Biosynthesis of sesterterpenes, head-to-tail triterpenes, and sesquarterpenes in Bacillus clausii: Identification of multifunctional enzymes and analysis of isoprenoid metabolites

Article abstract of DOI:10.1002/cbic.201500138

We performed functional analysis of recombinant enzymes and analysis of isoprenoid metabolites in Bacillus clausii to gain insights into the biosynthesis of rare terpenoid groups of sesterterpenes, head-to-tail triterpenes, and sesquarterpenes. We have identified an (all-E)-isoprenyl diphosphate synthase (E-IDS) homologue as a trifunctional geranylfarnesyl diphosphate (GFPP)/hexaprenyl diphosphate (HexPP)/heptaprenyl diphosphate (HepPP) synthase. In addition, we have redefined the function of a tetraprenyl-β-curcumene synthase homologue as that of a trifunctional sesterterpene/triterpene/sesquarterpene synthase. This study has revealed that GFPP, HexPP, and HepPP, intermediates of two isoprenoid pathways (acyclic terpenes and menaquinones), are biosynthesized by one trifunctional E-IDS. In addition, GFPP/HexPP and HepPP are the primary substrates for the biosynthesis of acyclic terpenes and menaquinone-7, respectively. Multifunctional terpene biosynthetic enzymes: Geranylfarnesyl diphosphate (GFPP), hexaprenyl diphosphate (HexPP), and heptaprenyl diphosphate (HepPP) are intermediates in two isoprenoid pathways and are biosynthesized by one trifunctional (all-E)-isoprenyl diphosphate synthase. GFPP/HexPP and HepPP are mainly utilized for the biosynthesis of acyclic terpenes and menaquinone-7, respectively.

