hairy roots, callus-like or thin roots without branching had higher growth rates compared with untransformed roots. In particular, hairy roots can produce the highest biomass and ginsenoside levels, such as Rg group ginsenoside. The mechanism of higher biomass and ginsenoside production in transgenic roots is explained by the significant role of auxin-responsive genes in the morphologies of P. ginseng transformed roots (Liu et al., 2001; Mallol et al., 2001). Further, overexpression of the A. rhizogenes rolC gene in P. ginseng callus causes the formation of tumors which can form roots. These regenerated roots can be used for large-scale ginsenoside production (Gorpenchenko et al., 2006). Although the culture of adventitious roots has been used for production of ginsenosides, the total ginsenoside contents of cultured adventitious roots is around tenth of that in 6-year-old ginseng root (Kim et al., 2013b). Recently, Kim et al. (2013b) reported the mutagenesis by γirradiation and ginsenoside production was enhanced up to 16-fold in induced P. ginseng 19

ACCEPTED MANUSCRIPT adventitious root culture as well as growth ratio compared with normal culture (Kim et al., 2013b), and about 1.6-fold more accumulation of ginsenosides in mutated adventitious root cultures compared with that in 6-year-old ginseng root. The possible mechanism is that this mutation causes an expression increase of the DDS gene (Kim et al., 2013b).

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In addition, the hydroponic cultivation of ginseng is another example of raw ginseng production within a shorter period under a controlled environment (Kim et al., 2010b; 2012). Hydroponic

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systems also can produce pesticide-free ginseng roots and leaves with high ginsenoside contents using shorter time. Ginseng grown hydroponically contained higher levels of ginsenoside PPT type, especially up to 60% higher level of ginsenoside Re, and a special type of ginsenoside Rh1, which cannot be detected in soil-cultivated ginseng (Han et al.., 2004; Kim et al., 2010b; Oh et al., 2014a).

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High light levels increased PPT-type ginsenosides in the leaves of ginseng plants (Kim et al., 2012); however, continuous dark treatment for 2 to 3 days increased ginsenoside level in both leaves and

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roots (Kim et al., 2014b) and in adventitious roots (Li et al., 2013). Correspondingly, it has been proposed that the whole MEP pathway is light-stimulated, most likely via phytochrome signaling,

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4.2. Chemical elicitors

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and that the MVA pathway is largely light-inhibited (Hemmerlin et al., 2012).

Ginsenoside accumulation in plant cells is stimulated by biotic and abiotic elicitors. To improve

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ginsenoside biosynthesis, elicitation by various molecules or treatments, called elicitors, has been investigated (Yendo et al., 2010) (Fig.5). Various biotic and abiotic elicitors, such as CuSO4 (Ali et

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al., 2006b), vanadate (Huang et al., 2013), NiSO4, sodium chloride, nitric oxide, ethephone (Hu et al., 2003; Jeong and Park, 2006), ultrasound (Wu and Lin, 2002), oligogalacturonic acid (Hu et al., 2003), osmotic stress (Wu et al., 2005), chitosan (Jeong and Park, 2005), polyunsaturated fatty acids (Wu et al., 2009; Dewir et al., 2010), and compounds such as geranium (Yu et al., 2005b), SA (Jeong et al., 2005), jasmonic acid (JA) and its derivatives (Ali et al., 2006a; Bae et al., 2006; Hu and Zhong, 2007; Qian et al., 2004; Yu et al., 2002), have been optimized as effective approaches for enhancing ginsenosides production in cultured ginseng cells or adventitious roots. These elicitors stimulate ginsenoside biosynthesis via the activation of phenylalanine ammonia lyase, a key enzyme required for biosynthesis of defense compounds (Qian et al., 2004) or signal transducer nitric oxide or reactive oxygen species such as hydrogen peroxide (Ali et al., 2006b; Hu et al., 2003; Huang et al., 2013). Among these elicitors, MJ has shown to be a strong effective elicitor to stimulate the biosynthesis of ginsenoside in vivo in all cell lines P. ginseng (Kim et al., 2007; 2009; Murthy et al., 2014; Paek et al., 2009; Wang and Zhong, 2002; Wang et al., 2005). Exposure to MJ at 100 µM in

ACCEPTED MANUSCRIPT hairy roots of P. ginseng induced expression of genes involved in ginsenoside biosynthesis, such as HMGR, SS, SE and DDS, with a slight decrease of CAS (Kim et al., 2007; 2009; Lee et al., 2004; Wang et al., 2013). PPD-type of saponins were increased 5.5- to 9.7- times by MJ treatment for 7 days, whereas the PPT-type of saponins increased 1.9- to 3.8- times. Addition of 200 µM MJ also

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increased individual ginsenosides heterogeneously, about 9-times for PPD-type and 2-fold for the PPT-type ginsenosides in P. notoginseng (Wang and Zhong, 2002). JA also increased the

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production of ginsenoside Rb1 group, indicating that this elicitor might trigger the synthesis of the PPD-type of ginsenosides (Yu et al., 2002). Exposure to MJ of the root system of whole plants stimulated ginsenoside accumulation, especially the PPD-type, in the inner root body at the highest level, but not in the epidermis, which again indicates the response of ginsenoside biosynthesis takes

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place in the vasculature rather than in the epidermis (Oh et al., 2014a).

SA and JA are signaling molecules that play key functions in plant defense regulation (Gutjahr

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and Paszkowski, 2009). The enhancement of ginsenoside yields by MJ in plants and cell culture supports the hypothesized role of ginsenosides in plant defense mechanisms. Exogenous MJ could induce the lipoxygenase, via the alpha-linoleic pathway, and lead to endogenous JA biosynthesis

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(Melan et al., 1993). JA, as a signal transducer, may play an important role in the accumulation of the PPD-type of ginsenosides (Hu and Zhong, 2007). Further, signals transferred via cyclic

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oxylipins to the nucleus activate transcription factors, including WRKY and MYB, which may be involved in ginsenoside biosynthesis gene regulation (De Geyter et al., 2012; Subramaniyam et al.,

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2014). Ginsenoside accumulation by heavy metal treatment is also possibly induced by promoting endogenous JA biosynthesis as the signal molecule (Huang et al., 2013).

