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

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; P45

by expressed sequence tag mining and functional

expression assay

Masaaki Shibuya1, Masaki Hoshino1, Yuji Katsube1, Hiroaki Hayashi2, Tetsuo Kushiro1, and

Yutaka Ebizuka1

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: yebiz@mol.f.u-tokyo.ac.jp

(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-3b,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.

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-3 β,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.

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, http://www.tigr.org/tigr-scripts/tgi/

T_index.cgi?species¼ 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-hy4-hy-droxylase

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.

(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 14C-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

C D

E

R 3 D E

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.

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.

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-soyasapogetri-O-acetyl-soyasapoge-nol 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.

1D-NMR (1H- and13C-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.

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 otriter-pened

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 free extract; E: using 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.

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

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,

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

using Ex Taq DNA polymerase (Takara Bio Inc, Shiga,

Japan) with template (the cDNA pool described above) and

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,

1 day Then, 1 mL of 40% galactose solution (final concen-tration 2%), 0.2 mL of hemin chloride solution (final

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

for 2 min, then temperature increased at the rate of

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

was added (final concentration 2%) The cells were incuba-ted under the same conditions for 1 day, harvesincuba-ted 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,

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

enzyme solution and the substrate (b-amyrin or sophoradiol

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,

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

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

pESC-PSY-CYP93E1 was inoculated in 1 L of SCR-U medium

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

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,

frac-tions containing the products were combined, evaporated

(1.4 mg), and again applied on a silica gel column (2 g of

77.00 in13C) as an internal standard

s), 0.88 (3H, s), 0.93 (3H, s), 1.13 (3H, s), 1.25 (3H, s), 3.34

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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