Tài liệu Báo cáo khoa học: Identification of b-amyrin and sophoradiol 24-hydroxylase by expressed sequence tag mining and functional expression assay docx

Identification of b-amyrin and sophoradiol 24-hydroxylase
by expressed sequence tag mining and functional
expression assay
Masaaki Shibuya
1
, Masaki Hoshino
1
, Yuji Katsube
1
, Hiroaki Hayashi
2
, Tetsuo Kushiro
1
, and
Yutaka Ebizuka
1
1 Graduate School of Pharmaceutical Sciences, The University of Tokyo, Japan
2 Gifu Pharmaceutical University, Japan
Triterpene saponins are glycosides of cyclic C30 terpe-
nes and include a number of active constituents of
medicinal plants, as exemplified by glycyrrhizin in
Glycyrrhiza glabra, ginsenosides in Panax ginseng, sai-
kosaponins in Bupleurum falcatum, etc. [1]. Extensive
pharmacological studies on triterpene saponins from
medicinal plants revealed their important biological
activities. For example, ginsenosides and ⁄ or their agly-
cones show various activities including central nervous
system-stimulating (or -suppressing) activity, and anti-
cancer activity, etc. [2]. Their distribution is not limited
to medicinal plants. They are rather ubiquitously distri-
buted in the plant kingdom. Legumes such as Glycine

max, Pisum sativum, and Medicago sativa are known
as rich sources of triterpene saponins [1]. Recently,
avicins, saponins isolated from the Australian desert
tree Acacia victoriae (Leguminosae), have been repor-
ted to induce apoptosis in tumor cells (Jurkat human T
cell line) by affecting mitochondrial function and are
promising anticancer agents [3]. Despite these promis-
ing activities for medicinal use, great difficulties in
obtaining sufficient quantities of these triterpene
Keywords
b-amyrin 24-hydroxylase; CYP93E1; Glycine
max; P450; sophoradiol 24-hydroxylase
Correspondence
Y. Ebizuka, Graduate School of
Pharmaceutical Sciences, The University of
Tokyo, Hongo Bunkyo-ku, Tokyo 113–0033,
Japan
Fax: +81 3 5841 4744
Tel: +81 3 5841 4740
E-mail:
(Received 28 October 2005, revised 12
December 2005, accepted 23 December
2005)
doi:10.1111/j.1742-4658.2006.05120.x
Triterpenes exhibit a wide range of structural diversity produced by a
sequence of biosynthetic reactions. Cyclization of oxidosqualene is the ini-
tial origin of structural diversity of skeletons in their biosynthesis, and sub-
sequent regio- and stereospecific hydroxylation of the triterpene skeleton
produces further structural diversity. The enzymes responsible for this
hydroxylation were thought to be cytochrome P450-dependent mono-

oxygenase, although their cloning has not been reported. To mine these hy-
droxylases from cytochrome P450 genes, five genes (CYP71D8, CYP82A2,
CYP82A3, CYP82A4 and CYP93E1) reported to be elicitor-inducible genes
in Glycine max expressed sequence tags (EST), were amplified by PCR, and
screened for their ability to hydroxylate triterpenes (b-amyrin or sophora-
diol) by heterologous expression in the yeast Saccharomyces cerevisiae.
Among them, CYP93E1 transformant showed hydroxylating activity on
both substrates. The products were identified as olean-12-ene-3 b,24-diol
and soyasapogenol B, respectively, by GC-MS. Co-expression of CYP93E1
and b-amyrin synthase in S. cerevisiae yielded olean-12-ene-3b,24-diol. This
is the first identification of triterpene hydroxylase cDNA from any plant
species. Successful identification of a b-amyrin and sophoradiol 24-hydroxy-
lase from the inducible family of cytochrome P450 genes suggests that other
triterpene hydroxylases belong to this family. In addition, substrate specific-
ity with the obtained P450 hydroxylase indicates the two possible biosyn-
thetic routes from triterpene-monool to triterpene-triol.
Abbreviations
EST, expressed sequence tags.
948 FEBS Journal 273 (2006) 948–959 ª 2006 The Authors Journal compilation ª 2006 FEBS
saponins from natural sources and ⁄ or by chemical syn-
thesis prevent them from being used in clinical trials. If
triterpene saponins are to be developed as therapeutic
agents, the problem of supply must be resolved. As the
practical supply of triterpene saponins by chemical syn-
thesis is difficult both in terms of quantity and cost,
biological production has been considered to be an
alternative method to obtain them in sufficient quanti-
ties. Production by plant cell or hairy root cultures as
a source of triterpene saponins has been attempted for
decades, but without practical success so far [4–6]. In

