Molecular cloning and characterization of isomultiflorenol synthase, a new triterpene synthase from Luffa cylindrica , involved in biosynthesis of bryonolic acid Hiroaki Hayashi 1 , Pengyu Huang 1 , Kenichiro Inoue 1 , Noboru Hiraoka 2 , Yasumasa Ikeshiro 2 , Kazufumi Yazaki 3 , Shigeo Tanaka 4 , Tetsuo Kushiro 5 , Masaaki Shibuya 5 and Yutaka Ebizuka 5 1 Gifu Pharmaceutical University, Japan; 2 Niigata College of Pharmacy, Japan; 3 Graduate School of Biostudies, Kyoto University, Japan; 4 Graduate School of Pharmaceutical Sciences, Kyoto University, Japan; 5 Graduate School of Pharmaceutical Sciences, University of Tokyo, Japan An oxidosqualene cyclase cDNA, LcIMS1, was isolated from cultured cells of Luffa cylindrica Roem. by heterologous hybridization with cDNA of Glycyrrhiza glabra b-amyrin synthase. Expression of LcIMS1 in yeast lacking endogenous oxidosqualene cyclase activity resulted in the accumulation of isomultiflorenol, a triterpene. This is consistent with LcIMS1 encoding isomultiflorenol synthase, an oxidosqualene cyclase involved in bryonolic acid biosynthesis in cultured Luffa cells. The deduced amino- acid sequence of LcIMS1 shows relatively low identity with other triterpene synthases, suggesting that isomultiflorenol synthase should be classified into a new group of triterpene synthases. The levels of isomultiflorenol synthase and cycloartenol synthase mRNAs, which were measured with gene-specific probes, correlated with the accumulation of bryonolic acid and phytosterols over a growth cycle of the Luffa cell cultures. Isomultiflorenol synthase mRNA was low during the early stages of cell growth and accumulated to relatively high levels in the late stages. Induction of this mRNA preceded accumulation of bryonolic acid. In contrast, cycloartenol synthase mRNA accumulated in the early stages of the culture cycle, whereas phytosterols accumulated at the same relative rate throughout the whole growth cycle. These results suggest independent regulation of these two genes and of the accumulation of bryonolic acid and phytosterols. Keywords: bryonolic acid biosynthesis; isomultiflorenol synthase; Luffa cylindrica; triterpene synthase. Higher plants have the capacity to accumulate a wide range of chemically diverse triterpenes in addition to sterols [1]. Sterols and triterpenes share a common biosynthetic intermediate, 2,3-oxidosqualene, which is cyclized by various oxidosqualene cyclases (OSCs) to give polycyclic skeletons. OSCs are situated at the putative branch point capable of channeling biosynthesis toward sterols or various triterpenes in higher plants [2]. In contrast, lanosterol synthase, the only OSC found in animals, plays a crucial role in cholesterol biosynthesis. As part of a continuing study on the evolution and reaction mechanism of OSCs in higher plants, cDNAs for b-amyrin synthase (EC 5.4.99 ) [3–6], lupeol synthase (EC 5.4.99 ) [7,8], multifunctional triter- pene synthase (EC 5.4.99 ) [5,9,10], and cycloartenol synthase (EC 5.4.99.8) [3,11– 14] were cloned, and functionally characterized. However, more than 90 different triterpene skeletal types have been found in nature [15], suggesting the existence of additional OSCs. As part of an effort to understand the molecular mechanism of the triterpene cyclization reactions, as well as the molecular evolution of these proteins in higher plants, we have a continuing program to isolate as many of the different triterpene synthases as possible. Cell suspension cultures of Luffa cylindrica Roem. (Cucurbitaceae) are capable of producing a large amount of bryonolic acid (D:C-friedoolean-8-en-3b-ol-29-oic acid) [16,17], a friedooleanane-type pentacyclic triterpene, which exhibits antiallergic activity against cutaneous anaphylaxis and contact dermatitis [17,18]. Bryonolic acid was first isolated from the roots of Bryonia dioica (Cucurbitaceae) [19], and was later shown to accumulate exclusively in the roots and cultured cells of various cucurbitaceous plants [20]. The biosynthetic pathway for bryonolic acid was elucidated by tracer and enzymological experiments using cultured watermelon cells [21]. 