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FEBS Letters 580 (2006) 5143–5149

Dammarenediol-II synthase, the first dedicated enzyme for biosynthesis, in Panax ginseng

Pimpimon Tansakul, Masaaki Shibuya, Tetsuo Kushiro, Yutaka Ebizuka* Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan

Received 20 July 2006; revised 11 August 2006; accepted 18 August 2006

Available online 1 September 2006

Edited by Ulf-Ingo Flu¨gge

more than 4–5 years, and needs many labors and extra efforts Abstract Panax ginseng produces called , which are classified into two groups by the skeleton such as protection from direct sunlight. Isolation and purifica- of aglycones, namely type and type. Dam- tion of each ginsenoside also needs a lot of tasks. Chemical marane-type ginsenosides dominate over oleanane type not only synthesis is not practical in terms of both quantity and cost. in amount but also in structural varieties. However, their sapoge- Various biological systems, including a large-scale bioreac- nin structure is restricted to two aglycones, protopanaxadiol and tor system (1–10 tons) of adventitious root cultures [5,6], for protopanaxatriol. So far, the genes encoding oxidosqualene cy- production of ginsenosides have been developed and solved clase (OSC) responsible for formation of dammarane skeleton some problems associated with cultivation in the field. How- have not been cloned, although OSC yielding oleanane skeleton ever, ginsenoside productivity did not exceed that of the origi- (b- synthase) has been successfully cloned from this plant. nal plant. Genetic engineering of biosynthesis might be one of In this study, cDNA cloning of OSC producing dammmarane the choices to improve productivity. Overexpression of the bio- triterpene was attempted from hairy root cultures of P. ginseng by homology based PCR method. A new OSC gene (named as synthetic genes in ginseng plant would improve ginsenoside PNA) obtained was expressed in a lanosterol synthase deficient contents. Alternatively, expression of the genes for biosyn- (erg7) Saccharomyces cerevisiae strain GIL77. LC-MS and thetic enzymes in heterologous host organisms, hopefully in NMR analyses identified the accumulated product in the yeast fast growing microbes such as yeast or Escherichia coli, would transformant to be dammarenediol-II, demonstrating PNA to lead to ginsenoside production in quantities. The latter ap- encode dammarenediol-II synthase. proach would also solve the problems associated with isola- 2006 Federation of European Biochemical Societies. Published tion, as the target compounds are produced with negligible by Elsevier B.V. All rights reserved. background metabolites. Genetic engineering discussed above seems to be the most promising way to obtain ginsenosides Keywords: Ginsenoside; Dammarenediol-II; (20S)- in large quantities and for that purpose thorough understand- dammarenediol; Oxidosqualene cyclase; cDNA cloning; Panax ginseng ing of ginsenoside biosynthesis and cloning of the relevant genes are highly expected. Ginsenosides are classified into two groups by the structure of aglycones, dammarane type and oleanane type. Major ginseno- sides are dammarane type, including G-Rb1 and G-Rg1 whose genuine aglycones are protopanaxadiol and protopanaxatriol, 1. Introduction respectively. Oleanane type is represented by only one minor , G-Ro, whose aglycone is . Ginsenosides Panax ginseng is one of the most famous oriental medicinal are biosynthesized from oxidosqualene by a sequence of three plants used as crude drugs in Asian countries for more than steps, namely cyclization, hydroxylation and glycosidation 5000 years. The major pharmacologically active constituents (Fig. 1). Cyclization of oxidosqualene is a biosynthetic branch- of ginseng are triterpene saponins called ginsenosides [1]. Until ing point not only for phytosterols and , but also for now, more than 30 ginsenosides (hereafter abbreviated as dammarane and oleanane type ginsenosides. In our previous G-Xmn), such as G-Rb1,-Rg1,-Ro, etc., have been isolated [2] study, three OSC (oxidosqualene cyclase) activities yielding and studied for their biological activities. Slight difference of cycloartenol, b-amyrin and dammarenediol-II were detected in structure causes drastic change of pharmacological activity. microsomal fraction prepared from P. ginseng hairy root cul- For example, G-Rg1 shows central nervous stimulating activ- tures [7]. cDNAs of cycloartenol synthase and b-amyrin syn- ity, while G-Rb1 suppressing activity. Recently, G-Rg3,G- thase were successfully cloned by homology based PCR from Rh1 and G-Rh2 were reported to show anti-cancer activity the same source [8,9] and also from other plant species [10– and considered to be promising anti-cancer drugs [3,4]. In spite 13]. However, dammarenediol-II synthase cDNA has not been of these prospects, difficulty of their supply in pure form in obtained from any plants including P. ginseng. quantity has prevented them from development for clinical To design and materialize biological systems for efficient gin- medicines. Cultivation of P. ginseng takes long time, generally senoside production by recombinant technology as an ultimate goal, cDNA cloning of dammarenediol-II synthase, the first *Corresponding author. Fax: +81 3 5841 4744. committed enzyme for the biosynthesis of genuine E-mail address: [email protected] (Y. Ebizuka). of dammarane type ginsenosides, has been attempted in this study. Abbreviations: OSC, oxidosqualene cyclase; G-Xmn, ginsenoside