Full text of DOI:10.1002/cbic.201500138

Accepted Article
Title: Biosynthesis of Sesterterpene, Head-to-tail Triterpene, and Sesquarterpene in
Bacillus clausii: Identification of Multifunctional Enzymes and Analysis of Isop-
renoid Metabolites
Authors: Daijiro Ueda; Hiroaki Yamaga; Mizuki Murakami; Yusuke Totsuka; Tetsuro
Shinada; Tsutomu Sato
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To be cited as: ChemBioChem 10.1002/cbic.201500138
Link to VoR: http://dx.doi.org/10.1002/cbic.201500138
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ChemBioChem
10.1002/cbic.201500138
FULL PAPER
Biosynthesis of Sesterterpene, Head-to-tail Triterpene, and
Sesquarterpene in Bacillus clausii: Identification of
Multifunctional Enzymes and Analysis of Isoprenoid Metabolites
[
a]
[a]
[a]
[b]
[b]
Daijiro Ueda, Hiroaki Yamaga, Mizuki Murakami, Yusuke Totsuka, Tetsuro Shinada, and
[
a]
Tsutomu Sato *
Abstract: We performed functional analysis of recombinant
enzymes and analysis of isoprenoid metabolites in Bacillus clausii to
gain insights into the biosynthesis of rare terpenoids of
sesterterpenes, head-to-tail triterpenes, and sesquarterpenes. We
have identified an (all-E)-isoprenyl diphosphate synthases (E-IDS)
1), is a new type of terpene cyclase with no sequence homology
[
3d]
to any known terpene synthase. HepPP is biosynthesized with
IPP and farnesyl diphosphate (FPP; C15) by heterodimeric E-IDS
(HepS/T), and is utilized to produce menaquinone-7 (MK-7)
(Scheme 1). In addition, B. megaterium tetraprenyl-β-curcumene
cyclase (TC) is a bifunctional enzyme that acts as an onoceroid
homolog
as
GFPP)/hexaprenyl diphosphate (HexPP)/heptaprenyl diphosphate
HepPP) synthase. In addition, we have redefined the function of
homolog as trifunctional
a
trifunctional
geranylfarnesyl
diphosphate
[
3e, 4]
(
(
synthase (Scheme 1).
We recently reported that the TS homolog from Bacillus clausii
(Bcl-TS) catalyzes the conversion of (all-E)-geranylfarnesyl
diphosphate (GFPP) and (all-E)-hexaprenyl diphosphate
(HexPP) into β-geranylfarnesene (1) and β-hexaprene (2),
respectively, in vitro (Scheme 2) and that both compounds are
tetraprenyl-β-curcumene synthase
a
sesterterpene/triterpene/sesquarterpene synthase. The present
study revealed that GFPP, HexPP, and HepPP, intermediates of two
isoprenoid pathways (acyclic terpenes and menaquinones), are
biosynthesized by one trifunctional E-IDS. In addition, GFPP/HexPP
and HepPP are the primary substrates for biosynthesis of acyclic
terpenes and menaquinone, respectively.
[5]
produced in B. clausii. Thus, we proposed that Bcl-TS is a
sesterterpene (C25)/triterpene (C30 synthase, and that TS
)
homologs in many bacteria represent a new family of terpene
synthases that form sesterterpene and triterpene as well as
[
5]
sesquarterpene. However, it remains unclear whether GFPP
and HexPP, which are the substrates for Bcl-TS, are
biosynthesized in B. clausii. Biosynthesis of sesterterpenes and
head-to-tail triterpenes is not fully understood. Only one
sesterterpene synthase (ophiobolin F synthase), which utilizes
Introduction
Isoprenoids (terpenoids) make up the largest group of natural
products, including more than 50,000 structurally diverse
GFPP as a substrate, has been identified in addition to Bcl-
[
1]
compounds. They are categorized by the number of C
5
[6]
TS.
Many tail-to-tail triterpene synthases, squalene, and
isoprene units: hemi- (one C
sesqui- (C15; three C units), di- (C20; four C
five C units), tri- (C30; six C
eight C
5
unit), mono- (C10; two C
5
units),
oxidosqualene cyclases, have been found from animal, plant,
5
5
units), sester- (C25
5
units), and tetra-terpenes (C40
;
;
[7]
fungi, and bacteria. However, a head-to-tail triterpene synthase
5
that uses HexPP as a substrate has never been found, except
for Bcl-TS. We recently synthesized unnatural head-to-tail
pentacyclic triterpenes by the in vitro enzymatic reaction of TC
[
1]
5
units).
Isoprenoid biosynthesis begins with the
condensation of isopentenyl diphosphate (IPP) into dimethylallyl
diphosphate (DMAPP) by isoprenyl diphosphate synthases
[8]
with acyclic triterpene 2 (Scheme 2).
[
2]
(
IDSs). (all-E)- and E,Z-mixed isoprenyl diphosphates are
[9]
[10]
All reported E-IDS types of GFPP, HexPP,
and HepPP
formed by E-IDSs and Z-IDSs, respectively, which have no
sequence similarity with each other. Recent studies have
revealed a unique biosynthetic pathway for sesquarterpenes, the
new family name for C35 terpenes. Tetraprenyl-β-curcumene
synthase (TS), which catalyzes the conversion of a linear C35
isoprenoid, (all-E)-heptaprenyl diphosphate (HepPP), into a
monocycle in Bacillus subtilis and Bacillus megaterium (Scheme
[11]
synthases have been described as mono-functional enzymes.
However, they form several products in vitro,
[
2]
[9-11]
and isoprenoid
metabolites in vivo have not been reported. In this paper, the
biosynthesis of rare terpenoid groups of sesterterpene, head-to-
tail triterpene, and sesquarterpene are described based on the
identification of multifunctional enzymes and analysis of
isoprenoid metabolites.
[
3]
[
a]
D. Ueda, H. Yamaga, M. Murakami, Prof. Dr. T. Sato,
Department of Applied Biological Chemistry, Faculty of Agriculture,
and Graduate School of Science and Technology, Niigata University,
Ikarashi 2–8050, Nishi-ku, Niigata 950–2181, Japan
E-mail: satot@agr.niigata-u.ac.jp
Results and Discussion
Cloning, expression, and purification of E-IDS1, E-IDS2-S,
and E-IDS2-L
In order to identify GFPP and HexPP in B. clausii, which is
[
b]
Y. Totsuka, Prof. Dr. T. Shinada
Graduate School of Science, Osaka City University
Sugimoto, Sumiyoshi-ku, Osaka 558-8585, Japan
essential
for
demonstrating
that
Bcl-TS
is
a
sesterterpene/triterpene synthase, we performed functional
analysis of all E-IDS homologs in B. clausii. The B. clausii
genome possesses two E-IDS homologs, AB905204 and
Supporting information for this article is given via a link at the end of
the document.

