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

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

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

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-

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

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

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

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.

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

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-

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

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-

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,

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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