Biosynthetic gene-based secondary metabolite screening: A new diterpene, methyl phomopsenonate, from the fungus Phomopsis amygdali

Article abstract of DOI:10.1021/jo802319e

The presence of the geranylgeranyl diphosphate synthase (GGS) gene is a common feature of gene clusters for diterpene biosynthesis. We demonstrated identification of a diterpene gene cluster using homology- based PCR of GGS genes and the subsequent genome

Full text of DOI:10.1021/jo802319e

Biosynthetic Gene-Based Secondary Metabolite Screening:
A New Diterpene, Methyl Phomopsenonate,
from the Fungus Phomopsis amygdali
Tomonobu Toyomasu,† Akane Kaneko,† Tetsuo Tokiwano,‡,¶ Yuya Kanno,† Yuri Kanno,†,|
Rie Niida,† Shigeyoshi Miura,† Taiki Nishioka,† Chiho Ikeda,§ Wataru Mitsuhashi,†
,#
Tohru Dairi,§ Tomikazu Kawano, Hideaki Oikawa,*,‡ Nobuo Kato, and Takeshi Sassa†
Department of Bioresource Engineering, Yamagata UniVersity, Tsuruoka, Yamagata 997-8555, Japan,
DiVision of Chemistry, Graduate School of Science, Hokkaido UniVersity, Sapporo 060-0810, Japan,
Biotechnology Research Center, Toyama Prefectural UniVersity, Imizu, Toyoma 939-0398, Japan, and The
Institute of Scientific and Industrial Research, Osaka UniVersity, Ibaraki, Osaka 567-0047, Japan
hoik@sci.hokudai.ac.jp
ReceiVed October 16, 2008
The presence of the geranylgeranyl diphosphate synthase (GGS) gene is a common feature of gene clusters
for diterpene biosynthesis. We demonstrated identification of a diterpene gene cluster using homology-
based PCR of GGS genes and the subsequent genome walking in the fungus Phomopsis amygdali N2.
Structure determination of a novel diterpene hydrocarbon phomopsene provided by enzymatic synthesis
with the recombinant terpene synthase PaPS and screening of fungal broth extracts with reference to
characteristic NMR signals of phomopsene allowed us to isolate a new diterpene, methyl phomopsenonate.
The versatility of the gene-based screening of unidentified diterpenes is discussed in regard to fungal
genomic data.
Introduction
mycetes2 and fungi.3 Therefore, homology-based PCR screening
of the terpene cyclase gene should be useful for identifying gene
cluster of terpenoid biosynthesis. However, this approach is less
frequently used, although a similar approach is generally applied
for searching for gene clusters of polyketides4 and non-ribosomal
polypeptides5 whose biosyntheses involve highly homologous
modular enzymes for construction of molecular backbones. The
major reason for less frequent use of the PCR screening is that
microbial terpene cyclases show a low homology6 compared
with the modular enzymes described above. This is a clear
difference between plant and microbial terpene cyclases since
Terpenoids are a rich source of bioactive compounds and have
a large structural diversity with a molecular skeleton constructed
by the cyclization of oligoprenyl diphosphate with terpene
cyclase.1 Thus, terpene cyclase play a key role in the construc-
tion of characteristic skeletons of terpenoids. Clustering of the
genes responsible for biosynthesis of secondary metabolites is
a common feature in most microorganisms including strepto-
† Yamagata University.
‡ Hokkaido University.
§ Toyama Prefectural University
(2) (a) Ikeda, H.; Ishikawa, J.; Hanamoto, A.; Shinose, M.; Kikuchi, H.; Shiba,
T.; Sakaki, Y.; Hattori, M.; Omura, S. Nat. Biotechnol. 2003, 21, 526–531. (b)
Oliynyk, M.; Samborskyy, M.; Lester, J. B.; Mironenko, T.; Scott, N.; Dickens,
S.; Haydock, S. F.; Leadlay, P. F. Nat. Biotechnol. 2007, 25, 447–453.
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Osaka University.
¶ Current address: Department of Biotechnology, Faculty of Bioresource
Sciences, Akita Prefectural University, Akita 010-0195, Japan.
| Current address: Plant Science Center, RIKEN, Yokohama, Kanagawa 230-
0045, Japan.
# Current address: School of Pharmacy, Iwate Medical University, Yahaba,
Shiwa-gun, Iwate 028-3694, Japan.