ChemBioChem
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FULL PAPER
AB735676. AB905204 and AB735676 are referred to as E-IDS1
and E-E-IDS2-L, respectively, in this paper. For example,
multiple sequence alignments of E-IDS1 and FPP synthase from
and COSY. The methylidene protons of 3 at δ
Hz) and δ 5.11 (d, J = 11.0 Hz) exhibited a strong HMBC
correlation with C-2 (δ 139.4, d) and C-3 (δ 146.5, s),
indicating that these protons are assignable to H-1. H-2 (δ 6.50)
H
5.34 (d, J = 17.7
H
C
C
[
12]
Geobacillus stearothermophilus
(identity: 52%) and E-IDS2-L
H
[
11b]
and HepT from B. subtilis
(identity: 53%) are shown in Fig.
exhibited a dd splitting pattern (J = 17.6 and 10.7 Hz) due to the
S1 and S3, respectively. The heterodimer type, which dimerizes
with the E-IDS homolog (HepT, large subunit or component II)
and non-homolog (HepS, small subunit or component I), was
spin-spin couplings with H-1a (trans) and H-1b (cis). The
methylidene protons at δ
assigned to H-29 because of the clear cross-peak with C-2, C-3,
and C-4 (δ 31.9, t) in the HMBC spectrum. Thus, the right side
H H
5.12 (s) and δ 5.11 (s) were
[
11b]
identified in B. subtilis (Scheme 1).
B. clausii possesses a
C
HepS homolog (AB735675: E-IDS2-S) in the gene cluster
including E-IDS2-L. Multiple sequence alignments of E-IDS2-S
of 3 shown in Fig. 5B contains an isoprene moiety. We
determined the structure of the isobutenyl moiety on the left side
of the compound, as shown in Fig. 5B (C-26, C-27, C-28, and C-
35), by HMBC spectral analysis. HMBC data showed five
isopentenyl moieties (represented by squares with broken lines
in Fig. 5B) linked to each other. The complete structure of 3 was
elucidated by connecting the three partial structures obtained
[11b]
and HepS from B. subtilis
(identity: 19%) are shown in Fig.
S2. Therefore, we also used E-IDS2-S to analyze the reaction.
E-IDS1, E-IDS2-S, and E-IDS2-L were cloned into the pCold I
vector, expressed as a soluble protein, and purified by Ni affinity
chromatography (Fig. 1).
from HMBC cross peaks between H-5 (δ
(δ = 124.6–125.0 ppm) and those between H-25 (δ
2.35 ppm) and C-27 (δ = 131.1 ppm). No NOE was observed
for H-6 (δ 5.37–5.43)/H-30 (δ 1.69, s), H-10 (δ 5.37–5.43)/H-
31 (δ 1.74, s), H-14 (δ 5.37–5.43)/H-32 (δ 1.74, s), H-18 (δ
5.37–5.43)/H-33 (δ 1.74, s), and H-22 (δ 5.37–5.43)/H-34 (δ
H
= 2.40 ppm) and C-6
Analysis of products formed by E-IDS1, E-IDS2-S, and E-
IDS2-L
C
H
= 2.30–
C
To characterize the pathway from DMAPP, which is a starter
allylic compound for isoprenoid biosynthesis in all organisms, to
H
H
H
H
H
H
H
H
1
4
GFPP and HexPP, the reaction of DMAPP and [1- C]IPP with
E-IDS1, E-IDS2-S, and E-IDS2-L was analyzed by radio-TLC
H
H
1.74, s), indicating that all prenyl residues in 3 are arranged in
the E configuration. Comparison of the chemical shifts (δ 40.2
ppm) of C-8, C-12, C-16, C-20, and C-24 with those of
(
Fig. 2). DMAPP was converted into FPP (C15
)
and
C
geranylgeranyl diphosphate (GGPP; C20) by E-IDS1, and into
GFPP (C25) and HexPP (C30) by E-IDS2-S/L (mixture of E-IDS2-
S and E-IDS2-L) (Fig. 2 and Fig. 3A).
Next, the reaction of FPP or GGPP, produced from DMAPP
by E-IDS1, with E-IDS2-S/L was analyzed by radio-TLC (Fig. 2).
E-IDS2-S/L produced GFPP (C25) and HexPP (C30) from FPP or
GGPP (Fig. 2). Thus, GFPP and HexPP are also biosynthesized
from FPP and GGPP by E-IDS2-S/L in B. clausii (Fig. 3A).
We thus demonstrated that GFPP and HexPP, the Bcl-TS
substrates, are biosynthesized by E-IDS2-S/L in B. clausii (Fig.
[
13]
ficaprenol (Z, ca. 32.0 ppm; E, ca. 40.0 ppm) further supports
1
13
the E geometry. The complete assignments of H and C NMR
data for 3 unambiguously supported this structure (Table 1).
While β-myrcene (C10), β-farnesene (C15), β-springene (C20), 1,
and 2 have been identified as acyclic terpene hydrocarbons
containing a similar isoprene moiety, compound 3 appears to be
a novel sesquarterpene. We propose that 3 be named β-
heptaprene.
To determine the amount of 1–3 produced by B. clausii, we
performed GC analysis of n-hexane extracts. As shown in Fig.
3B, the amounts of 1–3 per 1 g dry cell weight were 0.96, 0.66,
and 0.065 mg, respectively. The production ratios (%) of
compounds 1–3 were as follows: 1:2:3 = C25:C30:C35 = 57:39:4
3A). E-IDS2-S or E-IDS2-L alone formed no product from
DMAPP, FPP, and GGPP (data not shown); thus, formation of
GFPP and HexPP is mediated by heteromeric E-IDS2-S/L in B.
clausii.
(
Fig. 3A).
Isolation and structural analysis of novel acyclic
sesquarterpene 3
Analysis of menaquinones in B. clausii
(all-E)-Isoprenoids with chain lengths ranging from C25 to C35 are
essential in cell maintenance as the hydrophobic side chains of
quinones. B. subtilis produces MK-7, which possesses a C35
isoprenoid side chain; thus, we hypothesized that B. clausii may
also produce menaquinone. To gain further insights into the
biosynthetic pathways of C25, C30, and C35 isoprenoids, we
performed LC-ESI-MS and HPLC of n-hexane extracts from B.
clausii to assess menaquinone production. Since E-IDS2-S/L
formed GFPP (C25) and HexPP (C30) as the major products (Fig.
2), and compounds 1 (C25) and 2 (C30) were mainly produced as
acyclic terpenes (Fig. 3B), we expected MK-5 and MK-6, which
possess C25 and C30 isoprenoids, are the primary products of B.
clausii. However, MK-7 was the major menaquinone (Fig. 3B).
The amounts of MK-5, MK-6, and MK-7 per 1 g dry cell weight
were 0, 0.035, and 0.365 mg, respectively (Fig. 3B). The
production ratios (%) of isoprenoids in the acyclic terpene and
Unexpectedly, C35 HepPP production was detected in the in vitro
reaction of E-IDS2-S/L (Fig. 2 and Fig. S4). To search for the
sesquarterpene metabolite, n-hexane extracts of B. clausii were
re-analyzed by GC-MS. Injection of n-hexane extracts 10-fold
[
5]
larger than those previously reported
yielded minor peak 3
(
Fig. 4A). We detected no sesquiterpene (C15) or diterpene (C20).
To determine the structure of 3, B. clausii was cultured in 20
flasks containing 1 L medium each, and compound 3 was
isolated by silica gel column chromatography and SiO HPLC to
2
yield 1.1 mg of purified 3.
1
13
The structure of 3 was determined by NMR ( H, C, DEPT,
COSY, NOESY, HSQC, and HMBC) and EI-MS (Fig. 5 and
Table 1). The molecular formula was identified by HR-EI-MS as
C
35
H56. The partial structure (right side in Fig. 5B) of 3 (C-1–C-5
and C-29) was assigned according to data obtained from HMBC

