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Proc Natl Acad Sci U S A. Sep 16, 2008; 105(37): 14204–14209.
Published online Sep 8, 2008. doi:  10.1073/pnas.0803876105
PMCID: PMC2532699
Plant Biology

Licorice β-amyrin 11-oxidase, a cytochrome P450 with a key role in the biosynthesis of the triterpene sweetener glycyrrhizin


Glycyrrhizin, a major bioactive compound derived from the underground parts of Glycyrrhiza (licorice) plants, is a triterpene saponin that possesses a wide range of pharmacological properties and is used worldwide as a natural sweetener. Because of its economic value, the biosynthesis of glycyrrhizin has received considerable attention. Glycyrrhizin is most likely derived from the triterpene β-amyrin, an initial product of the cyclization of 2,3-oxidosqualene. The subsequent steps in glycyrrhizin biosynthesis are believed to involve a series of oxidative reactions at the C-11 and C-30 positions, followed by glycosyl transfers to the C-3 hydroxyl group; however, no genes encoding relevant oxidases or glycosyltransferases have been identified. Here we report the successful identification of CYP88D6, a cytochrome P450 monooxygenase (P450) gene, as a glycyrrhizin-biosynthetic gene, by transcript profiling-based selection from a collection of licorice expressed sequence tags (ESTs). CYP88D6 was characterized by in vitro enzymatic activity assays and shown to catalyze the sequential two-step oxidation of β-amyrin at C-11 to produce 11-oxo-β-amyrin, a possible biosynthetic intermediate between β-amyrin and glycyrrhizin. CYP88D6 coexpressed with β-amyrin synthase in yeast also catalyzed in vivo oxidation of β-amyrin to 11-oxo-β-amyrin. CYP88D6 expression was detected in the roots and stolons by RT-PCR; however, no amplification was observed in the leaves or stems, which is consistent with the accumulation pattern of glycyrrhizin in planta. These results suggest a role for CYP88D6 as a β-amyrin 11-oxidase in the glycyrrhizin pathway.

Keywords: expressed sequence tag, medicinal plant, secondary metabolite, monooxygenase, isoprenoid

Triterpene saponins belong to a class of natural plant products that includes various bioactive compounds found in medicinal plants (1). The roots and stolons of Glycyrrhiza plants (G. uralensis Fisch. and G. glabra L., Fabaceae), which are among the most important crude drugs in the world (2), and contain a large amount (2–8% of the dry weight) of glycyrrhizin, an oleanane-type triterpene saponin.

Because of its sweet taste, glycyrrhizin is used worldwide as a natural sweetener and flavoring additive. Moreover, various pharmacological activities of glycyrrhizin, including anti-inflammatory (3), immunomodulatory (4), antiulcer (5), and antiallergy activities (6), have been reported. Glycyrrhizin also has antiviral activity against various DNA and RNA viruses (reviewed in ref. 7), including HIV (8, 9) and severe acute respiratory syndrome (SARS)-associated coronavirus (10). Glycyrrhizin has been used in Japan for more than 20 years as a hepatoprotective agent for chronic hepatitis (11, 12). Therefore, a large amount of licorice and its extracts are on the world market as sweetening agents and medicinal materials; in fact, the annual value of global trade in licorice root is estimated at over US $42.1 million (13).

Although the various pharmacological properties of glycyrrhizin have been extensively studied, its biosynthesis remains poorly understood. The biosynthesis of glycyrrhizin (Fig. 1) involves the initial cyclization of 2,3-oxidosqualene, a common precursor of both triterpenes and phytosterols (14), to the triterpene β-amyrin, followed by a series of oxidative reactions at positions C-11 (two-step oxidation) and C-30 (three-step oxidation) and glucuronylation of the C-3 hydroxyl group, although the precise order of intermediate production is unknown.