(1) Cane, D. In Isoprenoids Including Carotenoids and Steroids; Vol. 2 in
ComprehensiVe Natural Products Chemistry; Barton, D., Nakanishi, K., Meth-
Cohn, O., Eds.; Elsevier: Amsterdam, 1999.
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10.1021/jo802319e CCC: $40.75
Published on Web 01/22/2009
2009 American Chemical Society
J. Org. Chem. 2009, 74, 1541–1548 1541

Toyomasu et al.
plant terpene cyclases have a relatively high homology and
homology-based PCR is useful for cloning of the corresponding
genes.7
During our search for novel diterpene syntheses from fungi,
we and others found that the occurrence of geranylgeranyl
diphosphate synthase (GGS) on the gene clusters is a common
feature in several biosynthetic diterpene gene clusters such as
gibberellin8 and aphidicolin.9 In addition, GGS was also found
in the gene cluster for the biosynthesis of the indole diterpene,
paxilline.10 Fungal GGS possessed relatively high homology
to design degenerated primers, suggesting that diterpene gene
clusters would be securely cloned by identification of both
constitutive and cluster-specific GGS genes and subsequent
genome walking. Recently, homology-based PCR cloning was
successfully applied to clone diterpene gene clusters11-13 and
was also used to identify a gene cluster for terpenoids with an
isoprene unit more than C20.14
The plant pathogenic fungus Phomopsis amygdali produces
diterpene phytotoxin fusicoccins15 whose structurally related
derivative cotylenin is a promising drug lead as a differentiation-
inducing agent on cancer cells.16 We recently found the partial
biosynthetic gene cluster of fusicoccins in P. amygdali, using
homology-based PCR cloning and genome walking.11 This study
allowed us to find a P. amygdali fusicoccadiene synthase (PaFS),
which is responsible for construction of the fusicoccane skeleton.
PaPS constitutes two domains, a terpene cyclase domain at the
N-terminus and a prenyltransferase domain at the C-terminus.11
During our genome walking, we unexpectedly found another
chimera cyclase, PaFS homologue. In this paper, we describe a
typical example of biosynthetic gene-based screening of novel
diterpenes phomopsene and methyl phomopsenonate.
FIGURE 1. Chromosome map around PaPS and the primary structure
of PaPS. (A) Chromosome map. A filled reverse triangle indicates the
start point of genome walking. The restriction sites for Dra I, Eco RV,
PVu II, and Stu I used in genome walking are represented by the letters
D, E, P, and S, respectively. The direction and deduced region of
transcription are represented by arrows. The deduced orf1, orf2, and
orf3 represent P450 monooxygenase, peroxiredoxin, and transporter
like genes, respectively. Homology searches were performed using
BLAST (http://www.ncbi.nlm.nih.gov/BLAST/). (B) Schematic of two
putative domains of PaPS. Black and gray bars indicate the terpene
cyclase domain and the prenyltransferase domain, respectively. Reverse
open triangles indicate the DDxxD motives of both domains. The
borders between the terpene cyclase domain and the prenyltransfer-
ase domain were deduced by the homology with other fungal aris-
tolochene synthases or GGSs.
the diterpene gene cluster in P. amygdali, degenerate primers
designed to amplify GGS genes essential for the biosynthesis
of the diterpene metabolites were used to amplify the corre-
sponding homologues.11 Previously, we reported six unique
GGS gene fragments (PaGGS1, 2, 3, 4, 5, and 6) among the
amplimers cloned and sequenced.11 When PaGGS3 was used
for genome walking, we found the partial sequence of a terpene
cyclase-like gene.
Results
Cloning and Sequencing of the Partial Diterpene Gene
Cluster Including PaFS Homologue. In the study identifying
The nucleotide sequence of the full-length cDNA was
determined by a rapid amplification of cDNA ends (RACE)
using gene-specific primers. This contained the predicted 2178-
bp open reading frame (ORF), encoding a product of 725 amino
acids, indicating that PaGGS3 is fused to the terpene cyclase-
like gene at the 3′ end, like PaFS.11 This gene contained five
intron insertion sites, on the basis of comparison with the
corresponding genomic DNA sequence. According to the results
shown later, we named this fused-type gene as a P. amygdali
phomopsene synthase gene (PaPS). The predicted peptide
constitutes two possible domains, the putative terpene cyclase
domain (position 1-327) at the N-terminus and the putative
prenyltransferase domain (position 328-725) at the C-terminus
(Figure 1B). The enzyme encoded by PaPS exhibited homology
with PaFS (35% identitiy and 53% similarity). The DDxxD
motives, proposed to coordinate with the Mg2+ ion,6a,7 was
conserved at both domains: DDLYD (position 94-98) and
DDVQD (position 484-488) (Figure 1B).
