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Biol. Pharm. Bull. 24(8) 912—916 (2001)
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Cloning and Characterization of a cDNA Encoding b -Amyrin Synthase Involved in Glycyrrhizin and Soyasaponin Biosyntheses in Licorice Hiroaki HAYASHI,*,a Pengyu HUANG,a Ara KIRAKOSYAN,a,1) Kenichiro INOUE,a Noboru HIRAOKA,b Yasumasa IKESHIRO,b Tetsuo KUSHIRO,c Masaaki SHIBUYA,c and Yutaka EBIZUKAc Gifu Pharmaceutical University,a 5–6–1 Mitahora-higashi, Gifu 502-8585, Japan, Niigata College of Pharmacy,b 5–13–2 Kamishin-ei-cho, Niigata 950–2081, Japan, and Graduate School of Pharmaceutical Sciences, The University of Tokyo,c 7–3–1 Hongo, Bunkyo-ku, Tokyo 113–0033, Japan. Received March 7, 2001; accepted April 27, 2001 An oxidosqualene cyclase cDNA, termed GgbAS1, was isolated from cultured cells of licorice (Glycyrrhiza glabra) by heterologous hybridization with cDNA of Arabidopsis thaliana LUP1 lupeol synthase. The yeast transformed with an expression vector containing the open reading frame of GgbAS1 produced b -amyrin, indicating that GgbAS1 encodes b -amyrin synthase involved in the glycyrrhizin and soyasaponin biosyntheses in licorice. Northern blot analysis showed that the level of b -amyrin synthase mRNA was drastically changed in the cultured licorice cells, whereas the mRNA level of cycloartenol synthase was relatively constant. Key words Glycyrrhiza glabra; cDNA cloning; b -amyrin synthase; glycyrrhizin; soyasaponin
Higher plants produce not only phytosterols, which play a role as indispensable membrane constituents, but also various triterpenoids derived from six isoprene units.2,3) Although structural elucidation of triterpenoids has been studied extensively, our understanding of the regulation of triterpenoid biosynthesis is quite limited.4—7) Oxidosqualene cyclases (OSCs) catalyze the cyclization of 2,3-oxidosqualene, a common biosynthetic intermediate of both triterpenoids and phytosterols. This step is situated at the critical branching point for phytosterol and triterpenoid biosyntheses,8) and our interest is focused on the regulation of various OSCs in higher plants. Up to now, three cDNAs of b -amyrin synthase (EC 5.4.99.–) from Panax ginseng9,10) and Pisum sativum,11) three cDNAs of lupeol synthase (EC 5.4.99.–) from Arabidopsis thaliana,12) Olea europaea13) and Taraxacum officinale,13) and a cDNA of multifunctional triterpene synthase from P. sativum11) have been functionally identified as triterpene synthases, which participate in the biosynthesis of triterpenoids. In addition, cDNAs of cycloartenol synthase (EC 5.4.99.8), an OSC involved in phytosterol biosynthesis, have been cloned from different plant species.9,13—19) Although mechanistic studies on plant triterpene synthases have been reported,20,21) the regulatory role of these OSCs in the triterpenoid biosynthesis has not been elucidated at the molecular level. We are using the cultured cells of Glycyrrhiza glabra L. (licorice) as a model system to investigate the regulation of triterpenoid biosynthesis in higher plants. The roots and stolons of G. glabra, one of the most important crude drugs in the world,22) contain a large amount (2—8% of dry weight) of glycyrrhizin, a sweet oleanane-type triterpenoid saponin. It is noteworthy that cultured licorice cells fail to form glycyrrhizin but produce two structurally different triterpenoid constituents, soyasaponins and betulinic acid.23,24) Both soyasaponins and glycyrrhizin share a common intermediate b amyrin, and betulinic acid is a lupane-type triterpene derived from lupeol (Fig. 1). cDNA cloning of cycloartenol synthase from cultured licorice cells has been reported.19) In the present study, we isolated an OSC cDNA of G. glabra by heterologous hybridization with A. thaliana LUP1 lupeol synthase cDNA.12) ∗ To whom correspondence should be addressed.