Fig. 1.
Proposed pathway for the biosynthesis of glycyrrhizin. A cartoon depiction of the proposed pathway for the biosynthesis of glycyrrhizin. The structures of the possible biosynthetic intermediates between β-amyrin (1) and glycyrrhizin (9) are shown: ...

Two genes that encode enzymes involved in the early stages of triterpene skeleton formation, namely squalene synthase (SQS) and β-amyrin synthase (bAS), have been functionally isolated from G. glabra (15, 16); however, no progress has been made toward the identification of genes involved in the multiple oxidations and glycosylations leading from β-amyrin to glycyrrhizin.

Cytochrome P450s play critical roles in oxidative reactions during the biosynthesis of diverse natural plant products, including terpenoids. Recent gene discovery efforts using functional genomic-based approaches identified several P450s responsible for the production of terpenoids with particular importance for human health, such as Taxol, a highly effective anticancer drug derived from Taxus species (17, 18), and artemisinin, an antimalarial sesquiterpene lactone from Artemisia annua (19, 20).

As a resource for gene discovery in glycyrrhizin biosynthesis, we have generated an EST library from the stolons of G. uralensis plants, comprising approximately 56,000 cDNAs, which were assembled into 10,372 unique sequences and annotated (H. Sudo, H. Seki, N. Sakurai, H. Suzuki, D. Shibata, A. Toyoda, Y. Totoki, Y. Sakaki, O. Iida, M. Kojoma, T. Shibata, T.M., and K.S., unpublished work). In this study, we used our collection of licorice ESTs to search for glycyrrhizin-biosynthetic genes. Mining of our EST dataset for putative P450 genes and subsequent transcript profiling-based selection of the candidate P450s led to the identification of CYP88D6. CYP88D6 was shown in vitro and in vivo (in bAS-expressing yeast) to catalyze two sequential oxidation steps in the glycyrrhizin pathway: the conversion of β-amyrin (compound 1 in Fig. 1) to 11-oxo-β-amyrin (compound 4 in Fig. 1) via 11α-hydroxy-β-amyrin (compound 2 in Fig. 1). CYP88D6 represents a unique subfamily of CYP88 enzymes that appears to be restricted to the Fabaceae. The results obtained in this study not only provide insight into the production of a wide variety of triterpene saponins within the Fabaceae, they also reveal the potential of using yeast cells in the production of valuable triterpenoids.


Selection of Candidate P450s.

Keyword searches of our annotated ESTs identified 37 unique P450 contigs (H. Sudo, H. Seki, N. Sakurai, H. Suzuki, D. Shibata, A. Toyoda, Y. Totoki, Y. Sakaki, O. Iida, M. Kojoma, T. Shibata, T.M., and K.S., unpublished work). To narrow down the candidate P450s, a transcript profiling approach was taken. The expression of each P450 gene was monitored both in the glycyrrhizin-accumulating underground organs (stolons and roots) and the aboveground organs (leaves and stems) where no glycyrrhizin can be detected. Five candidate P450s representing four distinct families (CYP72, CYP83, CYP88, and CYP714) were initially selected because their expression profiles were consistent with the organ-specific accumulation pattern of glycyrrhizin.

In Vitro CYP88D6 Enzymatic Activity Assay.

We first examined the potential β-amyrin oxidase activity of all five candidate P450s using in vitro assays. For this purpose, microsomes from Spodoptera frugiperda 9 (Sf9) insect cells expressing each of the candidate P450s were assayed in vitro with β-amyrin (1) followed by gas chromatography–mass spectrometry (GC-MS). Of the five candidates, only CYP88D6 (according to the recommendation of the Cytochrome P450 Nomenclature Committee) showed clear β-amyrin-oxidizing activity.