2,3-Oxidosqualene, the common precursor derived from mevalonic acid, is cyclized by an OSC to form isomultiflorenol, the pentacyclic triterpene intermediate, which is then converted into bryonolic acid. Hence, the OSC isomultiflorenol synthase is a potential regulatory enzyme controlling the biosynthesis of the bryonolic acid skeleton (Fig. 1). We have been studying the regulation of bryonolic acid biosynthesis in L. cylindrica as a means of investigating Correspondence to H. Hayashi, Gifu Pharmaceutical University, 5-6-1 Mitahora-higashi, Gifu 502-8585, Japan. Fax: þ 81 58 237 5979, Tel.: þ 81 58 237 3931, E-mail: hayashih@gifu-pu.ac.jp Enzymes: isomultiflorenol synthase (EC 5.4.99 ); b-amyrin synthase (EC 5.4.99 ); lupeol synthase (EC 5.4.99 ); cycloartenol synthase (EC 5.4.99.8). Note: The nucleotide sequence reported in this paper has been deposited in the DDBJ/EMBL/GenBank under the accession number AB058643. (Received 6 June 2001, revised 8 October 2001, accepted 9 October 2001) Abbreviations: DIG, digoxigenin; LC-APCIMS, liquid chromatography-atmospheric pressure chemical ionization mass spectrometry; OSC, oxidosqualene cyclase. Eur. J. Biochem. 268, 6311–6317 (2001) q FEBS 2001 how triterpenoid formation is controlled relative to the sterol biosynthesis pathway, which is necessary for cell growth. So far, two OSC cDNAs, LcCAS1 [13] and LcOSC2 [22], have been isolated from cultured Luffa cells by heterologous hyridization with pea cycloartenol synthase cDNA. LcCAS1 encodes a functional cycloartenol synthase involved in sterol biosynthesis [13], whereas expression of LcOSC2 in yeast does not result in any triterpene accumulation [22]. The relatively low identity of the deduced LcOSC2 protein with other pentacyclic triterpene synthases suggested that LcOSC2 did not encode isomultiflorenol synthase [22]. Furthermore, as the reaction mechanism proposed for triterpene formation by isomultiflorenol synthase is more similar to that by b-amyrin synthase than to that by cycloartenol synthase, an isomultiflorenol synthase is expected to share greater sequence similarity with b-amyrin synthase. In this study, we used the Glycyrrhiza glabra GgbAS1 b-amyrin synthase cDNA [6] to isolate cDNA for a new OSC family member from the Luffa cDNA library by heterologous hybridization. Functional expression of this cDNA in yeast indicated that the newly obtained cDNA coded for the isomultiflorenol synthase involved in bryonolic acid biosynthesis. Furthermore, the pattern of expression of isomultiflorenol synthase was compared with that of the cycloartenol synthase responsible for sterol biosynthesis in cultured Luffa cells, which showed sophisticated regulation of triterpenoid biosynthesis. MATERIALS AND METHODS Chemicals Authentic isomultiflorenol and bryonolic acid were kindly provided by W. Kamisako (Mukogawa Women’s University, Nishinomiya, Japan). Other chemicals were purchased from Wako Pure Chemicals (Osaka, Japan) and Nakalai Tesque (Kyoto, Japan). Customized oligonucleotide primers were synthesized by Amersham Pharmacia Biotech. Plant material and culture conditions Cell suspension cultures of Luffa [16] were maintained in a 300-mL Erlenmeyer flask containing 60 mL Linsmaier and Skoog medium [23], supplemented with 100 n M 1-naphthal- eneacetic acid in the dark at 25 8C, and subcultured at intervals of 4 weeks. For the time course experiments, the cells (1 g fresh weight) were cultured in a 100-mL Erlenmeyer flask containing 30 mL of the above medium. Harvested cultured cells were frozen with liquid nitrogen and stored at 2 80 8C. Construction of the L. cylindrica cDNA library Total RNA was prepared from 10-day-old-cultured Luffa cells by guanidine thiocyanate/hot phenol extraction [24]. Poly(A)-rich RNAwas purified by an mRNA purification kit (Pharmacia), and a Luffa cDNA library was constructed using a lZAP-cDNA synthesis kit (Stratagene). Cloning of a new triterpene synthase