ChemBioChem
10.1002/cbic.201500138
FULL PAPER
menaquinone family were largely different: MK-5:MK-6:MK-7 =
C25:C30:C35 = 0:9:91 (Fig. 3A). In addition, the amount of MK-7
(
(
0.365 mg) was approximately 5.6-fold higher than that of 3
0.065 mg) (Fig. 3B). These results suggest GFPP (C25) and
HexPP (C30) produced by E-IDS2-S/L are mainly utilized for
acyclic terpene (1 and 2) biosynthesis, while HepPP (C35) is
used for MK-7 synthesis (Fig. 3A).
.
Multiple functions of Bcl-TS, E-IDS2-S/L, and E-IDS1
To demonstrate that compound 3 is formed from HepPP by Bcl-
TS, chemically synthesized HepPP was incubated with purified
Bcl-TS. As shown in Fig. 4B and 4C, product 3 was detected by
GC-MS. Thus, Bcl-TS produces 3 as a minor product (Fig. 3B).
We thus have redefined the function of Bcl-TS as a trifunctional
sesterterpene/triterpene/sesquarterpene synthase. Since this
study revealed that Bcl-TS can accept HepPP (C35) as a
substrate, mutational analysis targeting residues that differ in TS,
[
3d]
which cyclizes HepPP (C35) to a monocycle (Scheme 1), will
help establish the mechanism that controls the formation of
acyclic and cyclic structures.
Detection of 3 and MK-7 in B. clausii cells and in vitro
conversion (DMAPPHepPP3) indicated that HepPP is
biosynthesized by E-IDS2-S/L in B. clausii (Fig. 3A). The
amounts of C25, C30, and C35 metabolites (sum of acyclic
terpenes and MKs) did not differ substantially (Fig. 3B). Thus, E-
IDS2-S/L is the first identified trifunctional GFPP/HexPP/HepPP
synthase. We have identified the first bifunctional Z-IDS, which
forms Z-HepPP (C35) and Z-decaprenyl diphosphate (C50), from
Scheme 1. Biosynthetic pathway of tail-to-tail triterpenes (C30),
sesquarterpenes (C35), and MK-7 in B. subtilis and B. megaterium. The
conversion of squalene to onoceroid by TC occurs only in B. megaterium.
HepS/T: heterodimeric heptaprenyl diphosphate synthase; TS: tetraprenyl-β-
curcumene synthase; TC: tetraprenyl-β-curcumene cyclase; MK-7:
menaquinone-7.
[
3h]
biosynthetic study of mycobacterial sesquarterpenes.
contrast, all reported E-IDS types of GFPP, HexPP,
In
and
[
9]
[10]
[
11]
HepPP synthases
enzymes. Since they form several products in vitro,
also be multifunctional. Further study of the mono-functional E-
have been described as mono-functional
[9-11]
they may
[
9-11]
IDSs
the discovery of multifunctional enzymes and novel compounds
such as sesterterpene, head-to-tail triterpene, and
sesquarterpene.
through analysis of isoprenoid metabolites may lead to
Most FPP and GGPP synthases produce single products in
vitro. In contrast, two E-IDSs from archaea and protozoans form
both FPP and GGPP in vitro and have been identified as
[
14]
bifunctional FPP/GGPP synthases.
Since E-IDS1 also
produced comparable amounts of FPP and GGPP (Fig. 2), we
propose that E-IDS1 be considered a bifunctional FPP/GGPP
synthase (Fig. 3A). To the best of our knowledge, this is the first
report of a bifunctional FPP/GGPP synthase in bacteria.
The differences in isoprenoid (acyclic terpenes and MKs)
distribution in B. clausii (Fig. 3B) may be determined by the
substrate
specificity
of
Bcl-TS,
E-IDS2-S/L,
and
prenyltransferase of the new menaquinone biosynthetic
Scheme 2. Proposed pathways for the biosynthesis of acyclic sesterterpene
and head-to-tail triterpene in B. clausii. Dotted square shows the in vitro
reaction. Bcl-TS: tetraprenyl-β-curcumene synthase homolog.
[
15]
pathway,
which has not been previously identified in any
organisms (Fig. 3A). Further experiments are required to
establish the regulatory mechanism of the acyclic terpene and
MK pathways.