Fig. 2A shows the CYP88D6-dependent formation of two GC-MS-detectable compounds. The retention time (Rt) and mass spectrum of each major (peak 2: Rt = 18.8 min; Fig. 2D) and minor product (peak 1: Rt = 15.7 min; Fig. 2C) showed excellent matches to those of authentic 11-oxo-β-amyrin (4) and 11α-hydroxy-β-amyrin (2) [supporting information (SI) Fig. S1], respectively. In contrast, the peaks were not detected in assays using microsomes from empty vector control Sf9 cells (Fig. 2B). These results indicate that CYP88D6 mainly catalyzes the sequential two-step oxidation of β-amyrin (1) at the C-11 position to yield 11-oxo-β-amyrin (4), a possible biosynthetic intermediate of glycyrrhizin.

Fig. 2.
In vitro oxidation of β-amyrin at C-11 by CYP88D6. GC-MS analysis (total ion chromatograms) of the reaction products resulting from in vitro assays containing β-amyrin (1) as the substrate and microsomal fractions isolated from (A) CYP88D6-expressing ...

The enzymatic activity of CYP88D6 was examined further using 30-hydroxy-β-amyrin (3) and 11-deoxoglycyrrhetinic acid (6) as potential substrates. When 30-hydroxy-β-amyrin (3) was used as the substrate, two compounds were formed within the CYP88D6-containing microsomes (Fig. 3A). The Rt and mass spectrum of each major (peak 1: Rt = 18.6 min; Fig. 3C) and minor product (peak 2: Rt = 22.6 min; Fig. 3D) showed excellent matches to those of authentic 11α,30-dihydroxy-β-amyrin (5) and 30-hydroxy-11-oxo-β-amyrin (7) (Fig. S1), respectively. In contrast, the peaks were not detected in assays using microsomes from empty vector control Sf9 cells (Fig. 3B). These results indicate that in vitro, CYP88D6 primarily catalyzes a single oxidation event when 30-hydroxy-β-amyrin (3) is used as the substrate. Moreover, no enzymatic activity was found when CYP88D6-containing microsomes were assayed with 11-deoxoglycyrrhetinic acid (6) as the substrate (data not shown).

Fig. 3.
In vitro oxidation of 30-hydroxy-β-amyrin at C-11 by CYP88D6. GC-MS analysis (total ion chromatograms) of the reaction products resulting from in vitro assays containing 30-hydroxy-β-amyrin (3) as the substrate and microsomal fractions ...

Because some members of the CYP88A subfamily, including those from Arabidopsis, barley, and pea, have been biochemically characterized as ent-kaurenoic acid (diterpenoid) oxidases that function in gibberellin biosynthesis (21, 22), we also examined the potential ent-kaurenoic acid oxidase activity of CYP88D6. However, CYP88D6-mediated conversion of ent-kaurenoic acid to further oxidized diterpene products was not observed in our in vitro enzymatic activity assays (data not shown).

In Vivo CYP88D6 Enzymatic Activity Assay.

To verify the results of our in vitro assays (Fig. 2), we examined the β-amyrin oxidase activity of CYP88D6 in an engineered yeast strain that produces β-amyrin endogenously. For this purpose, bAS from Lotus japonicus (LjOSC1) (23) was expressed under the control of a constitutive promoter (ADH1) in the wild-type yeast strain BJ2168. Subsequently, CYP88D6 was co-expressed with cytochrome P450 reductase (CPR) from L. japonicus (named LjCPR1, GenBank accession no. AB433810; T. Akashi, T. Nakao, T. Aoki, and S. Ayabe, unpublished work) as a redox partner in LjOSC1-transformed yeast. The expression of LjCPR1 and CYP88D6 was under the control of the galactose-inducible promoters GAL10 and GAL1, respectively. The overexpression of a plant-derived CPR is important for efficient electron transfer to plant P450s in recombinant yeast (24).

Following the culture of the transgenic yeast strain in medium containing galactose to induce the expression of LjCPR1 and CYP88D6, ethyl acetate extracts of the cells were analyzed.

