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

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)

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

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

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

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

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

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

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. Kamisako (Professor Emeritus,
Mukogawa Women’s University) for the gift of authentic isomulti-
florenol and bryonolic acid.
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