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A
Fig. 1. SDS-PAGE of the overexpressed and purified recombinant enzymes.
Lane
1:
molecular
weight
marker;
lanes 2, 4, and 6: total soluble proteins from BL21(DE3)/pColdI-E-IDS1,
BL21(DE3)/pColdI-E-IDS2-S, and BL21(DE3)/pColdI-E-IDS2-L, respectively;
lanes 3, 5, and 7: purified E-IDS1, E-IDS2-S, and E-IDS2-L, respectively.
B
1
.8
.6
.4
.2
0
0
0
0
0
1
MK-5
2
MK-6
3
MK-7
(C25) (C25) (C30) (C30) (C35) (C35)
Fig. 3. Proposed pathways for the biosynthesis of acyclic terpenes and
menaquinones (A) and acyclic terpenes and menaquinone production by B.
clausii (B). Major and minor pathways are represented by solid and dotted
arrows, respectively in A. Main metabolites (1, 2, and MK-7) are shown by
gray boxes in A. The information provided in parentheses in B indicates
isoprenoid carbon numbers.
Fig. 2. TLC analysis of radiolabeled products prepared with recombinant E-
IDSs. Lane 1: reaction products synthesized by incubation of DMAPP and [1-
14
C]IPP with E-IDS1; lanes 2–4: reaction products synthesized by incubation
of allylic substrate (lane 2: DMAPP; lane 3: FPP; lane 4: GGPP) and [1-
14
C]IPP with E-IDS2-S/L. Only E-IDS2-S and E-IDS2-L formed no product
14
from the allylic substrate (DMAPP, FPP, and GGPP) and [1- C]IPP. Ori.:
origin; S.F.: solvent front. This figure represents the reaction with 133.6 µM [1–
14
14
C]IPP. All reaction products of 15 µM [1- C]IPP with E-IDS1 or E-IDS2-S/L
produced similar results. In Fig. S4, Fig.2 (lanes 2–4) with enhanced intensity
has been provided; in this figure, the C35 spot can be seen more clearly.