cDNA from L. cylindrica A 1227-bp digoxigenin (DIG)-labeled DNA probe, corre- sponding to amino-acid residue numbers 353–729 of G. glabra GgbAS1 b-amyrin synthase [6], was prepared by PCR using GgbAS1 as a template, Taq DNA polymerase (Takara Shuzo, Kyoto, Japan), the primers 5 0 -GAAGCATA TCCACTATGAAGATGA-3 0 and 5 0 -TGAATACTCCCGTG ATTTCCTGTTG-3 0 , and DIG–dNTP mixture (Roche Diagnostics), according to the manufacturer’s manual. The cDNA library prepared from cultured Luffa cells was screened with the DIG-labeled probe under conditions of low stringency as previously reported [14]. The hybridized DIG-labeled probe was detected using a DIG-nucleic acid detection kit (Roche Diagnostics), according to the manufacturer’s manual. One positive clone was subcloned into pBluescript SK(–) (Stratagene) by in vivo excision, and was sequenced in both strands by the dideoxy chain- termination method using a model 373A DNA sequencer (PE Biosystems). Nucleotide and amino-acid sequences were analysed by Genetyx-Mac software (Software Development, Tokyo, Japan). This clone was designated LcIMS1. Fig. 1. Pathways of bryonolic acid biosynthesis in Luffa cylindrica. 6312 H. Hayashi et al.(Eur. J. Biochem. 268) q FEBS 2001 Functional expression in yeast mutant GIL77 To construct an expression plasmid for yeast, Kpn I and Xba I sites were introduced into the 5 0 and 3 0 -termini of the deduced ORF of LcIMS1 cDNA by PCR (25 cycles with 40 s at 94 8C, 40 s at 50 8C and 2 min at 72 8C) with Pfu DNA polymerase (Stratagene) used as the DNA polymerase. Two primers of 5 0 -CTGGTACCGATTGAGTTGAGGTG ATTG-3 0 (Kpn I site underlined) and 5 0 -CCTCTAGAGTA AAAGTCTCCAATC-3 0 (Xba I site underlined) were used to modify the cDNA. The PCR product was digested with Kpn I and XbaI, and ligated to the sites of pYES2 (Invitrogen), to obtain pYES2-LcIMS1 in which the ORF of the cDNA was ligated to the GAL1 promoter in the sense orientation. The nucleotide sequence of the inserted DNA was confirmed by sequencing. The ERG7-deficient yeast strain GIL77 [3] was transformed with pYES2-LcIMS1 by the lithium acetate method [25]. The protocols for the culture condition, induction by galactose, and preparation of triterpene mono-alcohol fraction have been described previously [3]. After purification by preparative TLC, the triterpene mono-alcohol fraction was analyzed by liquid chromatography–atmospheric pressure chemical ionization mass spectrometry (LC-APCIMS). LC-APCIMS was performed with an LCQ (Thermo Quest, Tokyo, Japan) under the following HPLC conditions: column, SUPER- ODS (diameter 4.6 mm, length 200 mm; Tosoh); solvent system, 95% acetonitrile aq.; flow rate, 1 mL·min 21 ; column temperature, 40 8C; detection, UV 202 nm; retention time for isomultiflorenol, 21.7 min Isomultiflorenol: MS m/z 409 [M þ H–H 2 O] þ , MS/MS (precursor ion at m/z 409) 313 (20%), 299 (41%), 245 (45%), 231 (100%), 217 (75%), 191 (56%). Northern-blot analysis The DIG-labeled RNA probes were prepared from Bam HI-digested LcCAS1 (probe length 1.3 kb) [13] and Bam HI-digested LcIMS1 (probe length 1.9 kb) using T7 RNA polymerase and DIG RNA Labeling Mix (Roche Diagnostics), according to the manufacturer’s manual. The two DIG-labeled RNA probes specifically hybridized to the respective cDNA under conditions of high stringency. Cultured Luffa cells frozen by liquid nitrogen were homogenized in a mortar to give total RNA by an Extract- A-Plant RNA isolation kit (Clontech). Five micrograms of total RNA (per lane) were separated on a 1% 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 [14]. The hybridized membrane was washed twice for 5 min at room temperature with 2 £ NaCl/Cit containing 0.1% SDS, and then twice for 10 min at 65 8C with 0.2 £ NaCl/Cit containing 0.1% SDS. The hybridized DIG probe was detected using the DIG Nucleic Acid Detection Kit according to the manufacturer’s manual. Quantitative analysis of bryonolic acid Freeze-dried powdered samples (50 mg) were extracted with ethyl acetate (2 mL twice, 60 8C for 1 h), and cholesterol (1 mg) was added to the extract as an internal standard. The dried sample was dissolved in pyridine (50 mL), and an aliquot (10 mL) of the pyridine solution was treated with N,O-bis(trimethylsilyl)acetamide (10 mL) at 50 8C for 1 h. An aliquot (1 mL) of the solution was applied to GC for the quantitative analysis of bryonolic acid. The rest of the pyridine solution was saponified with 6% (w/v) KOH in methanol (1 mL) at 90 8C for 1 h to hydrolyze steryl esters. Sterols were then extracted with n-hexane (3 mL). The dried extract was dissolved in a mixture of pyridine (40 mL) and N,O-bis(trimethylsilyl)acetamide (40 mL), and incubated at 50 8C for 1 h. An aliquot (1 mL) of the solution was analyzed by GC for total sterols. GC analysis was Fig. 2. LC-APCIMS analysis of the triterpene mono-alcohol fraction from GIL77 transformed with pYES2-LcIMS1. (A) HPLC profile of the triterpene mono-alcohol fraction of the pYES2-LcIMS1 transformant; (B) MS/MS (precursor ion at m/z 409) fragmentation patterns of the peak at 21.7 min of the pYES2-LcIMS1 transformant; (C) MS/MS (precursor ion at m/z 409) fragmentation patterns of authentic isomultiflorenol. q FEBS 2001 Cloning of isomultiflorenol synthase (Eur. J. Biochem. 268) 6313 performed under the following conditions: column, Ultra Alloy þ -17 capillary column (15 m £ 0.5 mm internal diameter; film thickness 1 mL; Frontier Laboratory Ltd, Koriyama, Japan); column temperature, 200–300 8C (20 8C·min 21 ); injector and detector temperature, 320 8C; carrier gas, He 3.5 mL·min 21 . The contents of bryonolic acid and total sterols were calculated from the peak area of their trimethylsilylated products relative to that of the internal standard. Sterol content was the total content of stigmasta-7,22-dien-3b-ol, stigmasta-7,25-dien-3b-ol, and stigmasta-7,22,25-trien-3b-ol, which are D 7 -phytosterols characteristic of cucurbitaceous plants [21]. RESULTS Cloning of a new OSC cDNA from L. cylindrica A lZAP cDNA library constructed from 10-day-old cultured Luffa cells was screened using a b-amyrin synthase cDNA from G. glabra as a heterologous hybridization probe under low-stringent conditions. One positive clone was isolated from < 200 000 plaques of the cDNA library, from which the phagemid pBluescript SK(–) containing the cDNA insert was recovered by in vivo excision. This cDNA was 2551 nucleotides in length, and contained an ORF of 2277 nucleotides. A stop codon was present 9 bp upstream of the initial ATG codon in the frame. The cDNA sequence was divergent from those of LcCAS1 [13] and LcOSC2 [22], previously isolated from this plant species. Translation of the coding region yields a putative polypeptide of 759 amino-acid residues with a predicted molecular mass of 87.7 kDa. The QW motif, which occurs repeatedly in the sequence of all known OSCs [26], and the DCTAE motif, corresponding to an active site of the OSC family [27], were observed in the deduced amino-acid sequence. We presumed therefore that the new OSC cDNA was a candidate for isomultiflorenol synthase of L. cylindrica. Functional expression of the new OSC cDNA in the ERG7-deficient yeast mutant GIL77 To elucidate the function of the new OSC cDNA, its ORF was inserted into a yeast expression plasmid with a galactose-inducible GAL1 promoter. The completed plas- mid, designated pYES2-LcIMS1, was introduced into an ERG7-deficient yeast mutant GIL77 [3], which lacks a functional lanosterol synthase gene and produces no detectable endogenous lanosterol or other triterpene mono- alcohols. The transformed yeast was treated with galactose to induce expression of the recombinant protein, and was extracted with hexane. The extract of pYES2-LcIMS1 transformant gave a spot with an R f value corresponding to triterpene mono-alcohol on preparative silica gel TLC (data not shown), which was absent from the control pYES2 transformant [3]. This spot corresponding to triterpene mono-alcohol was extracted and further analyzed by LC-APCIMS. As shown in Fig. 2, a major peak at 21.7 min was