As shown in Fig. 4A, the LjOSC1/LjCPR1/CYP88D6-expressing yeast strain (OSC1/CPR/CYP88D6) converted β-amyrin to 11-oxo-β-amyrin (4) as the major product (peak 2: Rt = 21.5 min) and 11α-hydroxy-β-amyrin (2) as the minor product (peak 1: Rt = 17.8 min). Both compounds were unique to the yeast strain expressing LjOSC1, LjCPR1, and CYP88D6; the expression of LjOSC1 and LjCPR1 (OSC1/CPR) alone did not yield detectable levels of either compound (Fig. 4B).

Fig. 4.
In vivo formation of 11-oxo-β-amyrin in yeast cells co-expressing β-amyrin synthase (LjOSC1) and CYP88D6. GC-MS analysis (total ion chromatograms) of ethyl acetate extracts from yeast strains expressing (A) LjOSC1, LjCPR1, and CYP88D6 ...

We also analyzed the culture medium of the LjOSC1/LjCPR1/CYP88D6-expressing cells (Fig. S2A), and found that nearly 40% of the 11-oxo-β-amyrin synthesized was secreted into the medium, whereas only 4–8% of β-amyrin was present in the culture medium. The final yields of 11-oxo-β-amyrin and 11α-hydroxy-β-amyrin at 2 d of culture after galactose-induction were approximately 1.6 and 0.2 mg/l, respectively.

The yeast derived 11-oxo-β-amyrin (4) and 11α-hydroxy-β-amyrin (2) were purified on a silica gel column, and their identities were confirmed by NMR analyses (see SI Materials).

The identification of the LjOSC1/LjCPR1/CYP88D6-expressing yeast-derived products as 11-oxo-β-amyrin (4) and 11α-hydroxy-β-amyrin (2) indicates that CYP88D6 is capable, in two steps, of oxidizing β-amyrin to 11-oxo-β-amyrin (4), with 11α-hydroxy-β-amyrin (2) as a probable reaction intermediate. Small amounts of the reaction intermediate might be released as a general consequence of catalysis.

Expression of CYP88D6.

The organ specificity of CYP88D6 expression was analyzed by RT-PCR (Fig. 5). Transcripts of CYP88D6 were detected in the roots and stolons, whereas no transcripts were observed in the leaves or stems. These results are similar to those for bAS, and are consistent with the fact that glycyrrhizin accumulates predominantly in the underground parts of Glycyrrhiza plants. These observations, combined with the data from our functional analyses, strongly suggest a key role for CYP88D6 as a β-amyrin 11-oxidase in glycyrrhizin biosynthesis.

Fig. 5.
Expression pattern of CYP88D6. RT-PCR analysis of β-amyrin synthase (bAS) and CYP88D6 mRNA levels in the stolons, roots, leaves, and stems. β-tubulin was included as an internal control.

CYP88D Is a Fabaceae-Specific Subfamily.

Although the 493-residue sequence of CYP88D6 showed the highest degree of similarity to an unnamed protein product predicted from the genome of Vitis vinifera (53% identity; CAO70436) in a BLAST search, the P450 nomenclature committee (c/o Dr. D.R. Nelson) informed us that CYP88D6 showed 60% identity to CYP88D1, which corresponds to a predicted P450 from Medicago truncatula (ABE91093), although the record of ABE91093 was recently withdrawn from GenBank for unknown reasons.

In terms of enzymes of known function, CYP88D6 is most similar to the ent-kaurenoic acid oxidases CYP88A6 (50% identity) from pea (named PsKAO1) (22) and CYP88A4 (49% identity) from Arabidopsis (named AtKAO2) (21), both of which function in the biosynthesis of gibberellins.