order to improve the biological production method, a
detailed understanding of the biosynthesis of triterpene
saponins is required, including the enzymes catalyzing
the sequence of reactions and the genes encoding these
enzymes.
The biosynthesis of triterpene saponins involves the
initial cyclization of 2,3-oxidosqualene, a common pre-
cursor of all the sterol and triterpene biosyntheses, into
various cyclic triterpenes, followed by oxidative modifi-
cation of these carbon skeletons and transfer of the
sugar moiety (Fig. 1). More than 80 different types
of skeleton are generated at the cyclization step [7].
Successful cloning of oxidosqualene cyclases in recent
years has disclosed the molecular origin of the skeletal
diversity of triterpenes [8–24]. It is notable that multi-
functional triterpene synthases yielding more than two
products exist in plants in addition to single-product-
specific triterpene synthase and contribute to the
skeletal diversity of triterpenes. Subsequent regio- and
stereospecific hydroxylations of the skeleton produce
further structural diversity. In contrast to the rapid
progress in the research on skeletal formation, little is
known about the subsequent oxidation and sugar
transfer reactions. Enzymological studies indicated that
oxidation of inert methylene and methyl groups of tri-
terpene skeletons is mediated by cytochrome P450
monooxygenase (P450) [25,26]. However, no gene
encoding the triterpene-hydroxylating P450 has yet
been reported.
In general, purification of microsomal P450 enzymes

from higher plants for amino-acid sequencing is diffi-
cult because a number of P450 exist even in a single
plant species. For example, 272 P450 genes were found
in the Arabidopsis thaliana genome [27,28], whose
products may be very similar in physical properties
and therefore be difficult to separate from each other.
Therefore, the reverse genetic method is not practical
for cloning P450 involved in triterpene biosynthesis.
An alternative approach by functional analysis of
heterologously expressed P450 based on genomic
sequences or expressed sequence tags (EST) appeared
HO
β-Amyrin
HO
Sophoradiol
OH
HO
Olean-12-ene-3β,24-diol
HO
HO
Soyasapogenol B
OH
HO
RO
R= -GlcA-Gal, Soyasaponin III
etc.
OH
HO
O
2,3-Oxidosqualene

3
24
22
Fig. 1. Biosynthesis of soyasaponin.
M. Shibuya et al. CYP93E1
FEBS Journal 273 (2006) 948–959 ª 2006 The Authors Journal compilation ª 2006 FEBS 949
promising, which requires information on the reaction
catalyzed (what is substrate, and what is product, etc.).
However, phytochemical information on A. thaliana
metabolites is still lacking [29], and the details of tri-
terpene metabolism need further study. In a recent
review, some 50 terpenes were listed as A. thaliana
metabolites [29]. The 15 triterpenes in the list, however,
were not isolated from the plant itself, but they were
identified as the products of heterologously expressed
triterpene synthases [10,14,17,20,24]. Although the
presence of some triterpenes (lupeol, b-amyrin, etc.)
was confirmed in the whole plant by GC-MS analysis
(data not shown), none of hydroxylated triterpenes
and their glycosides has been identified. The lack of
understanding of triterpene metabolism in A. thaliana
makes it difficult to identify the function of each P450
from the A. thaliana genome, even though there are
not more than 272.
In this study, an alternative approach based on EST
information was taken to clone triterpene-hydroxylat-
ing P450 from soybean (Glycine max). G. max is one
of the most important crops in the world, and the
accumulated EST information revealed the existence
of more than 200 types of P450 genes (TIGR Soy-

bean Gene Index, />T_index.cgi?species ¼ soybean). Fourteen of them
have been obtained in full length, including cinnamic
acid 4-hydroxylase [30], isoflavone synthase [31], di-
hydroxypterocarpan 6a-hydroxylase [32], and flavonoid
6-hydroxylase [33].
G. max produces triterpene saponins known as
soyasaponins. More than 10 types of soyasaponin have
been isolated, all of which are glycosides of oleanene
triterpene [34]. Their aglycone structures are restricted
to two, soyasapogenols A and B (Fig. 2). Soyasapoge-
nol A has four hydroxyl groups at C-3, C-21, C-22,
and C-24, whereas soyasapogenol B has three at C-3,
C-22, and C-24 [34]. In addition to these two agly-
cones, soyasapogenols C and D (dehydrated or oxi-
dized soyasapogenol B at the C-22 hydroxyl group,
respectively) were reported [35]. However, saponins
with soyasapogenols C and D as aglycone have not
been isolated. Therefore, they are considered to be
artifacts during the isolation procedure [35]. This evi-
dence reduces the potential number of triterpene
hydroxylases responsible for soyasaponin biosynthesis.
Two possible routes from b-amyrin to soyasapogenol
B shown in Fig. 1 indicate the presence of four types
of hydroxylase. Biosynthetic route for soyasapogenol
A is not as simple as that for soyasapogenol B, and
the presence of additional hydroxylases must be
considered. Fortunately, the aglycone of the major
soyasaponins is soyasapogenol B, and glycosides of
soyasapogenol A are minor saponins in G. max. This
abundance ratio strongly suggests high level of tran-