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Furthermore, the biosynthesis of PPD-group ginsenosides can be more stimulated by MJ than that of PPT group-ginsenosides, whereas chilling treatment caused more accumulation of PPT-type ginsenosides than that of PPD-type ginsenosides (Oh et al., 2014a). Different elicitation effects of biotic and abiotic stresses on the levels of individual ginsenosides imply distinct defense strategy of different ginsenosides in plants, which may be associated with their different biological activities. The biosynthetic pathway of different ginsenosides has yet to be determined, and further studies to identify enzymes such as UGTs involved in biosynthesis of different individual ginsenosides will be helpful in elucidating the mechanism of ginsenosides biosynthesis responsive to biotic and abiotic stresses.

4.3. Transgenic plants

21

ACCEPTED MANUSCRIPT Although there have been many efforts on the production of ginsenosides by tissue and cell cultures, the productivity of ginsenosides is still relatively poor; therefore, overproduction of ginsenosides by metabolic engineering has been an attractive strategy to improve ginsenoside yield. Attempts to up-regulate or down-regulate genes involved in ginsenoside production, by genetic

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engineering of Panax species, to achieve a higher yield of ginsenosides have been reported in transgenic plants (Fig.5).

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Recently, we showed that PgHMGR1 is a key target for increasing the ginsenoside production because A. tumefaciens-mediated overexpression of PgHMGR1 driven by Cauliflower Mosaic Virus (CMV) 35S promoter caused an increased accumulation of triterpenes and sterols in plants, particularly, achieving 1.5- to 2-fold increase in the amount of ginsenosides without altering the

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ratio of individual ginsenoside in transgenic ginseng adventitious roots. Moreover, heterologous expression of PgHMGR1 caused 1.8- to 2-fold increase in phytosterols, including β-sitosterol,

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campesterol, cycloartenol, and 2- to 2.5-fold increase in triterpenes, including α-amyrin and βamyrin in transgenic Arabidopsis rosette leaves (Kim et al., 2014b). Consistently, a similar result was achieved by Kim et al. (2013a), and it was shown that the A. rhizogenes-mediated ectopic

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overexpression of PgHMGR1 caused 1.5- to 2.5-fold increase of triterpene and 1.1- to 1.6-fold higher phytosterols in Platycodon gradiflorum hairy root. These results again confirmed conserved

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role of PgHMGR1 in triterpene and phytosterol accumulation in plants. Similarly, overexpression of PgFPS increased the expression of two downstream genes, PgCAS

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and PgDDS, leading to about a 2.4-fold increase of total ginsenosides and phytosterol (campesterol, stigmasterol, and β-sitosterol) in transgenic hairy root compared with the control, whereas β-amyrin

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levels were decreased (Kim et al., 2014c). By contrast, PgMVD-overexpressiing lines had upregulated expression of PgCAS, leading to increased levels of physterol, but without any change of ginsenoside and β-amyrin (Kim et al., 2014c), suggesting its less imporance for ginsenoside biosynthesis.

SS is also one of key regulatory enzymes for metabolomic engineering for the production of both phytosterols and triterpenes. Overexpression of PgSS1 in P. ginseng caused 1.6- to 3- fold increase of total ginsenosides and 2-fold increase of phytosterols in transgenic adventitious root, accompanied by the up-regulation of downstream enzymes such as SE, β-AS, and CAS (Lee et al., 2004; Shim et al., 2010). The 2-fold reduction of squalene contents in the roots might be due to the rapid flux intermediates toward phytosterols (Lee et al., 2004). Consistently, ectopic expression of PgSS1 in Eleutherococcus senticosus also caused an increased in the amount of phytosterols and triterpene saponin (Seo et al., 2005). However, silencing of PgSE1 by RNAi in ginseng strongly upregulated PgSE2 and CAS and resulted in enhanced phytosterol accumulation, indicating that expression of PgSE1 and PgSE2 is regulated in a different manner, and PgSE1 might regulate

ACCEPTED MANUSCRIPT ginsenoside biosynthesis, rather than by PgSE2. Therefore, use of different isozymes could contribute to the synthesis of different ginsenosides. All three PgSS genes are positive up-regulated by MJ treatment. However, MJ enhances PgSE1 expression but suppress PgSE2 expression, which support the different regulatory role of PgSE1 and PgSE2, in triterpenes and phytosterol

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biosynthesis, respectively (Han et al., 2010).

Dammarenediol-II is a bioactive tetracyclic triterpenoid, a basic precursor for ginsenoside

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saponin production. Han et al. (2006) showed that DDS is a key gene for ginsenoside production, and suppression of the branching enzyme, such as CAS for phytosterol production, causing an increase in DDS enzyme activity, led to a 50-100% increase of ginsenoside content in hairy roots of P. ginseng (Liang et al., 2009). Reduction of CAS enzyme activity caused an increase of available

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2,3-oxidosqualene, the common precursor of phytosterols and ginsenosides. However, CAS-RNAi lines led to slow growth rate of transgenic hairy roots during early stage of culture (Liang et al.,

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2009), suggesting that supply phytosterols at initial growth stage is required for rapid growth to use this transgenic hairy root to overproduce ginsenosides. Hetero-overexpression of DDS produced dammarenediol-II about 20-30 μg/g in tobacco leaves, and increase resistance against virus,

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suggesting the functional role of pathogen resistance of dammarenediol (Lee et al., 2012). More recently, Han et al. (2014) established an effective suspension culture system of transgenic tobacco

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cell expressing PgDDS for the production of dammarenediol-II that can be utilized as a source for producing pharmacologically active medicinal materials. Transgenic tobacco plants containing

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PgDDS, under the control of CMV 35S promoter, were obtained from Agrobacterium-mediated transformation, and accumulated dammarenediol-II in an organ-specific manner, and the highest

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amount of dammarenediol-II in the roots was 157.8 μg/g DW. Due to the efficient cyclyzation of 2,3-oxidosqualene to dammarenediol-II, transgenic tobacco plants had reduced levels of phytosterols. The cell suspension culture derived from transgenic root segments produced 5.2 mg dammarenediol-II per liter (Han et al., 2014).