The functional expression in yeast showed that this cDNA encodes b -amyrin synthase responsible for the glycyrrhizin and soyasaponin biosyntheses. Furthermore, we examined the mRNA level of b -amyrin synthase in the soyasaponinproducing cultured cells and compared it with that of cycloartenol synthase. MATERIALS AND METHODS Plant Material and Culture Conditions Cultured licorice cells, strain RNS-1B, were subcultured in Linsmaier–Skoog (LS) medium25) containing 100 m M 1-naphthaleneacetic acid and 1 m M 6-benzyladenine (standard medium) at 4-week intervals as previously reported.24) For the soyasaponin production experiments, the cells (1 g fresh weight) were cultured in 30 ml of LS medium containing 1 m M 1-naphthaleneacetic acid and 10 m M 6-benzyladenine (soyasaponin-producing medium) for the stimulation of soyasaponin production. cDNA Cloning of a New OSC A 767-bp digoxigenin (DIG)-labeled DNA probe, corresponding to amino acid residue numbers 305—560 of A. thaliana LUP1 lupeol synthase,12) was prepared by polymerase chain reaction (PCR) using Taq DNA polymerase (Takara Shuzo, Japan), two primers of 59-TATGCAAAAGAAGACATGTA-39 and 59ACAAACTCCCGCTCGACCAT-39, DIG-dNTP mixture (Roche Diagnostics, Germany) and pOSCLUP 20) as a template, according to the manufacturer’s manual. A cDNA library prepared from cultured licorice cells26) was screened by the DIG-labeled probe under a low-stringency condition, as previously reported.19) The hybridized DIG-labeled probe was detected using a DIG Nucleic Acid Detection Kit (Roche Diagnostics) according to the manufacturer’s manual. Three positive clones, isolated from 23105 plaques, were subcloned into pBluescript SK(2) (Stratagene, U.S.A.) by in vivo excision. The longest clone, designated GgbAS1, was sequenced in both strands. The nucleotide sequences were determined by the dideoxy chain termination method using an ALFred DNA sequencer (Amersham Pharmacia Biotech, U.K.). Nucleotide and amino acid sequence analyses were conducted using Genetyx-Mac software (Software Development, Japan).
e-mail:
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© 2001 Pharmaceutical Society of Japan
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Fig. 1.
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Biosynthetic Pathways of Triterpenoids in Glycyrrhiza glabra L.
bAS, b -amyrin synthase; CAS, cycloartenol synthase; LUS, lupeol synthase.
Functional Expression of cDNA in Yeast PCR was carried out for 25 cycles of 40 s at 94 °C, 40 s at 50 °C and 2 min at 72 °C using GgbAS1 as a template, Pfu DNA polymerase (Stratagene) and two primers of 59-CCGGTACCATGTGGAGGCTGAAGATAGCGG-39 (KpnI site underlined) and 59-GCGGTACCCTGCAGCTTTTAAGTTAAACAAAC-39 (KpnI site underlined) corresponding to the 59 and 39-termini of the deduced open reading frame. The PCR product was digested with KpnI and ligated to KpnI sites of pYES2 (Invitrogen, U.S.A.) to construct an expression plasmid, pYEGgOSC2, in which the open reading frame of the cDNA was downstream of the GAL1 promoter of pYES2 in a sense orientation. The nucleotide sequence of pYEGgOSC2 was confirmed by sequencing in both strands. The yeast strain GIL779) was transformed with pYEGgOSC2 by the lithium acetate method.27) The protocols of the culture condition, induction by galactose and preparation of triterpene mono-alcohol fraction are exactly the same as described in the study by Kushiro et al.9) After purification by preparative TLC, triterpene mono-alcohol fraction was subjected to liquid chromatography-atmospheric pressure chemical ionization mass spectrometry (LC-APCIMS) analysis as previously reported.11) LC-APCIMS was measured with LCQ (Thermo Quest, U.S.A.) under the following HPLC conditions: column, SUPER-ODS (diameter: 4.6 mm, length: 200 mm) (Tosoh, Japan); solvent system, acetonitrile : water (95 : 5, v/v); flow rate, 1 ml/min; column temp., 40 °C; detection, UV 205 nm; retention time for b -amyrin, 20.8 min. b -amyrin: MS m/z 409 [M1H2H2O]1, MS/MS (precursor ion at m/z 409) 339 (15%), 299 (35%), 285 (92%), 271 (83%), 259 (100%), 231 (89%), 217 (67%), 203 (56%), 177 (58%). Genomic Southern Blot Analysis A 201-bp DIG-labeled DNA probe of GgbAS1, corresponding to exon 1 of the Arabidopsis LUP1 gene (GenBank accession no. AC002986), was prepared by PCR using GgbAS1 as a template, Taq DNA polymerase, two primers of 59-ATGTGGAGGCTGAAGATAGCGG-39 and 59-CTGAAAACGC-