scription of 22- and 24-hydroxylase genes.
Soyasaponin biosynthesis in cell suspension cultures
of G. glabra is reported to be induced by methyl
jasmonate [36]. In this study, functional analysis of
elicitor-inducible P450s already isolated as EST from
G. max, was carried out by heterologous expression
in yeast.
Results and Discussion
P450 genes induced by elicitors in the cell culture
of G. max
Accumulation isoflavones phytoalexin glyceollins, in
the seedling infected with Phytophythora sojae or in
the cell cultures treated with a glucan elcicitor from
this oomycete have been reported [37]. Their accumu-
lation was caused by transcriptional activation of
their biosynthetic genes [37]. Such activation has also
been reported in other legumes, Phaseolus vulgaris
[38] and Glycyrrhiza echinata [39]. Not only isoflavo-
noids, but also soyasaponins are induced by methyl
jasmonate in the cell cultures of G. glabra. The triter-
pene biosynthetic genes, namely squalene synthase
and b-amyrin synthase, are transcriptionally activated
[36], suggesting that triterpene hydroxylase genes
might also be elicitor inducible. Nine full-length cyto-
chrome P450 genes were isolated from G. max as the
genes inducible by the yeast extract elicitor [30], three
of which were shown to encode cinnamic acid 4-hy-
droxylase (CYP73A11) [30], flavonoid 6-hydroxylase
HO
Soyasapogenol B

OH
HO
HO
Soyasapogenol A
OH
HO
OH
HO
Soyasapogenol C
HO
HO
Soyasapogenol E
HO
O
3
24
21
22
Fig. 2. Sapogenols in Glycine max.
CYP93E1 M. Shibuya et al.
950 FEBS Journal 273 (2006) 948–959 ª 2006 The Authors Journal compilation ª 2006 FEBS
(CYP71D9) [33], and dihydroxypterocarpan 6a-hy-
droxylase (CYP93A1) [32], leaving the remaining
clones (CYP71A9, CYP71D8, CYP82A2, -3, and -4,
CYP93A3) unidentified. As none of CYP82A sub-
family member has been identified for their enzyme
function, they are chosen in this study with the expec-
tation of detecting triterpene hydroxylase activity.
Four genes (CYP82A2, -3, and -4 together with
CYP71D8) were cloned using RT-PCR based on the

reported sequences [30] and RNA prepared from
yeast extract-treated cell cultures of G. max. (data not
shown). The genes obtained were ligated into expres-
sion vector pYES2 (Invitrogen) and expressed in
S. cerevisiae strain INVSC2 (Invitrogen). However,
in vitro assay of the cell-free extracts of these trans-
formants with
14
C-labeled b-amyrin as a substrate did
not show any hydroxylase activity, and in vivo assay
by feeding the same substrate to the culture of each
transformant did not yield any detectable hydroxylat-
ed product (data not shown).
Function of genes belonging to the CYP93 family
The CYP93 family consists of five subfamilies inclu-
ding several members with identified functions. Elici-
tor-inducible CYP93A1, CYP93B1, and CYP93C2
encode dihydroxypterocarpan 6a-hydroxylase [32] (2S)-
flavanone-2-hydroxylase [40], and isoflavone synthase
[31], respectively. As the majority of CYP93 family
members are flavonoid biosynthesis-related genes,
other members of this family, including CYP93D1
(found in the A. thaliana genome sequence, EMBL
and GenBank accession number AB010697; protein id
BAB11147.1) and CYP93E1 (inducible by infection
with P. sojae and obtained together with CYP93C2
from G. max), were considered to be flavonoid bio-
synthesis-related genes. Despite extensive trials, failure
to detect isoflavone synthase activity in CYP93E1
suggests its monooxygenase activity towards other