0014-5793/$32.00 2006 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. doi:10.1016/j.febslet.2006.08.044 5144 P. Tansakul et al. / FEBS Letters 580 (2006) 5143–5149

O-Glc-Glc HO

HO HO Glc-Glc-O cycloartenol β cycloartenol -sitosterol ginsenoside Rb-1 synthase OH OH PNX OH OH 21 22 OH 12 20 PNA

dammarenediol-II 6 synthase HO HO O HO OH (20S)- protopanaxadiol 2,3-oxidosqualene dammarenediol-II (20S)-protopanaxatriol PNY, PNY2 (dammarane skeleton) β-amyrin synthase O-Glc HO CO-O-Glc

HO Glc-GlcA-O 24 HO β-amyrin ginsenoside Ro (oleanane skeleton) O-Glc ginsenoside Rg-1

Fig. 1. Proposed biosynthesis of ginsenosides in P. ginseng.

2. Materials and methods modifications [8]. Used primers were as follows. For 30-RACE; PNA627S (50-ACCCTATCWGGGTTTGCTTCT-30), PNA648S (50- 0 0 2.1. Panax ginseng hairy root culture GGTGTTAAATTTTTCCTTTCAACA-3 ). For 5 -RACE; PNA- 0 0 P. ginseng Agro- 633A (5 -AGAAGCAAACCCWGATAGGGT-3 ), PNA365A The hairy root culture of was generated by infecting 0 0 0 bacterium rhizogenes [14]. The culture was maintained, in a MS liquid (5 -CTCGGTGGCACCATAGCGCATTAG-3 ), PNA356A (5 -AA- CAACTCTTTTTAGACCTCTTTT-30) and PNA340A (50-TTTGAG- medium containing 3% sucrose, on a rotary shaker at 50 rpm and 0 25 C in the dark and subcultured every 4 weeks. GAATGGTTCACTAAAGTA-3 ). To obtain the full-length clones, nested PCR was carried out. The first PCR was carried out with PNA-50N-32 (50-GACACC- 2.2. PCR and sequence analysis ACATACCAACAAGAA-3) and PNA-30C + 36 (50-TGGAATT- Synthesis of oligo DNAs was done by Nihon Bioservice (Saitama, TAGCGCATTAATTCATC-30) primers. The second PCR was carried Japan). PCR was performed by Robo Cycler Gradient 40 (Strata- out with KpnI-PNA-N (50-GAAGAGGTACCATGTGGAAGCA- gene). Sequencing was carried out by DNA Sequencer Long Read GAAGGGTGCC-30 KpnI site in bold face) and XhoI-PNA-C (50- IR 4200 (LI-COR). AAGAACTCGAGTTAAATTTTGAGCTGCTGGTG-30 XhoI site in bold face) primers. PCR was carried out with the same condition 2.3. cDNA cloning described above, except that the annealing temperature was 52 C. RNA was extracted by phenol-SDS method from the 28 day old The 2.3-kb PCR product was digested with KpnI and XhoI and hairy root culture by the same method as previously reported [8]. ligated into the corresponding sites of pYES2 (Invitrogen) to con- RNA was reverse transcribed to produce cDNAs using reverse struct the plasmid pYES2-PNA. The obtained full length cDNA transcriptase (Superscript II, Invitrogen) and oligo dT primer, clone, PNA was sequenced in both strands. This sequence is available RACE32 (50-GACTCGAGTCGACATCGATTTTTTTTTTTTTT-30) under Accession No. AB265170 in DDBJ sequence-database and with dNTP (0.2 mM) in a volume of 20 ll following the manufacturer’s also available in Japanese patent applications (Jan. Kokai Tokkyo protocol. The resulting cDNA mixture was used as the template in the Koho) under application number 2002-223787 (applied date: July 31, following PCR. 2002). PCRs were performed with a set of primer, 463S (50- MGICAYATHWSIAARGGIGCITGG-30) and 711A (50-CKRTAY- TCICCIARIGCCCADATIGGRAA-30)(1lg of primers were used 2.4. Expression in Saccharomyces cerevisiae in all PCR procedures for cloning), using Ex Taq. DNA polymerase The plasmid pYES2-PNA was transferred to S. cerevisiae strain (Takara Biochemical) with dNTP (0.2 mM) in a final volume of GIL77 [8] using Frozen-EZ Yeast Transformation II kit (ZYMO 100 ll following the manufacturer’s protocol. PCR was carried out RESEARCH). The transformant was inoculated in 20 ml synthetic for 30 cycles with a program (94 C, 1 min, 42 C, 2 min, 75 C, complete medium without uracil (SC-U), containing ergosterol 3 min, and final extension at 75 C, 10 min). The PCR product (20 lg/ml), hemin chloride (13 lg/ml) and Tween 80 (5 mg/ml), and (860 bp) was subcloned to the plasmid vector (pT7-Blue, Novagen). incubated at 37 C for 2 days. Media were changed to SC-U with Seventeen colonies were picked up in total, and nucleotide sequence the same supplements and 2% galactose in place of glucose. Cells were was determined. A new gene (2 clones, named as PNA) was cloned collected and resuspended in the same volume of 0.1 M potassium together with known genes (PNX, PNY and PNZ). phosphate buffer (pH 7.0) supplemented with 2% glucose and hemin Based on the sequence of PNA, specific primers were designed to ob- chloride (13 lg/ml) and further incubated for 24 h at 30 C. Cells were tain the 30 and 50 end sequences. RACE PCRs were carried out follow- collected and refluxed with 2 ml of 20% KOH/50% EtOH aq. for ing the method described in our previous paper with some 5 min. After extraction with the same volume of hexane, the extract P. Tansakul et al. / FEBS Letters 580 (2006) 5143–5149 5145