observed in the HPLC profile. This main product was identified as isomultiflorenol by comparing its retention time and MS/MS (precursor ion at m/z 409) fragmentation patterns with those of authentic isomultiflorenol. As the control pYES2 transformant produced no triterpene mono-alcohol [3], accumulation of Fig. 3. Multiple alignment of deduced amino-acid sequences of isomultiflorenol synthase (LcIMS1), cycloartenol synthase (LcCAS1), and putative oxidosqualene cyclase (LcOSC2). Hyphens were inserted to maximize homology. Amino-acid residues identical in two out of the three protein sequences are boxed. The DCTAE motif is marked by a double underline. 6314 H. Hayashi et al.(Eur. J. Biochem. 268) q FEBS 2001 isomultiflorenol in the pYES2-IMS1 transformant indicates that the new OSC cDNA, designated LcIMS1, encodes isomultiflorenol synthase, the key enzyme in bryonolic acid biosynthesis in L. cylindrica. In addition to isomultiflorenol, a minor compound at 18.9 min, which, like isomultiflorenol, gave an ion at m/z 409, was also observed on LC-APCIMS analysis (Fig. 2). This compound, which seems to be a side product of LcIMS1 isomultiflorenol synthase, could not be identified. Sequence comparison Figure 3 shows the multiple alignments of deduced amino- acid sequences of three OSCs from L. cylindrica. The sequence of the Luffa LcIMS1 isomultiflorenol synthase exhibits 67–66%, 61–57%, 61%, 51% and 50% identities with those of b-amyrin synthases [3–6], lupeol synthases [7,8], Pisum PSM multifunctional triterpene synthase [5], Luffa LcCAS1 cycloartenol synthase [13], and Luffa LcOSC2 putative OSC [22], respectively. To clarify the evolutionary relationship of isomultiflorenol synthase among plant OSCs, the phylogenetic tree was constructed from the deduced amino-acid sequences of the LcIMS1 and other OSCs so far cloned from higher plants (Fig. 4). The relatively low identities (67–57%) of the LcIMS1 protein with other triterpene synthases suggest that isomultiflorenol synthase belongs to a new class of triterpene synthases in higher plants. Differential expression of OSC mRNAs in the cultured cells of L. cylindrica Isomultiflorenol synthase and cycloartenol synthase are situated at a putative branch point for biosynthesis of bryonolic acid and sterol, respectively (Fig. 1). Therefore, it was of interest to compare the expression patterns of isomultiflorenol synthase and cycloartenol synthase mRNAs in cultured Luffa cells over a culture growth cycle. Northern- blot analyses were performed with gene-specific DIG- labeled RNA probes for the two OSCs: isomultiflorenol synthase and cycloartenol synthase (LcCAS1) [13], so far cloned from Luffa. Figure 5 shows the time course of cell Fig. 4. Phylogenetic tree constructed from the deduced amino-acid sequences of LcIMS1 cDNA and other plant OSCs. The phylogenetic tree was constructed by the unweighted pair group method using arithmetric averages [29] with Genetyx-Mac software (Software Development, Japan). The GenBank database accession numbers used in this analysis are as follows, AB009030 (Panax PNY), AB014057 (Panax PNY2), AB037203 (Glycyrrhiza GgbAS1), AB034802 (Pisum PSY), AB034803 (Pisum PSM), U49919 (Arabidopsis LUP1), AC002986 (Arabidopsis MFS [9,10]), AB058643 (Luffa LcIMS1), AB025343 (Olea OEW), AB025345 (Taraxacum TRW), Y15366 (Medicago MtN18), AB009031 (Panax PNZ), AB025346 (Taraxacum TRV), AB033335 (Luffa LcOSC2), AB025968 (Glycyrrhiza GgCAS1), D89619 (Pisum PSX), AB025344 (Olea OEX), AB009029 (Panax PNX), U02555 (Arabidopsis CAS1), AB033334 (Luffa LcCAS1) and AB025353 (Allium AMX). bAS, b-amyrin synthase; CAS, cycloartenol synthase; LUS, lupeol synthase; MFS, multifunctional triterpene synthase; IMS, isomultiflorenol synthase. Fig. 5. Time course of bryonolic acid accumulation and levels of isomultiflorenol synthase and cycloartenol synthase mRNA in cultured Luffa cells. (A) Time course of accumulation of bryonolic acid and total sterol in cultured Luffa cells. Mean of three replicates. Bar indicates SD. (B) Time course of levels of isomultiflorenol synthase and cycloartenol synthase mRNA in cultured Luffa cells at different stages of growth. Total RNA (5 mg) was separated on 1% agarose gel containing formaldehyde, blotted to a positively charged nylon membrane, and then hybridized with the DIG-labeled RNA probe of LcIMS1 or LcCAS1. Ethidium bromide staining of the gel before transfer is shown below. q FEBS 2001 Cloning of isomultiflorenol synthase (Eur. J. Biochem. 