substrates [31]. As the production of not only
flavonoid but also of triterpene saponins is induced by
elicitation as mentioned above, triterpene hydroxylase
is one possible function of CYP93E1.
Feeding of b-amyrin and sophoradiol to
S. cerevisiae transformed with pESC-CYP93E1
As reported for brassinosteroid-6-oxidase (CYP85A1)
[41,42] and taxane 10b-hydroxylase [44], enzyme activ-
ities of heterologously expressed P450s were demon-
strated by feeding the substrate to the transformed
yeast. To examine hydroxylating activity toward
b-amyrin and sophoradiol, possible intermediates in
soyasapogenol B biosynthesis (Fig. 1), they were
administered to the transformant (INVSC2 ⁄ pESC-
CYP93E1) after induction of the GAL1 promoter.
Cells were harvested, disrupted by boiling with 20%
KOH ⁄ 50% aqueous methanol solution, and extracted
with hexane. After acetylation, products were analyzed
with GC-MS. Expecting the formation of olean-3b,24-
hydroxy-12-ene from b-amyrin, and soyasapogenol B
from sophoradiol, GC was monitored by the intensity
of the respective base peaks (m ⁄ z ¼ 218 or m ⁄ z ¼
216), retro-Diels–Alder fragments at the C-ring, as
shown in Fig. 3. The b-amyrin feeding experiments
generate a peak with the same retention time
(15.4 min) (entry B) as that of authentic sample (entry
A). The MS fragmentation pattern of this peak (B in
Fig. 4) was completely identical to that of the authen-
tic olean-3b,24-diacetoxy-12-ene (A in Fig. 4). This
peak was not observed in the negative controls

(C: without substrate, D: no induction of GAL1 pro-
moter, E: transformant with void vector). These results
confirm that CYP93E1 encodes b-amyrin 24-hydroxy-
lase. 24-Hydroxylase activity was also observed with
sophoradiol, as shown in Fig. 5, indicating that
R
1
R
1
= OAc, R
2
= R
3
= H :
O
-Ac-β-amyrin,
m/z
= 468
R
1
= R
2
= OAc, R
3
= H : 3β,24-diacetoxyolean-12-ene,
m/z
= 526
R
1
= R

3
= OAc, R
2
= H :
di
-
O
-Ac-sophoradiol,
m/z
= 526
R
1
= R
2
= R
3
= OAc :
tri
-
O
-Ac-soyasapogenol B,
m/z
= 584
R
3
R
2
AB
C
D

E
R
3
D
E
C
D
E
C
R
3
= H,
m/z
= 218
R
3
= OAc,
m/z
= 276
m/z
= 216
R
3
= OAc
- AcOH
Fig. 3. Base peaks of oleanene-type triterpenes due to retro-Diels-Alder fragmentation in GC-MS analysis.
M. Shibuya et al. CYP93E1
FEBS Journal 273 (2006) 948–959 ª 2006 The Authors Journal compilation ª 2006 FEBS 951
CYP93E1 has 24-hydroxylase activities for both b-am-
yrin and sophoradiol substrates.

b-amyrin and sophoradiol hydroxylase activities
in the cell-free extract of S. cerevisiae harboring
pESC-CYP93E1
To demonstrate in vitro activity, a cell-free extract was
prepared from the transformed yeast. b-Amyrin or
sophoradiol was incubated with the extract. After
extraction with hexane and acetylation, the products
were analyzed with GC-MS. As shown in Fig. 6, when
b-amyrin was incubated, a peak at 15.4 min corres-
ponding to olean-3b,24-diacetoxy-12-ene was found
in the complete assay mixture (entry B), but not in
the negative controls (C: without substrate, D: boiled
cell-free extract, E: no induction of GAL1 promoter,
E: void vector). The MS fragmentation pattern was
also identical to that of the authentic olean-3b,24-di-
acetoxy-12-ene, except for the presence of several back-
ground peaks (the amount of authentic sample was
adjusted to equalize the height of both peaks in GC).
When the extract was incubated with sophoradiol, a
peak was found at 19.5 min in the complete assay mix-
ture (entry B), as shown in Fig. 7. The major peaks
(m ⁄ z ¼ 201 and 216) in MS fragmentation were identi-
cal to those of the authentic tri-O-acetyl-soyasapogenol
B. To the best of our knowledge, this is the first
demonstration of in vitro hydroxylase activity for a
triterpene substrate in a heterologous expression sys-
tem, although activities of several diterpene hydroxy-
lases were demonstrated in vitro [45–48].
Fig. 4. GC-MS analysis of the extract from transformant fed with b-amyrin. GC was monitored based on intensity of the base peak (m ⁄ z
218), which was a fragment of the D,E-ring moiety due to retro-Diels–Alder fragmentation at the C-ring in olean-3b,24-diacetoxy-12-ene.