12 was concentrated and applied onto TLC plate (Merck #11798) which was developed twice with benzene:acetone = 19:1 and visualized with phosphomolybdic acid (Fig. 2). Oxidosqualene 2.5. Products determination by LC-MS and NMR The band migrating just below ergosterol was scraped off the plate and extracted with acetone. The extract was concentrated and applied Dioxidosqualene to LC-MS (LCQ, Thermo Quest) equipped with a HPLC (Agilent, 1100 series) and an ODS-80Tm column (4.6 · 250 mm, Tosoh) main- tained at 40 C, eluted with 90% CH3CN at 1.0 ml/min, and monitored by UV 202 nm absorbance (Fig. 3). Under this condition, (20S)-epimer (dammarenediol-II) can be separated from (20R)-epimer (dammarene- diol-I) with baseline resolution (Rts for 20S; 15 min and for 20R; 16 min) [7]. In APCIMS analysis, the authentic dammarenediol-II gave no molecular ion peak, but the ion peaks at m/z 427 and 409 which come Ergosterol from [M+H]+ by elimination of one and two water molecule(s), respec- tively (data not shown). MS/MS spectra with m/z 427 as the parent ion showed a dominant peak at m/z 409, dehydration fragment peak, with- PNA product out other significant peaks (data not shown). MS/MS spectra with m/z 409 as the parent ion showed a complex fragmentation pattern which is identical to that of the PNA product as shown in Fig. 4.

2.6. NMR analysis of the PNA products accumulated in GIL77 transformants For preparative scale culture of the transformant with pYES2-PNA, 6-L culture was prepared. Induction and resting cultures were per- Fig. 2. TLC analysis of PNA product. Hexane extracts from trans- formed as described above. After refluxing with 100 ml of 20% formed cells by void vector (lane 1) and by the vector harboring PNA KOH/50% EtOH aq. for 1 h, the mixture was extracted with 100 ml (lane 2) were applied onto a silica gel TLC plate, which was developed of hexane for three times, combined and concentrated. The extract twice with benzene–acetone (19:1) and visualized with phosphomo- was purified by silica gel column chromatography with benzene–ace- lybdic acid. tone as an eluent. The fractions showing a slightly polar spot than that of ergosterol on TLC were collected (1 mg).