4.4. Engineered yeast cells Currently, the major sources of ginsenosides are extracted from ginseng roots, whereas wild ginseng roots are scarce, thus most marketing ginseng roots are from the cultivated ginseng in fields. Because of time-consuming, labour-costive in cultivating ginseng plants, some researchers attempted the production of ginsenosides in yeast cells. Bakers’ yeast (Saccharomyces cerevisiae) has been recognized as an ideal host for high-efficient production of valuable products such as

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ACCEPTED MANUSCRIPT proteins and useful compounds, thus yeast cells provide an alternative and attractive approach for production of ginsenosides in comparision with traditional extraction methods (Fig.5). Successful production of PPD through co-expression of PgDDS and CYP716A47 in yeast (Han et al., 2011) might represent a promising way to produce useful dammarene-type triterpenes using

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genetic engineering approaches. Dai et al. (2014) metabolically engineered S. cerevisiae by introducing β-amyrin synthase from Glycyrrhiza glabra, oleanolic acid synthase from Medicago

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truncatula, DDS, PPDS and PPTS from P. ginseng and NADPH cytochrome CYP reductase from A. thaliana for the efficient production aglycons of ginsenosides. Additionally, overexpression of genes encoding a truncated HMGR, SS and 2,3-oxidosqualene synthase could form more precursors for improving aglycon production. Using this strategy, Dai et al. (2014) developed recombinant

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yeast strains capable of producing 17.2 mg/L PPD, 15.9 mg/L PPT and 21.4 mg/L oleanolic acid. Although dried P. ginseng roots contain about 4% ginsenosides, the main functional form

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observed in mammalian blood after oral consumption of ginseng or ginsenosides is compound K, which was shown to have bioactivities such as anti-inflammation, hepatoprotection, antidiabetes and anti-cancer (Yan et al., 2014). More recently, China Food and Drug Administration approved

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compound K for clinical trials to prevent and treat arthritis. As compound K has not been identified in Panax plants, currently, compound K is produced by bio-deglycosylation of major PPD-type

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ginsenosides, such as Rb1, Rb2, Rd and Rc (reviewed by Christensen, 2009). Extensive investigations have revealed that a large portion of the intact ginsenosides can be transformed into

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minor ginsenosides with more enhancing biological effects through gastrointestinal acids, enzymes, and intestinal bacteria, especially by β-glucosidase enzymes in human intestine (Christenson, 2009),

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therefore the enzymatic methods have been used to obtain minor ginsenosides. Recently, several studies have successed to conduct scale-up engineering for the bioconversion using a recombinant enzymes, particularly for ginsenoside Rg3 (Chang et al., 2009; Kim et al., 2013c; Oh et al., 2014b; Shin et al., 2014; Quan et al., 2012; 2013) (Table 2). However, due to the limited availability of ginsenosides for large scale compound K production, and the challenge of chemical synthesis caused by the difficulty of selective glycosylation of ginsenosides, other approaches have been explored. Yan et al. (2014) employed the expression of UGTPg1 together with the overexpression of truncated HMGR and UPC2.1, a semi-dominant mutant UPC2 which acts as a global transcription factor for the synthesis of sterols, and obtained a yeast strain with a yield of up to 1.4 mg/L for compound K. This milestone work provides an inexpensive way to manufacture compound K to meet potential clinical applications. Furthermore, Jung et al. (2014) expressed two newly identified PgUGTs: PgUGT74AE2 and PgUGT94Q2, together with PgDDS and PgPPDS and produced ginsenoside Rg3 in yeast cells. These works provide a new method for large-scale production of specific ginsenosides via yeast fermentation.

ACCEPTED MANUSCRIPT 5. Conclusions and future prospectives Ginsenosides are specialized triterpene saponins uniquely present in Panax species. Because of

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the medical importance of ginsenosides, investigations on their chemical structure, medical activities, biosynthetic enzymes and production improvement have received much attention. The biosynthetic pathway of ginseng saponins shares common and diversified enzymes with those of

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triterpenes in plants. To date, there are more than 150 known different ginsenosides with various numbers, linkage positions and types of sugar moiety, and most of them are the dammarane or oleanane-type. Current evidence suggests that gene expansion resulting from genome and/or gene

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duplication followed by gene fixation, via positive selection of sub- and neofunctionalized homologs, may provide the fundamental genetic base for ginsenoside biosynthesis and plant adaptation and species radiation. However, the mechanism(s) driving the formation of ginsenosides,

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as well as the events underlying the diversification of ginsenosides in Panax species have yet to be elucidated. Another area in need of further research relates to the mechanisms underlying the

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synthesis of the various ginseng saponins, within specific tissues, and their transport to the target tissues.

Ginseng saponins are assumned to act as defense molecules in plant stress and pathogen

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interactions. The pharamacological efficacy of ginsenosides relies on their structural basis, especially their hydroxyl groups and sugar moieties, interacting with membrane lipids. In the future,

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more insight underlying the medical effects of ginsenosides, such as crosstalk with hormone

functions.

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signaling pathways will allow structural modifications to achieve improved beneficial activities and

The time-consuming and labor-intensive cultivation of ginseng in the field has driven bioengineering aproaches, such as cell and tissue culture, chemical elicition during production, transgenic plants and engineered yeast systems to improve ginsenoside production. Particularly, transgenic plants over-expressing genes involved in ginsenosides synthesis, such as HMGR1, SS, CYPs and DDS have significant increased ginsenoside yields. Recently, engineered yeast cells expressing ginsenoside producing enzymes, resulted in the production of PPD, PPT and oleanolic acid as well as compound K. These breakthroughs provide an inexpensive and efficient industrial platform for the manufacture of ginsenosides for clinical applications. Identification of additional functional enzymes for biosynthesizing ginsenosides will definitely lead to more approaches for efficient and large-scale production of ginsenosides.