Entry A in the upper panel: 20 pmol of authentic olean-3b,24-diacetoxy-12-ene; B: complete conditions as described in Experimental proce-
dures; C: without feeding with b-amyrin; D: without induction of the GAL1 promoter; E: transformant with void vector. MS fragmentations
of entries A and B are shown in the lower panel.
CYP93E1 M. Shibuya et al.
952 FEBS Journal 273 (2006) 948–959 ª 2006 The Authors Journal compilation ª 2006 FEBS
Co-expression of PSY (b-amyrin synthase)
and CYP93E1 in lanosterol synthase-deficient
S. cerevisiae strain GIL77
As shown in Fig. 3, there is no doubt that one hydro-
xyl group is introduced into the A- or B-ring of
b-amyrin, but the present results do not exclude the
possibility that the product has a hydroxyl group at
positions other than C-24, as there is no information
on the retention time and MS fragmentation of such
compounds. Feeding of b-amyrin (50 nmol) to the cul-
tures (20 mL) of the yeast transformed with pESC-
CYP93E1 yielded about 0.2 nmol of hydroxylated
b-amyrin (Fig. 4). Based on this conversion ratio, the
yield of the product after b-amyrin (2.5 mmol, 1 mg)
feeding in 1-L cultures was estimated to be 10 nmol
(4.5 lg). If the efficiency of uptake of b-amyrin from
media is one of the reasons for the low conversion, it
would be improved by in situ supply of the substrate
through coexpression with b-amyrin synthase. In our
previous studies, more than 10 mg of b-amyrin was
produced by 1-L culture of yeast transformant with
the plasmid harboring P. sativum b-amyrin synthase
gene (PSY) [13]. CYP93E1 and PSY genes were sub-
cloned into the S. cerevisiae expression vector pESC
harboring two expression cassettes. Cells were harves-

ted from 1-L of induced culture, lysed by boiling with
20% KOH ⁄ 50% aqueous methanol solution, and
extracted with hexane. The product was purified on sil-
ica gel column to yield 1.0 mg of product as crystals.
Fig. 5. GC-MS analysis of the extract from the transformant fed with sophoradiol. GC was monitored based on the intensity of the base
peak (m ⁄ z 216), which was a fragment of the D,E-ring moiety due to retro-Diels–Alder fragmentation at the C-ring in tri-O-acetyl-soyasapoge-
nol B. Entry A in the upper panel: 20 pmol of authentic tri-O-acetyl-soyasapogenol B; B: complete conditions as described in Experimental
procedures; C: without feeding with b-amyrin; D: without induction of the GAL1 promoter; E: transformant with void vector. MS fragmenta-
tions of entries A and B are shown in the lower panel.
M. Shibuya et al. CYP93E1
FEBS Journal 273 (2006) 948–959 ª 2006 The Authors Journal compilation ª 2006 FEBS 953
1D-NMR (
1
H- and
13
C-NMR) spectra were completely
identical to those reported for olean-12-ene-3b,24-diol
[49], and correlations observed in 2D-NMR (HMQC,
HMBC, and NOESY) further confirmed its identity
(data not shown).
The agylcone of the major soyasaponins in G. max is
soyasapogenol B, which is biosynthesized via two hy-
droxlyations at C-22 and C-24 of b-amyrin. In this
study, CYP93E1 was demonstrated to hydroxylate the
methyl group (C-24) of both b-amyrin and sophoradiol.
This result indicates that CYP93E1 has substrate specif-
icity for the 3-hydroxyolean-12-ene structure, and a
hydroxyl group at C-22 was not recognized. Hydroxyla-
tion only at C-24 methyl group points to very strict
regiospecificity for hydroxylation. To further investigate

substrate specificity, CYP93E1 was coexpressed with
YUP8H12R.43, a multifunctional triterpene synthase
(protein ID; AAC17070.1, BAC clone, EMBL and Gen-
Bank accession number; AC002986) from A. thaliana
producing lupeol, butyrospermol, tirucalla-7,21-dien-
3b-ol, taraxasterol, b-amyrin, w-taraxasterol, bauerenol,
a-amyrin, and multiflorenol [14], in the same manner as
above. No hydroxylated products other than olean-
3b,24-diacetoxy-12-ene were detected in GC-MS ana-
lysis (data not shown). Among the nine triterpenes
with different skeletons produced by A. thaliana
YUP8H12R.43, only b-amyrin was hydroxylated, sug-
gesting the strict substrate specificity of CYP93E1 for
the 3-hydroxyolean-12-ene structure.
The 24-hydroxylase activity of CYP93E1 for b-amy-
rin and sophoradiol suggests that the biosynthesis
of soyasaponin might form a metabolic grid, via
Fig. 6. GC-MS analysis of in vitro reaction products with b-amyrin as a substrate. GC was monitored based on the intensity of the base peak
(m ⁄ z 218) as described in the legend to Fig. 4. Entry A in the upper panel: authentic olean-3b,24-diacetoxy-12-ene (injected amount was not
determined); B: complete conditions as described in Experimental procedures; C: removal of b-amyrin from complete conditions; D: using
boiled cell-free extract; E: using cell-free extract prepared from the transformant with no induction of the GAL1 promoter; F: using cell-free
extract prepared from the transformant with void vector. MS fragmentations of entries A and B are shown in the lower panel.
CYP93E1 M. Shibuya et al.
954 FEBS Journal 273 (2006) 948–959 ª 2006 The Authors Journal compilation ª 2006 FEBS
olean-12ene-3b,24-diol or sophoradiol from b-amyrin
to soyasapogenol B (Fig. 1), and the hydroxylation of
b-amyrin or olean-12-ene-3b,24-diol at the C-22 posi-
tion is catalyzed by other P450, probably an enzyme
similar to CYP93E1. A similar metabolic grid branch-
ing at the hydroxylation of campestanol by two inde-