15.05 A 0.25

0.20

0.15

0.10

UV absorbance (AU) 0.05

0.0 5 10 115 20 25 Time (min)

15.03 B 0.25

0.20

0.15

0.10

UV absorbance (AU) 0.05

0.0 5 10 15 20 25 Time (min)

Fig. 3. HPLC profile of PNA product. After separated by TLC, PNA product (A) was analyzed by HPLC with monitoring of UV absorbance at 202 nm. Profile of authentic dammarenediol-II was shown in (B). 5146 P. Tansakul et al. / FEBS Letters 580 (2006) 5143–5149

409.3 A 100

217.1

191.0 257.1

203.1 271.1 50 285.1 231.1

Relative intensity (%) 299.1 353.2

243.1

149.0 189.1 311.2 339.2 325.2 163.0177.0 367.1 133.2147.0

119.1 391.1408.6 410.1 0 150 200 250 300 350 400 450 500 m/z

409.3 B 100

217.1 191.1

257.1 271.1

50 203.1 285.1 231.1 Relative intensity (%) 299.1 245.1 353.2 189.1 311.1 177.0 325.2339.2 149.1 367.2 135.0 121.1 119.2 408.7 381.0 415.8 0 150 200 250 300 350 400 450 500 m/z

Fig. 4. LC-MS analysis of PNA product. MS/MS spectra from the peak of m/z = 409 as a parent ion were shown in (A) (PNA product) and (B) (authentic dammarenediol-II).

1 13 H and C NMRs were measured in CDCl3 (JEOL JSX-500) with 3. Results and discussion TMS as an internal standard. 1 H NMR (500 MHz, CDCl3): d 0.77 (3H, s), 0.84 (3H, s), 0.87 (3H, 3.1. RNA and cDNA preparation s), 0.96 (3H, s), 0.97 (3H, s), 1.14 (3H, s), 1.62 (3H, s), 1.70 (3H, s), 3.20 (1H, dd, J = 5.0, 10.5 Hz), 5.12 (1H, t, J = 7.0 Hz). As time course study of ginsenoside production in hairy root 13 C NMR (125 MHz, CDCl3): d 15.33, 15.47, 16.20, 16.43, 17.70, cultures showed that dammarane type ginsenosides start to 18.25, 21.51, 22.53, 24.79 (C-21), 25.38, 25.74, 27.38, 27.52, 27.98, accumulate on day 17 and reach their maximum on day 27 31.16, 35.20, 37.10, 38.96, 39.01, 40.34, 40.45 (C-22), 42.26, 49.82, after inoculation to a new medium (unpublished result), we ex- 50.28, 50.60, 55.82, 75.42, 78.96, 124.61, 131.61. All spectra were identical to those reported for dammarenediol-II pected the presence of ample transcripts of dammarenediol-II [15,16]. C-21 and C-22 of dammarenediol-II (underlined) resonate at synthase gene on day 21. Therefore, in our previous study, distinct chemical shifts from those, 23.5 (C-21) and 41.8 (C-22) ppm, RNA was prepared from cultures on day 21. Although cyclo- of dammarenediol-I [16]. artenol synthase (PNX) [8], b-amyrin synthase (PNY [8] and P. Tansakul et al. / FEBS Letters 580 (2006) 5143–5149 5147