Acknowledgements 25

ACCEPTED MANUSCRIPT We thank Professor Fengwu Bai, Professor Jianjiang Zhong, Professor William J. Lucas, and Professor Donald Grierson for helpful comments and reading this paper. This work was supported by the funds from Ministry of Science and technology (MOST; 2015DFG32560), China; Basic

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Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2013R1A1A2064430) and Young Scientist Exchange Program between

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Republic of Korea (NRF) and The People’s Republic of China (MOST) (YJ Kim); China Innovative Research Team, Ministry of Education; 111 Project (B14016) (DB Zhang); iPET (312064-03-1-HD040), and Korea Institute of Planning and Evaluation for Technology in Food,

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Agriculture, Forestry and Fisheries, Republic of Korea (DC Yang).

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ACCEPTED MANUSCRIPT

Organism

Abbreviation

Accesion no. (protein)

3-hydroxy-3methylglutaryl coenzyme A synthase 3-hydroxy-3methylglutaryl coenzyme A reductase

P. ginseng

PgHMGS

GU565098 (ADI80347)

ORF (amino acid) 1407 bp (468 aa)

P. ginseng

PgHMGR1

Kim et al., 2014b

PgHMGR2

Sequence and transcript analysis

Kim et al., 2014b

P. ginseng

PgHMGR1 homologue

Overexpression into Platycodon gradiflorum

Kim et al., 2013a

P. ginseng

PgHMGR2 homologue

Sequence and transcript analysis

Luo et al., 2013

P. quinquefolius

PqHMGR

Sequence and transcript analysis

Wu et al., 2010

P. notoginseng

PnHMGR

Sequence reported in NCBI

-

Mevalonate kinase

P. notoginseng

PnMVK

Sequence and transcript analysis

Guo et al., 2012

Mevalonate-5pyrophosphate decarboxylase Farnesyl diphosphate synthase

P. ginseng

PgMVD

1722 bp (573 aa) 1785 bp (594 aa) 1722 bp (573 aa) 1770 bp (589 aa) 1770 bp (589 aa) 1725 bp (574 aa) 1164 bp (387 aa) 1263 bp (420 aa)

Overexpression into Arabidopsis and P. ginseng

P. ginseng

KM386694 (AIX87979) KM386695 (AIX87980) GU565097 (ADI80346) JX648390 (AGL08682) FJ755158 (ACV65036) KJ578757 (AHZ59734) JQ957844 (AFN02124) GU565096 (ADI180345)

Overexpression into P. ginseng

Kim et al., 2014c

P. ginseng

PgFPS

P. quinquefolius

PqFPS

Kim et al., 2010b Kim et al., 2014c -

PnFPS1

Sequence and transcript analysis

Niu et al., 2014

P. notoginseng

PnFPS2

Sequence reported in NCBI

-

P. ginseng

PgSS1

1029 bp (342 aa) 1029 bp (342 aa) 1029 bp (342 aa) 1032 bp (343 aa) 1248 bp (415aa)

Overexpression into Centella asiatica Overexpression into P. ginseng Sequence reported in NCBI

P. notoginseng

DQ087959 (AAY87903) GQ401664 (ADJ68004) KC953034 (AGS79228) DQ059550 (AAY53905) AB115496 (BAA24289)

Overexpression into P. ginseng, tobacco and Eleutherococcus

P. ginseng

PgSS2 PgSS3

Sequence and transcript analysis

Kim et al., 2011

P. quinquefolius

PqSS1

1248 bp (415aa) 1248 bp (415aa) 1248 bp (415aa)

Sequence and transcript analysis

P. ginseng

GQ468527 (ACV88718) GU183406 (ACZ71037) GU997681 (AED99863)

Lee et al., 2004; Seo et al., 2005; Shim et al., 2010 Kim et al., 2011

Sequence reported in NCBI

-

Squalene synthase

Sequence reported in NCBI

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MA

AC CE

Functional study

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Genes

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Table 1. Lists of identified genes involved in ginsenoside biosynthesis in Panax species Reference

-

ACCEPTED MANUSCRIPT

PnSS

Squalene epoxidase

P. ginseng

PgSE1

Squalene epoxidase

P. ginseng

PgSE2

P. quinquefolius

PqSE

P. notoginseng

PnSE1

P. notoginseng

PnSE2

P. notoginseng

PnSE1 homologue

P. ginseng

PNX

P. notoginseng

PnCS

Lanosterol synthase

P. ginseng

PNZ

ß-amyrin synthase

P. ginseng

PNY1

P. ginseng

PNY2

P. quinquefolius

Pqß-AS1

P. quinquefolius

Pqß-AS2

P. ginseng

PNA

P. ginseng

DDS

P. quinquefolius

PqDDS

P. notoginseng

PnDDS

Dammarendiol synthase

43

AC CE

Cycloartenol synthase

-

Sequence reported in NCBI

-

Sequence and transcript analysis

Niu et al., 2014

Silencing in P. ginseng

Han et al., 2010

Sequence and transcript analysis

Han et al., 2010

Sequence reported, but mistake information as squalene synthase Sequence and transcript analysis

-

Sequence and transcript analysis Sequence and transcript analysis

Niu et al., 2014; Luo et al., 2011 He et al., 2008

Ectopic expression in yeast

Kushiro et al. 1998a

Sequence reported in NCBI

-

Ectopic expression in yeast

Suzuki et al., 2006

Ectopic expression in yeast

Kushiro et al. 1998a

Ectopic expression in yeast

Kushiro et al. 1998b

Sequence reported in NCBI

Wu et al., 2014

2286 bp (761 aa) 2310 bp (769 aa) 2310 bp (769 aa) 2310 bp (769 aa) 2310 bp (769 aa)

Sequence reported in NCBI

Wu et al., 2014

Ectopic expression in yeast

Tansakul et al. 2006

Silencing in P. ginseng; overexpression in tobacco

Han et al. 2006; Lee et al., 2012 Wang et al., 2014a

PT

P. notoginseng

Sequence reported in NCBI

SC RI

PqSS3

1248 bp (415aa) 1248 bp (415aa) 1248 bp (415aa) 1611 (536 aa) 1638 (545 aa) 1629 bp (542 aa) 1614 bp (537 aa) 1638 bp (545 aa) 1614 bp (537 aa) 2274 (757 aa) 2277 bp (758 aa) 2343 bp (780 aa) 2292 bp (763 aa) 2286 bp (761 aa) partial