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FULL PAPER
6 6
Table 1. NMR assignments for 3 in C D
No.
1

H
(mult., J in Hz)
C
 (mult.)
H
H
a
: 5.34 (1H, d, 17.7)
: 5.11 (1H, d, 11.0)
113.1 (t)
b
2
3
4
5
6
7
8
9
6.50 (dd, 17.6, 10.7)

139.4 (d)
146.5 (s)
2.38 (m)
31.9 (t)
2.40 (m)
27.1–27.3 (t)
124.6–125.0 (d)
135.0–135.4 (s)
40.2 (t)
5.37–5.43 (m)

2.21–2.25 (m)
2.30–2.35(m)
5.37–5.43 (m)

27.1–27.3 (t)
124.6–125.0 (d)
135.0–135.4 (s)
40.2 (t)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
2.21–2.25 (m)
2.30–2.35(m)
5.37–5.43 (m)

Fig. 4. GC-MS analysis of acyclic terpenes. A) n-Hexane extract from B.
clausii cells. B) Reaction product formed by incubation of HepPP with purified
Bcl-TS. C) Reaction product formed by incubation of HepPP with heat-
denatured Bcl-TS. MK-5, MK-6, and MK-7 were not detected under these
conditions.
27.1–27.3 (t)
124.6–125.0 (d)
135.0–135.4 (s)
40.2 (t)
2.21–2.25 (m)
2.30–2.35 (m)
5.37–5.43 (m)

27.1–27.3 (t)
124.6–125.0 (d)
135.0–135.4 (s)
40.2 (t)
2.21–2.25 (m)
2.30–2.35(m)
5.37–5.43 (m)

27.1–27.3 (t)
124.6–125.0 (d)
135.0–135.4 (s)
40.2 (t)
2.21–2.25 (m)
2.30–2.35 (m)
5.37–5.43 (m)

27.1–27.3 (t)
124.6–125.0 (d)
131.1 (s)
1.81 (3H, s)
5.11 (1H, s)
25.8 (q)
115.9 (t)
5.12 (1H, s)
30
31
32
33
34
35
1.69 (3H, s)
1.74 (3H, s)
1.74 (3H, s)
1.74 (3H, s)
1.74 (3H, s)
1.69 (3H, s)
16.1 (q)
16.1 (q)
16.1 (q)
16.1 (q)
16.1 (q)
17.7 (q)
Fig. 5. Structure of 3 (A) and key correlations in the 2D-NMR data of 3 (B).