PNY2 [9]) and lanosterol synthase (PNZ) [17] genes were MS. A peak at 15.05 min, the same retention time with authen- cloned from this RNA preparation by PCRs, dammarene- tic dammarenediol-II (Fig. 3), showed the same MS fragmen- diol-II synthase cDNA was not obtained. One of the possible tation pattern (Fig. 4), indicating the accumulated product to reasons for this might be that the transcription of dammarene- be dammarenediol-II. diol-II synthase gene has already started on day 21 but has not In order to obtain rigorous proof of structure, the product exceeded those of other OSCs in quantity. In this study, there- (1 mg) was prepared by silica gel column chromatography fore, we waited one more week for RNA preparation, from from 6 L culture, and subjected to 1H and 13C NMR analyses. day 21 to day 28, to clone dammarenediol-II synthase. The results clearly proved that the product is dammarenediol- II, and thus PNA to encode a dammarenediol-II synthase. 3.2. Amplification of DNA fragment using degenerate primers Both stereoisomers, 20S and 20R, of dammarenediol have PCRs with four combinations (162S-623A, 162S-711A, been reported as natural products. Most ginsenosides have S 463S-923A and 463S-711A) of degenerate primers designed configuration at C-20. Our previous study demonstrated that from the conserved sequences of the known oxidosqualene cyc- dammarenediol produced by microsomal fraction prepared lases [8] were carried out. PCR products were subcloned to from P. ginseng hairy root cultures is a 20S isomer and no plasmid vector, and sequenced. Among the PCR products trace of 20R isomer was detected [7]. However, this result amplified with 463S and 711A primers, one new sequence was obtained with crude microsomal proteins and there left (860 bp in length and named PNA) was found, which shows some ambiguity as regard the presence of dammarenediol-II 65% identities to PNY (b-amyrin synthase of P. ginseng)in synthase in this plant. Successful cloning of dammarenediol- amino acid level. II synthase cDNA in this study provided a rigorous proof for the presence of dammarenediol-II synthase in P. ginseng. 3.3. Sequence of PNA As mentioned above, PNA yielded dammarenediol-II, (20S)- Sequence of full length PNA was obtained by RACE meth- isomer, as a sole product, demonstrating that water addition to od [18]. It contained an ORF of 2310 nucleotides encoding a dammarenyl cation is stereospecific. It is important to note polypeptide of 770 amino acids with a predicted molecular that quenching of carbocation by water addition, which termi- mass of 88.3 kDa. Among the known triterpene synthases, P. nates cyclization reaction, is precisely controlled by the en- ginseng b-amyrin synthase (PNY) shows the highest identity zyme. So far, three OSCs including PNA have been cloned value (57%) to PNA. However, high identity of 82% between which terminate the reaction by water addition. LUP1 from two b-amyrin synthase isozymes, PNY and PNY2, of P. gin- Arabidopsis thaliana, a low fidelity synthase, produces seng suggested the enzyme function of PNA to be different lupanediol (Fig. 5) in addition to lupeol in nearly equal from b-amyrin synthase. amount [19]. Although its product specificity is leaky, water addition to lupenyl cation to produce lupanediol is stereospe- 13 3.4. Expression of PNA in Saccharomyces cerevisiae cific as demonstrated by feeding of [1,2- C2]acetate to LUP1 The DNA fragment of ORF was amplified by PCR with yeast transformant [20]. Recently, another triterpene synthase primers based on the obtained sequence and ligated into the from A. thaliana was identified to produce arabidiol, a tricyclic multi-cloning site of yeast expression vector pYES2 (Invitro- triterpene-diol [21]. Arabidiol (Fig. 5) is a single diastereomer gen) under the control of GAL1 promoter to yield pYES2- of (3S,13R)-malabarica-17,21-dien-3,14-diol, although its con- PNA. Lanosterol synthase deficient S. cerevisiae strain figuration at C-14 has not been determined. It is noteworthy GIL77 [8] was transformed with pYES2-PNA. that these three triterpene-diol synthases strictly control the The transformant was cultured for 2 days. After induction of direction of water addition to produce only one diastereomer. GAL1 promoter by addition of galactose, cells were further In other words, in the active site of these enzymes, the position incubated in 0.1 M potassium phosphate for 1 day. Then, cells of water that terminates the reaction, is rather restricted. Un- were collected, lysed and extracted with hexane. The hexane ique mechanism of these clones to terminate the cyclization by extract was analyzed by silica gel TLC. As shown in Fig. 2, water addition without hydride and methyl shifts and skeletal a new spot with the same Rf value with that of authentic rearrangements might suggest a common evolutional path for dammarenediol-II appeared just below ergosterol. these enzymes. However, these three OSCs did not form a branch but are dispersed in a phylogenetic tree constructed 3.5. Product identification by LC-MS and NMR with the known OSCs (data not shown). Further structural The band corresponding to the PNA product was scraped studies would disclose the intricate mechanism displayed by off the plate, extracted with acetone, and analyzed by LC- these enzymes.