NU

P. quinquefolius

AM182456 (CAJ58418) AM182457 (CAJ58419) DQ186630 (ABA29019) AB122078 (BAD15330) FJ393274 (ACJ24907) KC524469 (AGK62446) KC953033 (AGS79227) JX625132 (AFV92748) DQ386734 (ABE60738) AB009029 (BAA33460) EU342419 ABY60426 AB009031 (BAA33462) AB009030 (BAA33461) AB014057 (BAA33722) JX185490 (AGG09938 ) JX262290 (AGG09939) AB265170 (BAF33291) AB122080 (BAD15332) KC316048 (AGI15962) KC953035 (AGS79229)

MA

PqSS2

PT ED

P. quinquefolius

Ectopic expression in yeast; overexpression into P. quinquefolius Sequence and transcript analysis

Niu et al., 2014

Niu et al., 2014

ACCEPTED MANUSCRIPT

P. notoginseng

PgOAS (CYP716A52v2) PnPPDS (PnCYP450)

P. notoginseng

PnPPTS (PnCYP450)

P. notoginseng

PnOAS (PnCYP450)

P. quinquefolius

PqPPDS (PqD12H)

P. quinquefolius

PqPPTS (PqCYP6H)

P. ginseng

UGTPg1

P. ginseng P. ginseng P. ginseng P. notoginseng

PgUGT71A27 PgUGT74AE2 PgUGT94Q2 PnUGRdGT

Ectopic expression in yeast

Han et al., 2011 Sun et al., 2013 Han et al., 2012

Ectopic expression in yeast

Han et al., 2013

Sequence reported, 99% similarity with PgPPDS

Luo et al., 2011

JX569336 (AFU93031) KC190491 (AGC31652) KF377585 (AIE12479) KM491309 JX898529 JX898530 GU997660 (AED99883)

1449 bp (482 aa) 1410 bp (469 aa) 1428 bp (475 aa)

PT

P. ginseng

Ectopic expression in yeast; silencing in P. ginseng

SC RI

PPTS (CYP716A53v2)

1461 bp (482 aa) 1410 bp (469 aa) 1446 bp (481 aa) 1503 bp (500 aa) 1446 bp (481 aa) 1410 bp (469 aa)

NU

P. ginseng

JN604537 (AEY75213) JX036031 (AF063031) JX036032 (AFO63032) GU997665 (AED99867) GU997666 (AED99868) GU997670 (AED99872)

MA

PPDS (CYP716A47)

PT ED

UDPglucosyltransferase

P. ginseng

AC CE

Cytochrome P450

1428 bp (475 aa)

Sequence reported, 98% similarity with PgPPTS

Luo et al., 2011

Sequence reported, 97% similarity with PgOAS

Luo et al., 2011

Silencing in P. quinquefolius

Sun et al., 2013

Ectopic expression in yeast

Wang et al., 2014b

Ectopic expression in yeast

Yan et al., 2014

Ectopic expression in yeast; identical with UGTPg1 Ectopic expression in yeast Ectopic expression in yeast Sequence report, 99% similarity with UGTPg1

Jung et al., 2014 Jung et al., 2014 Jung et al., 2014 Xiang et al., 2012

ACCEPTED MANUSCRIPT

Production of ginsenoside in different organisms Species

Strategies

Introduced gene or chemical

Plant

P. ginseng

SC flask culture

0.42 mM phosphate

SC flask culture with elicitation

P. notoginseng

643 mg/L

Liu and Zhong, 1998

275 mg/L

Akalezi et al., 1999

1130 mg/L

Wu et al., 2005

TG

b

TGb

5.5 mg/g DW, 55 mg/L

Huang and Zhong, 2013

10 μM N,N'-dicyclohexylcarbodiimide

TGa

3.33 mg/g DW, 36 mg/L

Huang et al., 2013

200 μM MJ for last 10 days of culture

TGa

8.82 mg/g DW, 100 mg/L

Thanh et al., 2005

TGb

960 mg/L

Liu and Zhong, 1998

1.5 g/L

1.25 mM phosphate

TG

a

b

TG

SC flask culture

-

TGa

29 mg/g DW, 197 mg/L

SC flask culture

1.25 mM phosphate

TGb

4% sucrose

AC CE

60 mM NO3-/NH4+(60:0)

30 mg/g DW, 980 mg/L

Zhong and Wang, 1998 Kochan and Chmiel, 2011 Zhong and Zhu, 1995

TG

b

177 mg/L

Zhang et al., 1996a

TG

b

850 mg/L

Zhang et al., 1996b

b

43 mg/g DW, 119 mg/L 20 or 28.9 mg/g DW, 167 or 241 mg/L

Zhong and Wang, 1996 Wang and Zhong, 2002; Hu and Zhong, 2008

SC flask culture with elicitation

6.0 μM copper

TG

SC flask culture with elicitation

200 μM MJ or HEJ

TGc

High density cell culture

TGb

30 mg/g DW, 2.1 g/L

Zhang and Zhong, 2004

200 μM MJ

TGa

SC centrifugal impeller bioreactor (30 L) SC bioreactor (1 L) with elicitation SC bioreactor (500 L)

Ad flask culture with elicitation Ad flask culture with elicitation Ad flask culture with elicitation Ad air-lift bioreactors (3, 5 L) with elicitation Ad air -lift bioreactors (5 L) with

45

TGb

40 mM NO3-/NH4+(40:0)

SC flask culture

P. ginseng

Reference

SC flask culture

SC flask culture

P. japonicus

Amount of ginsenosides produced

PT ED

P. quinquefolius

SC flask culture with elicitation SC bioreactor (5 L) with elicitation SC flask culture

NU

SC flask culture

6% sucrose 0.2 M sorbitol + 0.5 g/L casein hydrolysate 100 μM vanadate

MA

SC flask culture

Type of ginsenosi des

SC RI

Organ ism

PT

Table 2.