ChemBioChem
10.1002/cbic.201500138
FULL PAPER
Conclusions
2-E-IDS2-L-A
(XbaI),
5′-
CTCGTCTCTAGATTAGTATTTTCTCCGTCCTATG-3′. Amplified DNA
fragments were inserted into pGEM-T. Fragments (KpnI/XbaI or
EcoRI/XbaI) were inserted into pColdI. E. coli BL21(DE3) harboring
pColdI derivatives (pColdI-E-IDS1, pColdI-E-IDS2-S, and pColdI-E-IDS2-
Functional analysis of recombinant enzymes and analysis of
isoprenoid metabolites revealed that Bcl-TS, E-IDS1, and E-
IDS2-S/L
are
the
trifunctional
-1
L) were grown at 37°C in Luria-Bertani medium containing 100 µg mL
sesterterpene/triterpene/sesquarterpene synthase, bifunctional
FPP/GGPP synthase, and trifunctional GFPP/HexPP/HepPP
synthase, respectively (Fig. 3A). To our knowledge, this is the
ampicillin. Expression of the recombinant protein was induced by addition
of 0.1 mM IPTG at OD600 0.6, followed by culture for an additional 24 h at
15°C. E. coli expressing recombinant enzymes were harvested by
centrifugation and resuspended in buffer containing 20 mM Tris-HCl (pH
first
report
to
identify
the
synthase
trifunctional
and
7
.9), 10 mM imidazole, and 300 mM NaCl. Cells were disrupted by
sonication in a UP200s sonicator (Hielscher Ultrasonics GmbH) at 4–
0°C for 15 min, and then centrifuged at 10,000 × g for 20 min. The pellet
sesterterpene/triterpene/sesquarterpene
trifunctional GFPP/HexPP/HepPP synthase. In particular, the
present study suggests that isoprenoid metabolite analysis is
crucial for the identification of multifunctional E-IDSs. GFPP,
HexPP, and HepPP are intermediates in two isoprenoid
pathways (acyclic terpenes and menaquinones) and are
biosynthesized by one trifunctional E-IDS. In addition,
GFPP/HexPP and HepPP are the main substrates for
biosynthesis of acyclic terpenes and menaquinone, respectively
1
was discarded and the supernatant containing soluble His-tagged fusion
enzyme was purified according to manufacturer protocols (Takara).
Expression and purification of E-IDS1, E-IDS2-S, and E-IDS2-L were
analyzed by 10% SDS-PAGE (Fig. 1).
Analysis of products formed by E-IDS1, E-IDS2-S, and E-IDS2-L
(
Fig. 3A). The present study will promote the search for and
The reaction mixture for analyzing the chain length of products formed by
E-IDS1, E-IDS2-S, and E-IDS2-L contained 25 mM Tris-HCl buffer (pH
creation of novel sesterterpenes, head-to-tail triterpenes, and
sesquarterpenes.
8
.5), 10 mM dithiothreitol, 1 mM MgCl
2
, 25 mM NH
4
Cl, 133.6 or 15 µM
14
[
1– C]IPP, 15 µM allylic substrate (DMAPP, FPP or GGPP), and 3.0 µM
purified enzyme, in a total volume of 50 µL. The reaction was performed
at 37C for 1 h and terminated by boiling. Polyprenyl diphosphate
products were hydrolyzed to the corresponding alcohols by using potato
acid phosphatase (Sigma). A phosphatase reaction mixture (50 µL of
reaction mixture, 30 µL of 2-propanol, 15 µL of 1% Triton X-100, 53 µL of
Experimental Section
Materials
3
M acetate buffer, and 40 µg of potato acid phosphatase) was incubated
at 37C for 12 h. After addition of 150 µL MeOH, the n-hexane extract
100 µL) was analyzed by TLC on a reverse-phase RP-18 plate (Merck)
DMAPP, FPP, and GGPP were diphosphorylated from 3-methyl-2-buten-
(
1
-ol, farnesol, and geranylgeraniol, respectively, as described by
[
16]
with a solvent system of MeOH:acetone (8:2). Radioactive spots were
detected using an image analyzer (FLA-9000; Fujifilm).
Davisson et al.
described
. GFPP and HexPP were prepared as previously
[
5]
. HepPP was diphosphorylated from (all-E)-heptaprenol,
which was chemically synthesized as described in the Supporting
14
Information. [1- C]IPP was purchased from GE Healthcare, USA. All
other chemicals were of analytical grade.
Analysis of isoprenoids in B. clausii cells
B. clausii was cultured under shaker conditions at 37C for 4 d in 1 L
Cloning, expression, and purification of E-IDS1, E-IDS2-S, and E-
IDS2-L
Horikoshi-I
(http://www.jcm.riken.go.jp/). Cells were then collected by centrifugation
6,000 × g, 4C, 20 min). After lyophilization, cell extracts were made in
medium,
as
recommended
by
JCM
(
MeOH (3 200 mL) and the MeOH fractions were partitioned by using n-
hexane (3 600 mL). To analyze the acyclic terpenes 1–3, GC-MS
The following sets of primers and B. clausii JCM9138 genomic DNA were
used to PCR amplify three genes (E-IDS1: ABC2463, E-IDS2-S:
ABC1885; and E-IDS2-L: ABC1889), including approximately 200-bp
regions upstream and downstream of the open reading frames: E-IDS1-S,
(
injection temperature, 300C; oven temperature, 220–300C at an
−1
increment of 3C min
) was performed on a JMS-T100GCV
spectrometer (JEOL) equipped with a DB-1 capillary column (30 m  0.25
mm  0.25 µm; J&W Scientific, Inc.) by using the EI mode operated at 70
eV. GC analysis (injection temperature, 300C; oven temperature, 220–
5
3
′-actgaaagtcgagaagcagcttg-3′; E-IDS1-A, 5′-ggttcagactgcaggcaaacgctc-
′; E-IDS2-S-S, 5′-GGATACGGAGGAATCAGGCGC-3′; E-IDS2-S-A, 5′-
TCATTGTATCTTTACGCCAGC-3′;
CTGCTTCATCAGCCAACAAGC-3′;
E-IDS2-L-S,
E-IDS2-L-A,
5′-
5′-
−
1
300C at an increment of 3C min ) was performed twice to determine
the amounts of 1–3 on a Shimadzu GC-2014 chromatograph fitted with a
flame ionization detector (30 m  0.32 mm  0.25 µm; J&W Scientific,
Inc.). To analyze the menaquinones (MK-5, MK-6, and MK-7), LC-MS
was performed on a JMS-T100LP spectrometer (JEOL), using ESI mode.
HPLC (MeOH/2-propanol 6:1, flow = 1.0 mL/min) was performed twice to
determine the amount of menaquinones with an SPD-10A detector (270
nm) and LC-10AS pump (Shimadzu) equipped with a C18 reverse-phase
column (SHISEIDO, CAPCELL PAK C18). Authentic MK-5, MK-6, and
CGTTCCATTGTCTAACCTCGC-3′. Amplified DNA fragments were
inserted into pGEM-T (Promega) and the constructs were sequenced.
The DNA sequences of e-ids1, e-ids2-s, and e-ids2-l from B. clausii
JCM9138 were deposited in the DNA Data Bank of Japan under
accession numbers AB905204, AB735675, and AB735676. The amino
acid sequences of E-IDS1, E-IDS2-S, and E-IDS2-L from B. clausii
JCM9138 showed 96%, 95%, and 98% identities to those from B. clausii
KSM-K16, for which complete sequence data are available. The following
primer pairs and the pGEM-T derivatives of e-ids1, e-ids2-s, and e-ids2-l
were used to add restriction sites: 2-E-IDS1-S, (KpnI), 5′-
ggaggcggtaccATGAGCGCATTGTTCCAA ACG-3′; 2-E-IDS1-A, (XbaI),
[17]
MK-7 isolated from B. krulwichiae
were detected at 9.3, 13.6, and
+
20.6 min, respectively. Molecular ions [M+Na] of MK-5, MK-6, and MK-7
were m/z 535, 603, and 671, respectively. Isolated MK-6 and MK-7 from
B. clausii was found to have fragmentation patterns identical to those of
the authentic samples on performing direct-EI-MS, including the
characteristic m/z 225, which supported methylnaphtoquinone
substructure.
5′-cctgtttctagaTTAATGGTCACGCGTAACGATG-3′;
2-E-IDS2-S-S,
(EcoRI), 5′-gggacagaattcATGAGCAATACAACGTTGCCG-3′; 2-E-IDS2-
S-A (XbaI), 5′-tgggcctctagaCTACCTCCTTGCTTTTTCAAG-3′; 2-E-IDS2-
L-S (EcoRI), 5′-agggtggAAtTcATGAAGTTAGCAGATATTTATAAA G-3′;