HO

14 OH HO HO

lupanediol arabidiol

Fig. 5. Triterpene-diols directly produced by oxidosqualene cyclases. 5148 P. Tansakul et al. / FEBS Letters 580 (2006) 5143–5149

3.6. Dammarenediol-II synthase in P. ginseng Acknowledgements: A part of this research was supported by a Grant- Dammarenediol-II synthase cDNA (PNA) was successfully in-Aid for Scientific Research (S) (No. 15101007) to Y.E. from Japan cloned from P. ginseng hairy root cultures on day 28. RT– Society for the Promotion of Science, Japan. P.T. was a recipient of scholarship from the Ministry of Education, Culture, Sports, Science PCR with RNA prepared from those on day 21 with the spe- and Technology of Japan. cific primers amplified the PNA fragment, but its quantity was quite low (data not shown). Although a precise time course expression study is lacking, not enough accumulation of References PNA transcript on day 21 might be the reason why we failed to clone PNA using the degenerate primers in our previous [1] Shibata, S., Tanaka, O., Soma, K., Iida, Y., Ando, T. and study. Nakamura, S. (1965) Studies on saponins and sapogenin of ginseng, the structure of . Tetrahedron Lett. 6, 207– Recently, EST clone with identical sequence to PNA was iso- 213. lated from flower bud (GenBank, AB122080) and annotated as [2] Shibata, S. (2001) Chemistry and cancer preventing activities of b-amyrin synthase. Now that its function is established in this ginseng saponins and some related triterpenoid compounds. J. study, it should be corrected to dammarenediol-II synthase. Korean Med. Sci. 16 (s), s28–s37. [3] Shinkai, K., Akedo, H., Mukai, M., Imamura, F., Isoai, A., The presence of dammarenediol-II synthase gene in flower Kobayashi, M. and Kitagawa, I. (1996) Inhibition of in vitro buds well accounts for the isolation of dammarane type ginse- tumor cell invasion by ginsenoside Rg3. Jap. J. Cancer Res. 87, nosides from this part of the plant [22–24]. 357–362. In a recent report, four EST clones isolated from the library [4] Iishi, H., Tatsuta, M., Baba, M., Uehara, H., Nakaizumi, A., prepared from in vitro cultured seedlings are annotated as Shinkai, K., Akedo, H., Funai, H., Ishiguro, S. and Kitagawa, I. (1997) Inhibition by ginsenoside Rg3 of bombesin-enhanced OSC. High content of ginsenosides in this cultures suggests peritoneal metastasis of intestinal adenocarcinomas induced by the presence of PNA among them, although their sequences azoxymethane in Wistar rats. Clin. Exp. Metast. 15, 603–611. were not indicated in the paper [25]. [5] Choi, S.M., Son, S.H., Yun, S.R., Kwon, O.W., Seon, J.H. and Genome sequence analysis revealed the presence of 13 OSC Paek, K.Y. (2000) Pilot-scale culture of adventitious roots of ginseng in a bioreactor system. Plant Cell Tissue Organ Cult. 62, genes in A. thaliana. 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J. and protopanaxatriol, the aglycones of ginsenosides, could Biochem. 267, 3453–3460. be produced by yeast expression system if the genes of relevant [11] Hayashi, H., Huang, P., Kirakosyan, A., Inoue, K., Hiraoka, N., (C-12 and C-6) hydroxylases become available. Ikeshiro, Y., Kushiro, T., Shibuya, M. and Ebizuka, Y. (2001) In addition to production of ginsenoside aglycones by Cloning and characterization of a cDNA encoding b-amyrin synthase involved in glycyrrhizin and soyasaponin biosynthesis in heterologous expression, dammarenediol-II synthase cDNA licorice. Biol. Pharm. Bull. 24, 912–916. could be utilized for high production of ginsenosides in gin- [12] Zhang, H., Shibuya, M., Yokota, S. and Ebizuka, Y. (2003) seng plant itself. The aglycones of dammarane type ginseno- Oxidosqualene cyclases from cell suspension cultures of Betula sides, protopanaxadiol and protopanaxatriol, are rarely platyphylla var. japonica: molecular evolution of oxidosqualene cyclases in higher plants. 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