23 mg/g DW, 530 mg/L

Wang et al., 2005

TG

d

5 - 49 mg/g DW

Kochkin et al., 2013

TG

a

48 mg/g, 364 mg/L

Kim et al., 2007

TG

a

50 mg/g, 500 mg/L

Kim et al., 2007

2 mg/L JA

TG

a

28 mg/g, 255 mg/L

Polyunsaturated fatty acids

TGa

7.9 mg/g DW, 87 mg/L

50 μM ethephon + 100 μM MJ

TGa

30 mg/g, 270 mg/L

Yu et al., 2002 Dewir et al., 2010; Wu et al., 2009 Bae et al., 2006

100 μM MJ 25 μM IBA + 100 μM MJ

ACCEPTED MANUSCRIPT elicitation Ad air -lift bioreactors (5 L)

IBA

TGa

8.09 mg/g

Ad air -lift bioreactors (5 L)

5% sucrose

TGa

10.02 mg/g DW, 121.6 mg/L

Ad air -lift bioreactors (5 L)

18.25 mM NO3-/NH4+(18.5:0),

Ad air -lift bioreactors (5 L)

100 μM MJ

Ad air -lift bioreactors (5 L)

10 -25 μM Cu

Ad air r-lift bioreactors (1000 L)

100 μM MJ for last 8 days of culture

P. notoginseng

Ad flask culture

-

P. ginseng

Hairy root flask culture Hairy root culture with elicitation

40 mg/g DW, 378 mg/L

TGe

45 mg/g DW, 120 mg/L

Agrobacterium rhizogenes

TGa

2.5-5.4 mg/g DW

1.2% (w/v) Tween 80

TGa

27 mg/g DW

Kim et al., 2009

P. japonicus P. ginseng

Hairy root bioreactor culture (10 L) Hairy root flask culture Mutated Ad flask culture

P. ginseng

Transgenic ginseng Ad

P. quinquefolius Escherichia coli

SC RI

NU

Hairy root flask culture

MA

P. quinquefolius

TGa TGa

PT ED

200 μM MJ

PT TGa

Hairy root culture with elicitation Hairy root bioreactor culture (5 L) Hairy root bioreactor culture with elicitation

TG

a

Agrobacterium rhizogenes

TGa

10.5 mg/g, 133 mg/L

Yu et al., 2005a

Agrobacterium rhizogenes

TGa

145.6 mg/L

Palazón et al., 2003

Agrobacterium rhizogenes

TGa

3-10 mg/g DW

Mathur et al., 2010; Kochan et al., 2012; 2013

Agrobacterium rhizogenes

TGa

6 mg/g DW

Kochan et al., 2012

Agrobacterium rhizogenes γ-irradiation

Re TGa

60 mg/g DW 82 mg/g DW

Zhang et al., 2010 Kim et al., 2013b

PgHMGR1

TGa

10-13 mg/g DW

Kim et al., 2014b

a

15-30 mg/g DW

Lee et al., 2004

a

30-35 mg/g DW

Kim et al. 2014c

573 ug/g DW, 5.2 mg/L

Han et al., 2014

-

Wang et al., 2014a

0.44-47 mg/ml

Quan et al., 2012; 2013

AC CE

Transgenic ginseng Ad

Bacte ria

TGa

9.89 mg/g DW, 83.6 mg/L 40-50 mg/g DW, 160-480 mg/L 3-4 mg/g DW, 26 mg/L

Paek al., 2009 Sivakumar et al., 2005; Paek al., 2009 Paek al., 2009 Kim et al., 2004; Ali et al., 2006a; Paek al., 2009 Paek al., 2009 Paek al., 2009; Murthy et al., 2014 Gao et al., 2005 Mallol et al., 2001; Jeong et al., 2005 Liang et al., 2014

PgSS1

TG

Transgenic hairy root

PgFPS

Transgenic tobacco cell

PgDDS

Transgenic hairy root Bioconversion from ginsenoside Rb1 or Rb2 using recombinant enzyme

PqDDS

TG Dammare nediol-II TGa

β-GH from Microbacterium

Rg3

ACCEPTED MANUSCRIPT

Saccharomyces cerevisiae

Recombinant yeast strain for the production of aglycone Recombinant yeast strain for the production of ginsenoside Recombinant yeast strain for the production of ginsenoside

Kim et al., 2013c

Cellulase-12T

Rg3

1.49% in white ginseng extract

Chang et al., 2009

SC RI

PT

144 g / 5 L using 250 g PPD ginsenosides mixture (78% conversion ratio)

β-GH from Pyrococcus furiosus

Rg1, Rh1

0.4 g/L, 2.74 g/L

Oh et al., 2014b

β-GH from Gordonia terrae

Rg3, Rg2, Rh1

1.16 mg/ml, 1.47 mg/ml, 1.17 mg/ml in 10% (w/v) ginseng extract

Shin et al., 2014

PPD, PPT

17.2 mg/L, 15.9 mg/L

Dai et al., 2014

CK

1.4 mg/L

Yan et al., 2014

Rg3

1.3 mg/L

Jung et al., 2014

NU

Yeast

Rg3

PgDDS, PPDS, PPTS from P. ginseng, and NADPH cytochrome CYP reductase from A. thaliana UGTPg1, PgDDS, PgPPDS, and truncated PgHMGR1 and UPC2 PgUGT74AE2, PgUGT94Q2, PgDDS, PgPPDS

MA

Enzymatic conversion of white ginseng extract into ginsenoside Rg3 Bioconversion from PPT-type ginsenosides using recombinant enzyme Bioconversion from ginseng root extract using recombinant enzyme

β-GH from Flavobacterium, α-L-arabinofuranosidase from Leuconostoc

PT ED

Scale-up (5 L) bioconversion using recombinant two enzymes

SC- suspension cell; Ad- adventitious root; DW - Dry weight; MJ - methyl jasmonate; HEJ - 2-hydroxyethyl jasmonate

a

AC CE

β-GH - β-glucosidase PPD - protopanaxadiol; PPT - protopanaxatriol; CK - compound K