268) 6315 growth, accumulation of bryonolic acid and total sterol, and mRNA levels of isomultiflorenol synthase and cycloartenol synthase in the cultured Luffa cells. Although the total sterol level increased during all growth phases except the stationary phase, the bryonolic acid level increased during the late growth phase only [16,17]. The level of isomultiflorenol synthase mRNA was high at days 16 and 20 and low at days 4 and 8. The induction of this mRNA preceded the accumulation of bryonolic acid. In contrast, the level of cycloartenol synthase mRNAwas high at days 4 and 8 and decreased at later growth stages. Similar results were also obtained in the repeated time course experiment (data not shown). These results suggest independent regulation of these two genes and of the accumulation of bryonolic acid and phytosterols. It is noteworthy that the changes in their mRNA levels correlated with the accumulation of the respective end products. DISCUSSION In this study, a new OSC cDNA (LcIMS1) was cloned from cultured Luffa cells by heterologous hybridization with that of G. glabra (licorice) b-amyrin synthase. We tried to express the new OSC in the yeast mutant GIL77 [3], an ergosterol auxotroph lacking a functional lanosterol synthase gene, which lacks endogenous triterpene mono- alcohol and accumulates oxidosqualene inside the cells. As the triterpene synthase activity in a transformed yeast was too low to identify cyclization products, we sought to detect cyclization products in the yeast transformed with the LcIMS1 clone. Expression of LcIMS1 in the mutant yeast resulted in the accumulation of isomultiflorenol. This result is consistent with LcIMS1 encoding isomultiflorenol synthase, an OSC involved in bryonolic acid biosynthesis in cultured Luffa cells. Despite the use of licorice b-amyrin synthase cDNA as the hybridization probe, a cDNA for b-amyrin synthase, which produce b-amyrin as a main triterpene, was not obtained. The major triterpenoid in culured Luffa cells is bryonolic acid, a friedooleanane-type triterpenoid, and oleanane-type triterpenenoids derived from b-amyrin have not been isolated from cultured Luffa cells. As oleanane- type triterpene saponins, together with dammarane-type triterpene saponins, were isolated from the aerial parts of Luffa [28], additional OSC genes for b-amyrin synthase may be obtained if a cDNA library prepared from green tissues or a genomic library is screened. Although many cDNAs of OSCs have been cloned from higher plants, there are few reports [6,14] on their gene expression in the context of the physiological regulation of sterol and triterpenoid biosynthesis. Molecular cloning of the isomultiflorenol synthase gene will provide a useful tool not only for elucidating bryonolic acid biosynthesis in cultured Luffa cells, but also for studying the regulation of the branch point in triterpenoid biosynthesis to supply diverse triterpene molecules in plant cells. Further experiments are under way to confirm the regulation of the isomultiflorenol synthase gene. ACKNOWLEDGEMENT The authors are grateful to Dr W. 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Columbia University Press, New York. q FEBS 2001 Cloning of isomultiflorenol synthase (Eur. J. Biochem. 268) 6317 . Molecular cloning and characterization of isomultiflorenol synthase, a new triterpene synthase from Luffa cylindrica , involved in biosynthesis of bryonolic. genes and of the accumulation of bryonolic acid and phytosterols. Keywords: bryonolic acid biosynthesis; isomultiflorenol synthase; Luffa cylindrica; triterpene