pendent P450 (6-hydroxylase and 22-hydroxylase) and
joining at the formation of castasterone by the same
enzymes was proposed in brassinosteroid biosynthesis
[42].
Not only glycosides but also triterpene aglycones
show interesting biological activities. For example,
soyasapogenol B has hepatoprotective activity [50],
and oleanolic acid and ursolic acid show anti-inflam-
matory and antitumor-promoting activities [51], etc.
As the supply of triterpenes including oxygenated
derivatives through organic synthesis is not practical,
these compounds must be isolated from natural
sources. Successful production of olean-12-ene-3b,24-
diol by coexpression of b-amyrin synthase and triter-
pene hydroxylase in S. cerevisiae in this study opened
a way for the production of useful oxygenated triterpe-
nes through fermentation. This methodology will be
useful for the production of triterpene saponins after
cloning of the sugar transferases.
As all CYP93 family members thus far identified are
flavonoid biosynthesis-related enzymes, it was unex-
pected that CYP93E1 would encode b-amyrin and
sophoradiol 24-hydroxylase. The identification of
CYP93E1 as triterpene hydroxylase implies that the
function of other members of the CYP93E subfamily
are not necessarily related to flavonoid biosynthesis.
Fig. 7. GC-MS analysis of in vitro reaction products with sophoradiol as a substrate. GC was monitored based on the intensity of the base
peak (m ⁄ z 216) as described in the legend to Fig. 5. Entry A in the upper panel: authentic tri-O-acetyl-soyasapogenol B (injected amount was
not determined); B: complete conditions as described in Experimental procedures; C: removal of b-amyrin from complete conditions; D:
using boiled cell-free extract; E: using cell-free extract prepared from the transformant with no induction of the GAL1 promoter; F: using cell-

free extract prepared from the transformant with void vector. MS fragmentations of entries A and B are shown in the lower panel.
M. Shibuya et al. CYP93E1
FEBS Journal 273 (2006) 948–959 ª 2006 The Authors Journal compilation ª 2006 FEBS 955
Cloning of the full-length cDNA and functional analy-
sis of several other EST clones belonging to this sub-
family is in progress. On the other hand, sterol
oxygenases thus far identified do not show high
sequence homology and are assigned to different CYP
families (obtusifoliol 14a-demethylase; CYP51 [52],
brassinosteroid 6-oxidase; CYP85A1 [41,42], brassino-
steroid 22a-hydroxylase; CYP90B1 [43]). Therefore,
mining of triterpene hydroxylase from other members
of the CYP family must be continued.
Experimental procedures
RNA extraction and amplification of CYP93E1
cDNA
Soybean seeds (cultivar Wase–Edamame purchased from
Atariya Nouen, Tokyo, Japan) were germinated in a
growth cabinet under 16-h daylight and 8-h dark conditions
at 25 °C. After 2 weeks, the leaves (4.7 g) were detached,
immediately frozen in liquid nitrogen, and homogenized
with a mortar and pestle. RNA was extracted with the phe-
nol-chloroform method as reported previously [8], to give
98 lg of total RNA. A single-strand cDNA pool was pre-
pared with reverse transcriptase (Superscript II, Invitrogen,
Carlsbad, CA, USA) and 0.5 lg of oligo dT primer,
RACE32 (5¢-GACTCGAGTCGACATCGATTTTTTTTT
TTTTT-3¢) with dNTP (0.2 mm) in a volume of 20 lL fol-
lowing the manufacturer’s recommended protocol. The
resulting cDNA mixture was used as the template in subse-