Total ginsenosides measured by HPLC (Sum of major ginsenosides, Rb1, Rb2, Rc, Rd, Re, (Rf), and Rg1) Total ginsenosides measured by TLC colorimetric method c Total ginsenosides measured by HPLC (Sum of major ginsenosides Rb1, Rd, Re and Rg1) d Total ginsenosides measured by HPLC (Sum of major ginsenosides malonyl-Rb1, Rb1, Rb2, Rc, Rd, Re, Rg1, and R0) e Total ginsenosides measured by HPLC (Sum of major ginsenosides R1, Rb1, Re and Rg1) b

47

ACCEPTED MANUSCRIPT Figure Legends and Figures Figure 1. Life cycle of ginseng and accumulation of major ginsenosides. Bold arrow indicates life cycle of Panax ginseng plants from the seed to vegetative and reproductive stages with indicated special tissues by thin arrow. Embryo in ginseng seeds become

PT

maturation (generally 4 mm in length) and starts germination after stratification in humidified sand for three month at 5°C. One-year-old seedlings have single compound leaf with 3 leaflets attached

SC RI

in petiole, and height is about 5-10 cm. Two-year-old plants produce stem and generally have two palmate leaves and each with three to five leaflets. Subsequently, ginseng plants develop more leaves attached on longer petioles each year until the fourth to sixth growth year, and the height of mature plants is about 40-50 cm. Flowering generally starts to occur in the third year, and the

NU

berries start to develop in August and September. Usually, seeds are harvested from the fourth year, and roots are harvested between 4 and 6 years of age. Dashed red box with bold letter indicates

MA

major ginsenosides, and yellow and green color density indicates accumulation of total ginsenosides in organs during development of P. ginseng.

ED

Figure 2. Structures of different types of ginsenosides (A), and their classification based on glycosides attached (B).

PT

Glc, β-D-glucopyranosyl; Ara(p), α-L-glucopyranosyl; Ara(f), α-L-arabinofuranosyl; Rha, α-Lrhamncpyranosyl. Xyl, xylose; Ac, acetyl. +This ginsenoside is unique for processed red ginseng. Compound K, was only identified by in human tissues after oral administration.

CE

++

AC

Figure 3. Possible biosynthesis pathway for ginsenosides in major Panax species. Pg, Panax ginseng; Pq, Panax quiqnefolius; Pj, Panax japonicus; Pn, Panax notoginseng. Isopentenyldiphosphate (IPP) as triterpene structure can be formed from squalene though mevalonic acid (MVA) pathway, whereas the role of IPP from the 2-C-methyl-D-erytritol 4-phosphate (MEP) pathway in contributing to ginsenoside biosynthesis is still unclear. Blue letters in pink box indicate common enzymes in other plants and red one in green box indicates unique enzymes in ginseng. Green dashed lines represent the unique diversified pathway for each Panax species indicated as Pg, Pq, Pj, and Pn mentioned above. Major ginsenosides of each of the four types are indicated in green: protopanaxadiol (PPD)-type, protopanaxatriol (PPT)-type, oleanane-type and ocotillol-type. ++

Compound K (CK) and new compound 20S-O-β-(D-glucosyl)-dammarenediol II (DMG) were

produced in vitro in the yeast system using the identified P. ginseng glycosyltransferase (Yan et al., 2014). AACT, Acetyl-CoA C-acetyltransferase; HMG-CoA, 3-hydroxy-3-methylglutaryl-CoA; HMGS, 3-hydroxy-3-methylglutaryl-CoA synthase; HMGR, 3-hydroxy-3-methylglutaryl-CoA reductase; MVK, mevalonate kinase; MVP, mevalonate phosphate; PMK, phosphomevalonate

ACCEPTED MANUSCRIPT kinase; MVPP, diphosphomevalonate; MVD, mevalonate diphosphate decarboxylase; FPP, farsenyldiphosphate; DMAPP, dimethyl ally diphosphate; IDI, isopentenyl-diphosphate deltaisomerase; DXP, 1- deoxy-D-xylulose-5-phosphate; MEP, methylerythritol phosphate; FPS, farnesyl diphosphate synthase; SS, squalene synthase; SE, squalene epoxidase; DDS,

PT

dammarenediol synthase; β-AS, β-amyrin synthase; CAS, cycloartenol synthase; LAS, lanosterol synthase; PPDS, protopanaxadiol synthase; PPTS, protopanaxatriol synthase; CYP, cytochrome

SC RI

P450; GT, glycosyltransferase; UGT, UDP-glycosyltransferase; Dotted line displays putative pathway. Reported enzymes in ginseng are shown with NCBI accession numbers are indicated in Table 1.

NU

Figure 4. Distribution and cellular localization of ginsenoside in ginseng plants. Pink color indicates high accumulation of ginsenosides in cells. Ginsenosides are highly

MA

accumulated in epidermis in roots and vascular bundles. Ginsenosides are located in chloroplasts, peroxisomes, and cytoplasm of parenchymal cells in leaves, vacuoles and granules in vascular bundles in stem, vacuoles and vacuolar protein of parenchymal cells in roots. V = vacuole; N =

ED

nucleus.

PT

Figure 5. Approaches for the improvement of ginsenoside biosynthesis. Bioengineering strategies including suspension cell culture, adventitious root culture, chemical

CE

elicitors, transgenic plants and engineered yeast systems have been used in improving ginsenoside production. Most of these approaches can promote the expression of ginsenosides biosynthetic

AC

genes.

49

ACCEPTED MANUSCRIPT Figure 1

Berry

Inflorescence

Seed

SC RI

PT

Re

Re

Embryo Germination

NU

Re

ED

Compound leaves

PT

Rg1, Re, Rd Rg1,Re

Petiole

AC

1-year

Re, Rd, Rg1

Re, Rd, Rg1

Bud for next year sprout Rg1, Rhizome Rb1 Re

Stem Re, Rg1 Rb1

2-year

Re

Peduncle

Petiole

CE

Rg1, Re, Rb1

Re, Rd, Rg1

MA

Re

Main root

Rg1, Re, Rb1

3-year

Lateral root

4-year

70

80

90

100

110

120 mg/g

10

20

30

40

50

60 mg/g

Fine root

ACCEPTED MANUSCRIPT Figure 2 OH

A OH

20S

3

OH

12

12

R1O

20S

6

HO

3

B

6

HO

6

Oleanolic acid type R3

R4

-Glc2-1Glc

Ginsenoside Ra3

-Glc2-1Glc

Ginsenoside Rb1

-Glc2-1Glc

-

-Glc6-1Glc

-

Ginsenoside Rb2

-Glc2-1Glc

-

-Glc6-1Ara(p)