CAAAGGAGG-39 and DIG-dNTP mixture. Genomic DNA was extracted from the cultured licorice cells (strain RNS1B) as previously reported.28) Twenty micrograms of genomic DNA was digested with BamHI, EcoRI, HindIII, or PstI, separated on a 0.8% (w/v) agarose gel by electrophoresis, transferred to a positively charged nylon membrane (Roche Diagnostics) and hybridized with the DIG-labeled DNA probe of GgbAS1 at 50 °C in a hybridization buffer containing 0.5 M sodium phosphate buffer (pH 7.2), 7% sodium dodecyl sulfate (SDS) and 1 mM ethylenediaminetetraacetic acid. The membrane was washed twice for 5 min at room temperature with 2X SSC containing 0.1% SDS and then twice for 10 min at 60 °C with 0.5X SSC containing 0.1% SDS. The hybridized DIG probe was detected using the DIG Nucleic Acid Detection Kit according to the manufacturer’s manual. Northern Blot Analysis The DIG-labeled RNA probes were prepared from BamHI-digested GgbAS1 and BamHIdigested GgCAS119) using T7 RNA polymerase and DIG RNA Labeling Mix (Roche Diagnostics) according to the manufacturer’s manual. Plant materials were frozen by liquid nitrogen and homogenized in a mortar, and total RNA was isolated by the Extract-A-PlantTM RNA Isolation Kit (Clontech, U.S.A.). Five micrograms of total RNA (per lane) was separated on a 1% (w/v) agarose gel containing formaldehyde and blotted to a positively charged nylon membrane (Roche Diagnostics). The membrane was hybridized with the DIG-labeled RNA probe as previously reported.19) The membrane was washed twice for 5 min at room temperature with 2X SSC containing 0.1% SDS and then twice for 10 min at 65 °C with 0.2X SSC containing 0.1% SDS. The hybridized DIG probe was detected using the DIG Nucleic Acid Detection Kit according to the manufacturer’s manual. RESULTS Cloning and Functional Expression of b -Amyrin Synthase cDNA A lZAP cDNA library constructed from 14-
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d-old cultured licorice cells,26) was screened by A. thaliana LUP1 lupeol synthase cDNA,12) as a heterologous hybridization probe, under the condition of low stringency. Three positive clones were isolated from approximately 200000 plaques of the cDNA library, and the inserts were subcloned into pBluescript SK (2) vector by in vivo excision. Since partial sequencing of these positive clones revealed that all clones have an identical 39 non-coding sequence, except for variation in the length, the longest clone was sequenced in both strands. The 2695-bp cDNA contained a 307-bp 59 non-coding region, a 2295-bp open reading frame for a polypeptide (87.5 kD) of 765 amino acids, a 69-bp 39 non-coding region and a 24-bp poly(A)-tail. The DNA sequence is different from that of G. glabra cycloartenol synthase cDNA,19) but the coding region showed significant homology to OSC cDNAs, indicating that the cDNA encodes a new OSC of G. glabra. To determine the function of the new OSC cDNA, the ORF of the cDNA was amplified by PCR and ligated into a yeast expression plasmid, pYES2 (Invitrogen). The obtained plasmid, designated pYEGgOSC2, was introduced into a yeast mutant GIL77,9) which lacks lanosterol synthase. After the induction by galactose, the transformed yeast was extracted, and the triterpene mono-alcohol fraction was analyzed by LC-APCIMS. Only a peak corresponding to standard b -amyrin was observed in the HPLC (Fig. 2), and it produced an identical MS/MS (precursor ion at m/z 409) fragmentation pattern to that of authentic b -amyrin. Thus, the new cDNA, designated GgbAS1, encodes b -amyrin synthase of G. glabra.29) Figure 3 shows the alignment of the amino acid residues deduced from b -amyrin synthase cDNAs of G. glabra, P. sativum, and P. ginseng. The deduced amino acid sequence of GgbAS1 exhibits 90%, 82%, 81%, 79%, 70%, 63%, 63% and 58% identities to those of P. sativum PSY b -amyrin synthase,11) P. ginseng PNY b -amyrin synthase,9) P. sativum PSM multifunctional triterpene synthase,11) P. ginseng PNY2
Fig. 3.