At the 5′ flanking region of PaPS, we found a putative
cytochrome P-450 monooxygenase gene (orf1: Aspergillus
claVatus NRRL 1, identitiy 40%, similarity 58%) and a putative
peroxiredoxin (orf2: identity Taiwanofungus camphoratus61%,
similarity 75%). At the 3′ flanking region, partial ORF
(orf3Emericella nidulans) that encodes a putative self-resistance
protein was also found. Although sequence data for the whole
gene cluster is not currently available, the information shown
above strongly indicated that the gene cluster is responsible for
the biosynthesis of diterpene metabolites.
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Biosynthetic Gene-Based NoVel Diterpene Screening
SCHEME 1. Proposed Biosynthetic Pathway of Phomopsene
(1) and Methyl Phomopsenonate (2)a
FIGURE 2. GC-MS results for products converted from GGPP by
GST-PaPS. (A) Total ion chromatograms of the products after
incubation of GGPP with the recombinant GST-PaPS. (B) Total ion
chromatogram of the hydrocarbon fraction obtained from P. amygdali
N2. (C) Full-scan mass spectrum of an enzymatic reaction product 1.
(D) Full-scan mass spectrum of phomopsene (1) from P. amygdali N2.
a PaPS: phomopsene synthase.
(DMAPP), into GGPP (Figure 3). These results demonstrated
that the N-terminal 40-kDa peptide catalyzed the cyclization
of GGPP to 1, and the C-terminal 44-kDa peptide catalyzed
formation of GGPP.
Functional Analysis of PaPS. To study the function of the
PaPS, a full-length cDNA was ligated into a pGEX 4T-3 vector
and was expressed in Escherichia coli BL21trx as the glu-
tathione S-transferase (GST) fusion protein. In hexane extracts
of the reaction mixture obtained by incubation of purified GST-
PaPS with geranylgeranyl diphosphate (GGPP), an unidentified
hydrocarbon 1 was observed at a retention time of 12.2 min on
GC-MS analysis (Figure 2A and C), whereas the product was
not detected from either geranyl diphosphate (GPP) or farnesyl
diphosphate (FPP) (data not shown). The same product was
obtained in the incubation of the enzyme with isopentenyl
diphosphate (IPP) and FPP, indicating that the enzyme has both
GGS and diterpene cyclase activities. In the mycelial extracts
of P. amygdali, the diterpene hydrocarbon 1 was also found
(Figure 2B and D). The structure of metabolite 1 was determined
as a novel 5/6/5/5-tetracyclic diterpene hydrocarbon, named
phomopsene (Scheme 1). Details of the structure determination
of phomopsene are described later.
FIGURE 3. TLC autoradiography of the alcohols obtained by hy-
drolysis of the products derived by the recombinant C398. Ori indicates
origin. DMAPP, GPP, and FPP were incubated with [14C]IPP. Spots
of authentic standard alcohols are indicated by arrows: C10, geraniol;
C15, farnesol; C20, geranylgeraniol; C30, farnesylfarnesol; C45.
Structure of Novel Diterpene Hydrocarbon, Phomopsene.
To determine the structure of the diterpene hydrocarbon
phomopsene (1) detected in the enzymatic reaction of PaPS with
GGPP, the mycelial acetone extract of P. amygdali N2 was
partitioned with hexane-acetonitrile. The resultant hexane
extract was purified by silica gel column chromatography and
by reverse-phase HPLC to give 1, which was also obtained by
fermentation of the E. coli harboring pGEX-PaPS. The HR-
EIMS of the isolated hydrocarbon showed the molecular formula
of C20H32. Its 13C NMR and DEPT spectra indicated five
To demonstrate the function of both putative domains, N-
and C-terminal truncated mutants were prepared (Figure S2A,
Supporting Information). The N347 (position 1-347) mutant
exhibited cyclase activity, which was estimated by the produc-
tion of 1 from GGPP (Figure 1B and Figure S2B, Supporting
Information), and the C398 mutant (position 328-725) catalyzed
conversion of GPP or FPP, not dimethylallyl diphosphate
J. Org. Chem. Vol. 74, No. 4, 2009 1543

Toyomasu et al.