quent PCR. The open reading frame of CYP93E1 was
amplified by PCR (initial denaturing for 5 min at 94 °C, 30
cycle of 94 °C for 1 min, 65 °C for 2 min, and 72 °C for
3 min, and a final extension reaction for 10 min at 72 °C)
using Ex. Taq DNA polymerase (Takara Bio Inc, Shiga,
Japan) with template (the cDNA pool described above) and
primers (5¢-AAACACTAGTATGCTAGACATCAAAGG
CTAC-3¢, and 5¢-TTCAATCGATTCAGGCAGCGAACG
GAGTGAA-3¢), which were synthesized based on the
reported sequences. The obtained clone was sequenced in
both strands. This sequence has been submitted to the
DDBJ sequence database and is available under accession
number AB231332.
Construction of S. cerevisiae expression vector
pESC-CYP93E1 and S. cerevisiae transformant
The amplified cDNA fragment was ligated into the restric-
tion enzyme sites (SpeI and ClaI) of pESC-URA (Invitro-
gen) after digestion with these enzymes. The plasmid
obtained was designated pESC-CYP93E1. S. cerevisiae
strain INVSC2 (Invitrogen) was transformed with pESC-
CYP93E1 using a Frozen-EZ Yeast Transformation II kit
(Zymo Research, California, USA).
Feeding of b-amyrin and sophoradiol to the
transformant with pESC-CYP93E1
The transformant with pESC-CYP93E1 was inoculated in
20 mL of synthetic complete medium [53] without uracil,
containing hemin chloride (13 lgÆmL
)1
), and raffinose (2%)
in place of glucose (SCR-U), and incubated at 30 °C for

1 day. Then, 1 mL of 40% galactose solution (final concen-
tration 2%), 0.2 mL of hemin chloride solution (final
concentration 13 lgÆmL
)1
), and the substrate (50 nmol of
b-amyrin or sophoradiol, in methanol solution) were added.
Cells were incubated under the same conditions for 1 day
and then harvested by centrifugation at 500 g for 5 min.
After the addition of 0.25 mL of 40% potassium hydroxide
solution and 0.25 mL of methanol, the cell suspension was
boiled for 5 min. Products were extracted three times with
0.2 mL of hexane and evaporated. Pyridine and acetic
anhydride 0.01 mL each were added to the residue, and
then the mixture was left to stand at room temperature
overnight. The reaction was terminated by the addition of
0.1 mL each of methanol and water. The products were
extracted three times with hexane (0.2 mL). After evapor-
ation, the residue was dissolved in 0.02 mL of hexane, and
0.001 mL of hexane solution was used for GC-MS analysis
using a Shimadzu (Kyoto, Japan) GCMS-QP2010 with a
Restec Rtx-5MS glass capillary column (30 m in length,
0.25 mm in diameter, 0.25-lm film thickness) and He as a
carrier gas (45 cmÆmin
)1
) on the program (held at 230 °C
for 2 min, then temperature increased at the rate of
10 °CÆ min
)1
until 330 °C). The temperature of the ioniza-
tion chamber was 250 °C, and ionization was by electron

impact at 70 eV.
In vitro assay of b-amyrin and sophoradiol
hydroxylase using cell-free transformant extracts
The transformant with pESC-CYP93E1 was inoculated in
20 mL of SCR-U medium (described above), and incubated
at 30 °C for 1 day. Then, 1 mL of 40% galactose solution
was added (final concentration 2%). The cells were incuba-
ted under the same conditions for 1 day, harvested by cen-
trifugation at 500 g for 5 min, resuspended in 0.1 mL of
50 mm potassium phosphate buffer (pH 7.5, containing
10% sucrose, 5 mm EDTA, and 14 mm 2-mercaptoetha-
nol), and broken using a Beat-beater (Biospec Products,
Oklahoma, USA) with glass beads (0.4–0.6 mm diameter,
one-third of total volume) at 4 °C. Then an additional
0.4 mL of the same buffer was added to suspend broken
cells. Cell homogenates were centrifuged at 3000 g for
5 min. The supernatant (0.4 mL) was used as the enzyme
solution. With a solution (0.1 mL) of the NADPH-re-
generating system (10 mm NADPH, 38 mm glucose-6-phos-
phate, 2.5 UÆmL
)1
glucose-6-phosphate dehydrogenase), the
enzyme solution and the substrate (b-amyrin or sophoradiol
50 nmol) were incubated for 6 h at 30 °C. The reaction was
CYP93E1 M. Shibuya et al.
956 FEBS Journal 273 (2006) 948–959 ª 2006 The Authors Journal compilation ª 2006 FEBS
terminated by the addition of 0.5 mL of 40% potassium
hydroxide solution. After boiling for 5 min, products were
extracted twice with 1 mL of hexane. The hexane extract
was evaporated, acetylated, and analyzed using GC-MS fol-