b -amyrin synthase,10) A. thaliana LUP1 lupeol synthase,12) O. europaea OEW lupeol synthase,13) T. officinale TRW lupeol synthase,13) and G. glabra GgCAS1 cycloartenol synthase,19) respectively. Genomic Southern Analysis of b -Amyrin Synthase Gene in the Licorice Genome Multiple OSC genes were reported to exist in the genome of Arabidopsis thaliana (GenBank accession nos. Z97338, AC002986, AC005171), and two functional b -amyrin synthase cDNAs were cloned from hairy root cultures of P. ginseng.9,10) It was also suggested by genomic Southern analysis that at least two copies for cycloartenol synthase gene exist in the licorice genome.19) Since b -amyrin synthase participates in both glycyrrhizin and soyasaponin biosyntheses, we wanted to know whether a single b -amyrin synthase gene is responsible for the biosynthesis of both saponins. To estimate the copy number of the b -amyrin synthase gene in the licorice genome, we performed genomic Southern analysis using a DIG-labeled PCR fragment of GgbAS1. A 201-bp DIG-labeled DNA probe of
Fig. 2. HPLC Profile of the Triterpene Mono-alcohol Fraction from the Yeast Strain GIL77 Transformed with pYEGgOSC2 The peak at 20.8 min was identified as b -amyrin by LC-APCIMS analysis.
Alignment of the Amino Acid Sequences Deduced from b -Amyrin Synthase cDNAs
GgbAS1, Glycyrrhiza glabra b -Amyrin Synthase; PSY, Pisum sativum b -Amyrin Synthase; PNY, Panax ginseng b -Amyrin Synthase. Hyphens were inserted to maximize homology. The amino acid residues identical in two out of three protein sequences are boxed.
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soyasaponins I and II.24) Since b -amyrin synthase participates in soyasaponin biosynthesis, we examined the time course of mRNA levels of b -amyrin synthase and cycloartenol synthase in the soyasaponin-producing medium. Soyasaponin production in the soyasaponin-producing medium was two times higher than that in the standard medium,24) in which the cultured cells have been subcultured for more than 10 years. The level of b -amyrin synthase mRNA was high from day 4 to day 20 in the soyasaponinproducing medium, when soyasaponins were actively produced, whereas the mRNA level on day 24 at the stationary phase was much lower. The level of b -amyrin synthase mRNA on day 8 in the standard medium was also low. In contrast to b -amyrin synthase mRNA, the mRNA level of cycloartenol synthase was relatively constant in all lanes. Fig. 4. Southern Hybridization Analysis of Genomic DNA Isolated from Glycyrrhiza glabra Licorice genomic DNA (20 m g per lane) was digested with PstI (P), HindIII (H), EcoRI (E) or BamHI (B) separated on 0.8% (w/v) agarose gel by electrophoresis, transferred to a positively charged nylon membrane and then hybridized with the DIG-labeled PCR fragment of GgbAS1. A l -HindIII digest served as the molecular marker.
Fig. 5. Variation of mRNA Levels for b -Amyrin Synthase and Cycloartenol Synthase in the Cultured Licorice Cells of Different Growth Stages at Day 4 to 24 in the Soyasaponin-Producing Medium (PM) and at Day 8 in the Standard Medium (SM) Total RNA (5 m g per lane) was separated on 1% (w/v) agarose gel containing formaldehyde, blotted to a positively charged nylon membrane and then hybridized with the DIG-labeled RNA probe of GgbAS1 or GgCAS1. The ethidium bromide staining of the gel before transfer is shown at the bottom of the figure.