FIGURE 5. (A) Planar structure of methyl phomopsenonate (2)
deduced by 1H-1H COSY and HMBC data. (B) Stereostructure of
methyl phomopsenonate proposed by differential NOE data.
FIGURE 4. (A) Planar structure of phomopsene (1) deduced by 1H-1H
COSY and HMBC data. (B) Stereostructure of 1 proposed by
differential NOE data.
The HR-EIMS and NMR data of 2 showed the molecular
formula C21H30O3. The 13C NMR and DEPT spectra indicated
seven quaternary carbons including the R,ꢀ-unsaturated ketone
(199.22 ppm), the ester carbonyl (172.85 ppm), the olefin
(162.84 and 129.56 ppm), three CH, six CH2, and five CH3.
quaternary carbons including the olefin (140.7 and 123.1 ppm),
three CH, seven CH2, and five CH3. The H NMR spectrum
1
showed the presence of the doublet methyl group (0.88 ppm)
and the allylic methyl group (1.58 ppm). The 1H and 13C NMR
signals in CDCl3 were fully assigned by COSY, HMQC,
HMBC, and NOE experiments (Figure 4). In addition to
observation of COSY correlations of two spin systems H4-H5
and H8-H9-H10-H11, strong HMBC correlations between
methyl protons and neighboring carbons provided three sub-
structures as shown in Figure 4A. Additional HMBC correlations
of H1, H5, and H10 unambiguously determined the 5/6/5/5-
membered tetracyclic fused structure of 1 with the cyclohexene
component. The apparent NOEs supported this planar structure
and revealed the relative configuration of 1 (Figure 4B). The
C11/C15 ring junction was assigned as cis on the basis of strong
NOE correlations between H-11 and 18-Me/20-Me. The NOE
correlations between 16-Me and H-1ꢀ/H-10 and between H-10
and 19-Me showed special proximity of these protons and
allowed us to propose relative configurations at C2, C3, and
C10.
1
The H NMR spectrum showed the methyl ester (OMe, 3.74
ppm) and the allylic methyl group (1.74 ppm). The 1H and 13
C
NMR signals in CDCl3 were fully assigned by extensive analysis
(COSY, HMQC, and HMBC). In addition to COSY correlations
observed on three spin systems, H3-H4-H5, H9-H10-H11, and
H13-H14, strong HMBC correlations between methyl protons
and neighboring carbons allowed us to propose three substruc-
tures as shown in Figure 5A. Additional HMBC correlations
of H1, H3, H5, and H9 unambiguously determined the tetra-
cyclic structure of 2 as essentially identical to that of pho-
mopsene. The observed NOEs established the configuration of
2, which was the same as that of 1 (Figure 5B). Further
confirmation of the structure of 2 was obtained by the X-ray
crystallographic analysis shown in Figure 6. We named this
diterpene metabolite methyl phomopsenonate.
To assign the absolute configuration of 2, the modified
Mosher’s method18 was applied to the secondary allylic alcohol
3 prepared by reduction of the C-8 ketone (Scheme 2).
Treatment of 2 with NaBH4 in the presence of CeCl3 provided
the single diastereomer 3, which was converted to the corre-
sponding R- and S-R-methoxy-R-trifluoromethylphenylacetic
acid (MTPA) esters, 4a and 4b, respectively. The NOE analysis
of Mosher ester 4a allowed us to determine the stereochemistry
at C-8 as shown in Figure 7. Calculated ∆δ values were negative
for signals of H-5 and H-17 and nearly zero for H-4 signals
whereas ∆δ values were positive for signals of H-9, H-10, and
Next, we turned our attention to the isolation of the unidenti-
fied diterpene metabolite biosynthesized by this gene cluster.
The presence of modification enzymes such as a monooxygenase
on the gene cluster strongly indicated that a target molecule
should have polar oxygen functionality and essentially the same
molecular skeleton as that of 1, which has three singlet methyl
and a doublet methyl group in its NMR spectrum. Using these
characteristic NMR signals as markers, we screened oxidatively
modified phomopsene diterpene in the culture broth. Although
P. amygdali produces fusicoccin diterpene-glycosides as major
metabolites,17 characteristic signals originated from OMe and
a sugar moiety allowed us to eliminate fusicoccin derivatives.
Thus, unidentified diterpene 2, structurally related to pho-
mopsene (1), was isolated from the ethyl acetate extract of the
fermentation broth of the phomopsene-producing strain by silica
gel column chromatography and recrystallization.