lowing the same procedure as described above.
Production of olean-12-ene-3b,24-diol by
coexpression of CYP93E and b-amyrin synthase
in lanosterol synthase-deficient S. cerevisiae
strain GIL77
Two oligo DNAs (5¢-CTTCGTCGACAAGATGTGGAG
GTTGAAGATA-3¢ and 5¢-GTCCGCTAGCTCAAGGCA
AAGGAACTCTTCT-3¢), corresponding to the N- and
C-terminal sequences of b-amyrin synthase from P. sativum,
were synthesized. The open reading frame was amplified by
PCR with these primers and the plasmid pYES-PSY [13] as
a template under the same conditions as described above,
except that the annealing temperature was 58 °C. The
amplified DNA fragment was ligated into the restriction
enzyme sites (SalI and NheI) of pESC after digestion with
these enzymes. The plasmid obtained was designated pESC-
PSY. S. cerevisiae strain GIL77 [8] was transformed with
pESC-PSY. Production of b-amyrin by the resulting trans-
formant was confirmed following the reported method [13].
pESC-CYP93E1 and pESC-PSY were digested with restric-
tion enzymes (SalI and ClaI). The fragment containing
CYP93E1 and promoters (GAL1 ⁄ GAL10) from pESC-
CYP93E1 was separated upon agarose gel electrophoresis
and ligated into SalI and ClaI-digested and dephosphory-
rated pESC-PSY. The resulting plasmid was designated
pESC-PSY-CYP93E1. S. cerevisiae strain GIL77 was trans-
formed with this plasmid using the same method as
described above. The transformant with pESC-PSY-
CYP93E1 was inoculated in 1 L of SCR-U medium (des-
cribed above), and incubated at 30 °C for 1 day. Then,

50 mL of 40% galactose solution was added (final concen-
tration 2%). The cells were incubated under the same con-
ditions for 1 day, harvested by centrifugation at 500 g for
5 min, resuspended in 100 mL of 0.1 m potassium phos-
phate buffer (pH 7.0) supplemented with 2% glucose and
hemin chloride (13 lgÆmL
)1
), and further incubated at
30 °C for 24 h. Then, the cells were collected and suspen-
ded in 25 mL of 40% potassium hydroxide and 25 mL of
methanol and refluxed for 2 h. Products were extracted
with 50 mL of hexane. The hexane layer was washed with
25 mL of saturated sodium bicarbonate. Extraction was
repeated three times, and then the hexane layers were com-
bined, dehydrated with sodium sulfate, and evaporated.
The residue was applied on a silica gel column (4 g of
Wako FC-40, Wako Pure Chemical Industries, Osaka,
Japan) with the eluent (benzene:acetone ¼ 4 : 1). The frac-
tions containing the products were combined, evaporated
(1.4 mg), and again applied on a silica gel column (2 g of
Wako FC-40) with the eluent (hexane:ethyl acetate ¼ 4:1)
to isolate the product (1.0 mg) of GIL77 ⁄ pESC-PSY-
CYP93E1.
1
H- and
13
C-NMR spectra were measured in
CDCl
3
(ECA, JEOL, Tokyo, Japan,

1
H: 500 MHz,
13
C:
125 MHz) with the signal of CHCl
3
(7.26 p.p.m. in
1
H,
77.00 in
13
C) as an internal standard.
1
H-NMR (500 MHz, CDCl
3
): d 0.82 (3H, s), 0.87 (6H,
s), 0.88 (3H, s), 0.93 (3H, s), 1.13 (3H, s), 1.25 (3H, s), 3.34
(1H, d, J ¼ 11.5 Hz, C-24), 3.45 (1H, dd, J ¼ 5.5 Hz,
11.5 Hz, C-3), 4.21 (1H, d, J ¼ 11.5 Hz, C-24), 5.17(1H, t,
J ¼ 3.5 Hz, C-12).
13
C-NMR (125 MHz, CDCl
3
): d 16.0, 16.7, 18.4, 22.3,
23.7, 23.8, 25.9, 26.1, 26.9, 27.7. 28.4, 31.1, 32.5, 32.8, 33.3,
34.7, 36.6, 37.1, 38.3, 39.8, 41.7, 42.8, 46.8, 47.2, 47.7, 55.8,
64.5, 80.9, 121.5, 145.2.
Acknowledgements
The authors are grateful to Dr S. Nishimaya (Meiji
Seika Kaisha, Ltd) for the gift of authentic olean-12-

ene-3b,24-diol, sophoradiol, and soyasapogenol B. A
part of this research was financially supported by
a Grant-in-Aid for Scientific Research (S) (No.
15101007) to Y.E. from the Ministry of Education,
Culture, Sports, Science and Technology, Japan.
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