Different from the CYP88A subfamily of enzymes, which are distributed widely among plants, the CYP88D subfamily appears to be restricted to the Fabaceae and is not found in Arabidopsis or rice (25). Extensive searches of plant EST databases (see SI Methods) identified several partial cDNA sequences potentially encoding additional CYP88D subfamily members from M. truncatula and L. japonicus; however, no ESTs encoding CYP88D1 were identified. These partial sequences were extended to full-length by RACE PCR, which resulted in the identification of four ORFs encoding additional CYP88D subfamily members: CYP88D2 (62% sequence identity with CYP88D6) and CYP88D3 (79% identity) of M. truncatula, and CYP88D4 (71% identity) and CYP88D5 (72% identity) of L. japonicus. Phylogenetic analysis (Fig. 6) showed that CYP88D1-D6 are grouped separately from the CYP88A enzymes and other CYP88 family proteins of unknown function, including CYP88B1 from tomato and CYP88E1 from Selaginella moellendorffii (the primary sequences of CYP88B1 and CYP88E1 may be found at http://drnelson.utmem.edu/BiblioD.html).

Fig. 6.
Phylogenetic relationships among the CYP88 proteins. A phylogenetic tree was generated based on a comparison of the CYP88 primary sequences using the ClustalW program (26). The arrowhead indicates the P450 functionally defined in this study, and the bracket ...


In this study, we used an EST-based approach to identify CYP88D6, a P450 family gene that encodes β-amyrin 11-oxidase, which functions in the glycyrrhizin pathway.

Glycyrrhizin is biosynthesized via a series of oxidative reactions at the C-11 and C-30 positions of β-amyrin. The co-occurrence of two possible biosynthetic intermediates in planta, 11-oxo-β-amyrin (4) and 11-deoxoglycyrrhetinic acid (6) (K.O., K.S., and T.M., unpublished results), indicates that more than one biosynthetic route likely exists from β-amyrin to glycyrrhizin in terms of the order of oxidation at C-11 and C-30 (Fig. 1). Biochemical analyses suggest that CYP88D6 primarily catalyzes the sequential two-step oxidation of β-amyrin (1) at C-11 to yield 11-oxo-β-amyrin (4) (Figs. 2 and and4).4). Note that CYP88D6 readily catalyzed the hydroxylation of 30-hydroxy-β-amyrin (3) at C-11, but failed to convert the resultant compound, 11α,30-dihydroxy-β-amyrin (5), to the ketone 30-hydroxy-11-oxo-β-amyrin (7) with appreciable efficiency (Fig. 3). Moreover, CYP88D6 exhibited no enzymatic activity when 11-deoxoglycyrrhetinic acid (6) was used as the substrate (data not shown). These results suggest that the preferred route of glycyrrhizin biosynthesis involves oxidation at C-11 first; however, a more detailed and comparative biochemical characterization of CYP88D6 and the as yet unidentified enzyme(s) that catalyzes the oxidation at C-30 are necessary to verify this speculation.