GgbAS1, corresponding to exon 1 of the Arabidopsis LUP1 gene (GenBank accession no. AC002986), did not cross-hybridize to the GgCAS1 cDNA19) or to a lupeol synthase cDNA from G. glabra (Hayashi et al., unpublished results) under the same hybridization condition. As shown in Fig. 4, only one signal was observed in the EcoRI and HindIII lanes, however, two signals were detected in the BamHI and PstI lanes. The latter result suggests that an additional homologous gene to GgbAS1 may exist in the licorice genome. mRNA Levels of b -Amyrin Synthase and Cycloartenol Synthase in the Cultured Cells of G. glabra Northern blot analysis was performed to determine the mRNA levels of b -amyrin synthase and cycloartenol synthase under the condition of high stringency. The two DIG-labeled RNA probes hybridized specifically to the respective cDNA, and did not cross-hybridize to a lupeol synthase cDNA from G. glabra (Hayashi et al., unpublished results). The cultured licorice cells fail to form glycyrrhizin, but they produce another oleanane-type triterpenoid saponin,
DISCUSSION Although a sole OSC, lanosterol synthase, is responsible for cholesterol biosynthesis in mammals, multiple OSCs are situated at the critical branching point to determine the biosynthesis toward either phytosterols or various triterpenoids in higher plants.8) It is valuable to compare the regulation of various OSCs from the viewpoint of the evolution of triterpenoid biosynthesis. In the present study, a cDNA (GgbAS1) for b -amyrin synthase, involved in both glycyrrhizin and soyasaponin biosyntheses, was isolated from the soyasaponin-producing cultured licorice cells. Southern blot analysis suggested that an additional homologous gene to GgbAS1 might exist in the licorice genome. Since the DIG-labeled DNA probe of GgbAS1 used for the Southern blot analysis did not cross-hybridize to cycloartenol synthase cDNA19) or lupeol synthase cDNA (Hayashi et al., unpublished results) from G. glabra, this additional gene might be another b -amyrin synthase gene. Further experiments are required to clarify whether a single b -amyrin synthase gene is responsible for both soyasaponin and glycyrrhizin biosyntheses. Although the level of cycloartenol synthase mRNA was relatively constant in the cultured cells, the level of b -amyrin synthase mRNA was drastically changed in the cultured licorice cells. The level of b -amyrin synthase mRNA was very high in the cultured cells when soyasaponins were actively produced. These results suggest that the mRNA level of b -amyrin synthase gene plays a crucial role in the biosynthesis of soyasaponins in cultured licorice cells. In addition to b -amyrin synthase and cycloartenol synthase, the third OSC, lupeol synthase, participates in the betulinic acid biosynthesis in licorice.23) In the present study, however, no lupeol synthase cDNA was isolated from cultured licorice cells by heterologous hybridization with Arabidopsis LUP1 lupeol synthase cDNA. On the other hand, two lupeol synthase cDNAs, OEW and TRW, have been cloned from O. europaea and T. officinale, respectively.13) Although the amino acid sequence of OEW was 78% identical to that of TRW, both clones show only 57% amino acid sequence identity with Arabidopsis LUP1 lupeol synthase used in the present study as a heterologous hybridation probe. Recently, using OEW lupeol synthase cDNA as a heterologous hybridization probe, we have also cloned a lupeol synthase cDNA from licorice (Hayashi et al., unpublished results).
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Molecular cloning of b -amyrin synthase provides a useful tool to elucidate the glycyrrhizin and soyasaponin biosyntheses of G. glabra. Further experiments are under way to substantiate the regulation of the b -amyrin synthase gene during the development of the intact plants, especially to analyze the regulation of glycyrrhizin biosynthesis in the thickening roots and stolons.
11) 12) 13) 14) 15)
Acknowledgements This research was supported in part by a Grant-in-Aid for Scientific Research to H. H. (No. 09771930) from the Ministry of Education, Science, Sports, Culture and Technology, Japan.
16) 17) 18)
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