-

-Glc2-1Glc

-

-Glc6-1Xyl

-

-Glc2-1Glc

-

-Glc6-1Ara(f)

-

-Glc2-1Glc

-

-Glc

-

Ginsenoside Rg3+

-Glc2-1Glc

-

-H

-

Ginsenoside Rh2+

-Glc

-

-H

-

Ginsenoside F2

-Glc

-

-Glc

-

-Glc2-1Glc6-Ac

-

-Glc6-1Glc6-Ac

-

-H

-

-Glc

-

-

-Glc2-1Rha

-Glc

-

Ginsenoside Rf

-

-Glc2-1Glc

-Glc

-

Ginsenoside Rg1

-

-Glc

-Glc

-

Ginsenoside Rg2

-

-Glc2-1Rha

-H

-

Ginsenoside Rh1

-

-Glc

-H

-

-H

-Glc

-Glc2-1Xyl

-Glc

-Glc2-1Xyl

-H

MA

NU

Ginsenoside Ra2

ED

PT

Quinquenoside R1 K++

CE

Compound

AC

Ginsenoside Re

Ginsenoside F1 Notoginsenoside R1 Notoginsenoside R2

51

R2

3

-Glc2-1Glc

Ginsenoside Rd

Oleanolic acid type

R1O

6

COOR4

Ginsenoside Ra1

Ginsenoside Rc

Ocotillol-type

12 17

Ocotillol-type

R1

Ginsenoside Rb3

PPT type

3

OR2

Protopanaxatriol (PPT)-type

Ginsenoside

PPD type

20S

12

OR2

Protopanaxadiol (PPD)-type

O

PT

OH

24R

R3O

SC RI

R 3O

-

-Glc6-1Ara(p)4-1Xyl -Glc6-1Ara(f)2-1Xyl -Glc6-1Glc3-1Xyl

Majonoside R2

-Glc2-1Xyl

Pseudoginsenoside F11

-Glc2-1Rha

Ginsenoside RO

-Glc2-1Glc

-

-

-Glc

Ginsenoside ROA

-Glc2-1Glc

-

-

-Glc6-1Glc

ACCEPTED MANUSCRIPT Figure 3 Pq, Pn

Pg, Pq, Pj, Pn

OH

Ocotillol-type

MVA pathway

PPT-type

Glc-O OH

O OH

Cytoplasm HO O-Glc-Rha

O-Glc-Rha

Pg, Pq, Pj

Acetyl-CoA

GT?

GTs? HO OH

CO-O-Glc Glc-Glc- O

Ginsenoside RO

OH

Mevalonate (MVA)

HO OH

OH O HO

PMK

Oleanolic acid

HO

MVD IPP

IPP

ED

FPS HO

β-amyrin

UGTPg1

PPDS (CYP716A47)

CE

DDS

HO

LAS

Lanotsterol

Glc-O

HO

Glc-Glc-O

Rg3

UGTPg1

UGTPg1

Glc-O OH

CK++

Glc-O OH

UGT74AE2 PPDS Glc-O (CYP716A47)

DMG++

GTs? Glc-Glc-O OH

Glc-Glc- O

UGT94Q2 F2

Glc-Glc-O

Rd

PnUGRdGT GT?

Glc-Glc-O

Glc-Glc-O

Notoginsenoside I

Dammarenediol-type Sterols

Glc-O OH

HO

UGT71A27

Dammarenediol

HO

Cycloartenol

HO

AC

CAS

UGT94Q2

Glc-O

Rh2

HO

2,3-oxidosqualene

PPD-type HO OH

UGT74AE2

Protopanaxadiol

PT

β-AS

SE

O

Rf

HO OH

HO

Squalene

O-Glc-Glc

Rh1

HO OH

DMAPP

SS

HO O-Glc

PPTS (CYP716A53)

IDI

FPP

GT?

Protopanaxatriol

OAS (CYP716A52)

HO OH

HO

MA

MEP

GT?

HO OH

GT?

OH

MVPP

Rg2

GT?

NU

DXP

Rg1

Epoxidase?

MVP

O-Glc-Rha

O-Glc

GTs?

Chloroplast

HO

HO

HO

HMGR

MVK

HO OH

O

HMGS HMG-CoA

Glc-O OH

SC RI

Acetoacetyl-CoA

MEP pathway

Re

Pseudoginsenoside F11

Oleanane-type

AACT

PT

HO

Pn

Rb1 Glc-Glc-O

Arap-Glc-O OH

Araf-Glc-O OH

Rb2 Glc-Glc-O

Rc

GT?

ACCEPTED MANUSCRIPT Figure 4 Cuticle

Mitochondria V

Palisade parenchyma

Phloem Bundle sheath cells

Spongy mesophyll Stoma

Guard cells

Epidermis Cortex

N

Vascular bundle

SC RI

Lower epidermis

Xylem

Pith

N

ED

Endodermis

MA

Cortex

AC

CE

PT

Root hair

53

Periderm (Epidermis)

Granule

Vascular bundle

NU

Cambium

Peroxisome

Tubular element

V

Phloem

Pith

Chloroplast

Xylem

PT

Upper epidermis

V

Vacuolar protein

N Phloem Xylem Pericycle

Stele

Starch granule

ACCEPTED MANUSCRIPT Figure 5

Adventitious roots

Transgenic plants

NU

Suspension cells

SC RI

PT

Elicitators: Chitosan, SA, JA, MJ, Sucrose, Cu, Nitrate, NaCl

Yeast cells

HMG-CoA

MA

Ectopic or increased expression of key genes in ginsenoside synthetic pathway HMGR

MVA

FPS

ED

FPP

SS

Ginsenoside production

Squalene

SE

2,3-oxidosqualene

Ginsenoside Rh1

PT

DDS

Cycloartenol Dammarenediol

PPDS

AC

CE

Sterols

PPD

PPTS PPT

UGTs UGTs

Compound K