ChemBioChem
10.1002/cbic.201500138
FULL PAPER
Isolation and structural analysis of 3
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2
011, 133, 17540–17543. f) T. Sato, A. Kigawa, R. Takagi, T. Adachi, T.
B. clausii was cultured in 20 × 1 L medium at 37C for 4 d. Lyophilized
cells (dry weight, 37 g) were extracted using MeOH (3  800 mL), and
the MeOH fraction was concentrated to 800 mL and partitioned using n-
hexane (3  800 mL). The n-hexane layer (295.6 mg) was partially
purified by silica gel (20 g) column chromatography with n-hexane and
EtOAc. The fraction eluted with n-hexane (48.3 mg) was subjected to
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18338.
further silica gel (20 g) column chromatography with n-hexane and SiO
HPLC with n-hexane, yielding 1.1 mg of purified 3. The structure of 3 was
2
[
4]
5]
1
13
determined by NMR and MS. β-Heptaprene (3): Colorless oil; H and
C
[
T. Sato, H. Yamaga, S. Kashima, Y. Murata, T. Shinada, C. Nakano, T.
Hoshino, Chembiochem. 2013, 14, 822–825.
NMR assignments are shown in Table 1. EI-MS m/z (%): 69 (100), 81
(
65), 93 (32), 107 (20), 135 (17), 161 (15), 203 (10), 271 (1), 339 (1), 407
+
+
[6]
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1), 476 ([M] , 1). HR-EI-MS: m/z calcd for C35
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and S6-S10, respectively.
H56: 476.4390 [M] ; found:
[7]
4
Enzyme assay of Bcl-TS, using HepPP as the substrate
2
93–300.
[
5]
Bcl-TS was purified as described previously. The Bcl-TS assay reaction
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[
8]
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This work was supported by JSPS KAKENHI Grant Numbers
25450149 and 25108709. We thank Dr. M. Fujihashi (Kyoto
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Keywords: isoprenoids • isoprenyl diphosphate synthase •
prenyltransferase • terpene synthase • menaquinone
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2

ChemBioChem
10.1002/cbic.201500138
FULL PAPER
Entry for the Table of Contents
FULL PAPER
Geranylfarnesyl diphosphate (GFPP),
hexaprenyl diphosphate (HexPP), and
heptaprenyl diphosphate (HepPP) are
intermediates in two isoprenoid
pathways (acyclic terpenes and
Daijiro Ueda, Hiroaki Yamaga, Mizuki
Murakami, Yusuke Totsuka, Tetsuro
Shinada, and Tsutomu Sato *
Page No. – Page No.
menaquinones)
biosynthesized by one trifunctional
all-E)-isoprenyl diphosphate
and
are
Biosynthesis of Sesterterpene, Head-
to-tail Triterpene, and Sesquarterpene
in Bacillus clausii: Identification of
(
synthase. GFPP/HexPP and HepPP
are mainly utilized for the biosynthesis
of acyclic terpenes and menaquinone,
respectively.
Multifunctional
Enzymes
and
Analysis of Isoprenoid Metabolites