In this study, we revealed a function of CYP88 family proteins as triterpene-oxidizing enzymes. Some members of the CYP88A subfamily, including those from Arabidopsis, barley, and pea, have been biochemically characterized as ent-kaurenoic acid (diterpenoid) oxidases that function in gibberellin biosynthesis (2629, 22). Different from the CYP88A subfamily enzymes, which are found in a variety of plants, the CYP88D subfamily appears to be restricted to the Fabaceae. Based on extensive searches of plant EST databases and subsequent RACE PCR analyses, we identified two additional CYP88D subfamily members each from M. truncatula (CYP88D2 and 88D3) and L. japonicus (CYP88D4 and 88D5). The functional identification of CYP88D6 hints at the involvement of other P450s in the CYP88D subfamily in the biosynthesis of triterpene saponins. Because, to our knowledge, no oleanane-type triterpene aglycones containing a C-11 carbonyl group have been identified from M. truncatula or L. japonicus, these additional CYP88D subfamily members may each act on β-amyrin or related triterpene substrates with unique reaction specificity. Recently, Shibuya et al. (30) identified CYP93E1, a cytochrome P450 from soybean that participates in soyasaponin biosynthesis as a β-amyrin and sophoradiol 24-hydroxylase. By using a PCR-based method, we isolated a cDNA from G. uralensis encoding a homolog (named CYP93E3; 82% identity) of CYP93E1. Moreover, our in vitro enzymatic activity assay showed that CYP93E3 possesses β-amyrin 24-hydroxylase activity (Fig. S3). These observations clearly indicate that in G. uralensis and most probably in other legumes as well, the P450 enzymes involved in triterpene saponin biosynthesis are recruited in at least two very distant CYP families, CYP93 (CYP71 clan) of the A-type P450s and CYP88 (CYP85 clan) of the non-A type P450s. Plant P450s are generally assigned to two major clades: A-type and non-A type (31). Another example of the convergent evolution of P450s comes from the biosynthesis of gibberellins in higher plants. In Arabidopsis, CYP701A3 and CYP88As (CYP88A3 and A4) have been shown to catalyze very similar enzymatic reactions and two successive steps in the gibberellin biosynthetic pathway (21, 29, 32); however, CYP701A (CYP71 clan) is an A-type P450 whereas the CYP88As (CYP85 clan) are non-A type P450s. These examples clearly show that P450s involved in the same pathway or that catalyze similar reactions are not always phylogenetically related.

Licorice production depends on the collection of wild Glycyrrhiza plants, especially in China, and this has caused a decrease in licorice reserves and an increase in desertification (33). Moreover, the chemical synthesis of glycyrrhizin is difficult both in terms of quantity and cost. Therefore, the production of glycyrrhizin through plant tissue culture, hairy root culture (34), and cell culture (35) has been attempted, although the efforts were all unsuccessful. The possibility exists that CYP88D6 and additional genes could be used to engineer the production of glycyrrhizin in a plant and/or microbial host. In this study, we described the successful production of a glycyrrhizin pathway intermediate, 11-oxo-β-amyrin (1.6 mg/l), in a wild-type yeast background by the coexpression of bAS (LjOSC1), CYP88D6, and LjCPR1 as a redox partner for CYP88D6 (Fig. 4 and Fig. S4). Further genetic optimization to enhance the availability of 2,3-oxidosqualene as described by Kirby et al. (36), by overexpression of a truncated form of 3-hydroxy-3-methylglutaryl CoA (HMG-CoA) reductase and down-regulation of ERG7 (lanosterol synthase), may improve production. The conversion of 11-oxo-β-amyrin to glycyrrhizin will require further oxidation at the C-30 position, and this conversion could potentially involve other P450 enzymes. Functional analyses of the other CYP88D subfamily members that may exist in G. uralensis but which may be missing from our EST collection are thus a high priority.

Materials and Methods


The NMR spectra were recorded on a DSX-300 (Bruker) and ECA-500 (JEOL) spectrometer in CDCl3. β-Amyrin (1) was synthesized by a previously described method (37). Glycyrrhetinic acid (8) was purchased from Sigma. Other standard samples were synthesized from either β-amyrin or glycyrrhetinic acid as described in the SI Methods and Fig. S5. 1H and 13C NMR chemical shifts are reported as δ-values based on the internal TMS (δH = 0) and reference solvent signal (CDCl3 δC = 77.0).

In Vitro Enzymatic Activity Assay.

Heterologous expression of CYP88D6 using a baculovirus–insect cell system was performed as described in the SI Methods. The microsomal fraction of insect cells expressing CYP88D6 was prepared as previously described (38). The enzymatic activity of CYP88D6 was reconstituted by mixing CYP88D6-containing microsomes with purified Arabidopsis NADPH-P450 reductase (39). The assay was conducted in a total volume of 500 μl of 50 mM potassium phosphate buffer (pH 7.25) containing 1 mM NADPH, 0.1 U/ml NADPH-P450 reductase, 100 μg of microsomal protein, and 20 μM substrate. The samples were incubated for 2 h at 30 °C, and the reaction products were extracted three times with 0.5 volume of ethyl acetate. The organic phase was evaporated, trimethylsilylated, and analyzed by GC-MS.

Yeast in Vivo Assays.

The plasmids used in our yeast in vivo assays, pYES3-ADH-OSC1 (constitutive expression of bAS driven by the ADH1 promoter), pELC (galactose-inducible expression of LjCPR1 driven by the GAL10 promoter), and pDEST52-CYP88D6 (galactose-inducible expression of CYP88D6 driven by the GAL1 promoter) were constructed as described in the SI Methods. Saccharomyces cerevisiae BJ2168 (MATa, prc1–407, prb1–1122, pep4–3, leu2, trp1, ura3–52, and gal2; Nippon Gene) cells harboring both pYES3-ADH-OSC1 and pELC were transformed with pDEST52-CYP88D6 or pYES2 empty vector (Invitrogen) as a control. The recombinant cells were cultured in synthetic complete medium containing 2% glucose without uracil, tryptophan, or leucine (SC-U-W-L) for 2 d at 28 °C. The cells were then collected and resuspended in SC-U-W-L medium containing 13 μg/ml hemin and 2% galactose instead of glucose, and cultured at 28 °C for 2 d. The cells were then harvested and extracted with ethyl acetate, and portions of the extracts were analyzed by GC-MS after trimethylsilation.

GC-MS and NMR Analysis.

See SI Methods.

RT-PCR Analysis.

Total RNA was extracted from the roots and stolons using RNAwiz (Ambion Inc.), and from the leaves and stems with cetyltrimethylammonium bromide (CTAB)-containing extraction buffer essentially as described previously (40). Total RNA was treated with RNase-free DNase (TaKaRa Bio) and further purified using an RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's specifications. First-strand cDNA synthesis was carried out using a SMART RACE cDNA Amplification Kit (Clontech) with 1 μg of total RNA. PCR was performed using primers 1 and 2 for bAS, primers 3 and 4 for CYP88D6, and primers 5 and 6 for β-tubulin (for primer sequences see Table S1), with 33 cycles for bAS and CYP88D6, and 30 cycles for β-tubulin.

Isolation of CYP88D Subfamily Members from M. truncatula and L. japonicus.

See SI Methods.

Supplementary Material

Supporting Information:


We thank Drs. S. Ayabe (Nihon University) and Y. Ebizuka (University of Tokyo) for encouragement, and Drs. H. Hayashi (Iwate Medical University) and M. Kojoma (Health Sciences University of Hokkaido) for providing the plant materials and for valuable discussions. We thank Drs. N. Sakurai, H. Suzuki, and D. Shibata (Kazusa DNA Research Institute) and Drs. A. Toyoda, Y. Totoki, and Y. Sakaki (RIKEN GSC) for constructing the EST databases, and Drs. T. Shibata and O. Iida (Research Center for Medicinal Plant Resources) for providing the initial G. uralensis samples used to construct the cDNA library. We thank Dr. M. Shibuya (The University of Tokyo) for the gift of authentic 24-hydroxy-β-amyrin, and Dr. S. Yamaguchi (RIKEN PSC) for the gift of authentic ent-kaurenoic acid and GA12. We also thank Dr. S. Seo and Y. Toya (Tokiwa Phytochemical Co., Ltd.) for technical advice, and Drs. C. J. Arntzen (Arizona State Univ.) and M. Suzuki (Kihara Institute for Biological Research) for helpful discussions. T. Nakao (Nihon University) and T. Nishizawa (Kihara Institute for Biological Research) provided technical support. This study was supported in part by a grant from the New Energy and Industrial Technology Development Organization (NEDO), Japan, and by a Grant-in-Aid (No. 18780082) for Young Scientists (B) to H. S. from the Ministry of Education, Culture, Sports, Science and Technology.


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