(17) (a) Muromtsev, G. S.; Voblikova, V. D.; Kobrina, N. S.; Koreneva,
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1544 J. Org. Chem. Vol. 74, No. 4, 2009

Biosynthetic Gene-Based NoVel Diterpene Screening
P-450 ORF3. Subsequently, oxidation of the allylic position and
methylation of the carboxyl group may give 2. Although further
study is necessary to identify genes such as a monooxygenase
and a methyltransferase, the predicted functions of genes on
the cluster are correlated with the structure of 2.
Two fungal metabolites, diterpene conidiogenone19 and
sesterterpene mangicol A,20 have molecular skeletons closely
related to methyl phomopsenonate (2) as shown in Figure 8.
The biosynthetic pathway of mangicol A was proposed by
feeding experiments with 13C-labeled precursors.20 However,
its detailed cyclization mechanism was not elucidated. To
explore a versatile biosynthetic pathway of these metabolites,
we are currently investigating an enzymatic cyclization of the
chimeric diterpene cyclase PaPS.
Gene-based screening of diterpenes relies on two essential
factors: (1) versatility of homology-based PCR cloning and (2)
functional analysis of diterpene synthase. Recently, genomic
information on various fungi available from the database
(http://www.broad.mit.edu/annotation/genome/aspergillus_ter-
reus/Blast.html) allowed us to find a diterpene gene cluster based
on bioinfomatics. The first factor is relatively easy to examine.
For example, we can find more than five candidates, i.e., (1)
AO090023000070, AO090023000073; (2) AO090012000686,
AO090012000688; (3) AO090113000170, AO090113000171;
(4) AO090701000221, AO090701000222; and (5) AO090038
000495 (chimera)), in Aspergillus oryzae in which putative
diterpene synthase and GGS genes are located close to each
other. Identification of six GGS genes and four diterpene gene
clusters11,12 on P. amygdali provided strong support for the
reliability of a gene-based search of diterpene metabolites. For
the second factor, diterpene synthases are relatively small
proteins compared with modular enzymes such as polyketides
synthases and non-ribosomal peptide synthetases. From our
experiences with the handling of plant and fungal diterpene
synthases, most can be successfully expressed in E. coli, simply
using a GST-fusion protein expression system.21 This allowed
us to determine a structure of an enzymatic cyclization product.
FIGURE 6. ORTEP drawing of methyl phomopsenonate (2).
Another factor, which we have to consider for the reverse
genetic approach, is a screening marker for the diterpene
metabolite. In this study, we used characteristic 1H NMR signals
as markers. Alternatively, analytically sensitive 2H NMR signals
can be used for screening of diterpenes. Since fungal diterpenes
are biosynthesized via the mevalonate pathway, multiple methyl
2
FIGURE 7. ∆δ values obtained for (R)- and (S)-MTPA esters (4a and
4b) and NOE correlations of 4a.
groups of diterpenes would be labeled with H by feeding of
[C2H3]-acetate.22 In the 2H NMR spectra of fungal crude
extracts, these diagnostic methyl signals appear at the high field
region (0.8-1.0 ppm) and usually do not overlap with other
signals such as those of the polyketide metabolites. Thus,
isolation of a target diterpene should be achieved with these
NMR methods.
In conclusion, we have demonstrated biosynthetic gene-based
screening to identify novel diterpenes. Our approach is proven
to be effective for cloning the gene cluster of diterpene
biosynthesis from microorganisms whose genomic information
is not available. Conventionally, novel bioactive metabolites are
found by bioassay-guided screening. Gene-guided screening as
H-3 (Figure 7). Other ∆δ values were nearly zero within the
experimental error for NMR measurements. On the basis of
these results, the absolute configuration of 2 was proposed to
be 2R, 3S, 10S, 11R, and 15R. Since phomopsene (1) is
considered to be the putative biosynthetic precursor of methyl
phomopsenonate (2), it is reasonably assumed that both
metabolites have the same absolute configuration.
Discussion
On the basis of information regarding the gene cluster, the
biosynthetic pathway of methyl phomopsenonate (2) is proposed
in Scheme 1. At first, the universal precursor of diterpene GGPP
is provided and is cyclized by the unusual bifunctional enzyme
PaPS to give phomopsene (1). Since the oxidation of a methyl
group to a carboxyl group is frequently catalyzed by cytochrome
P-450,8 the C-16 methyl group would be oxidized by a putative
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(22) Seo, S.; Yoshimura, Y.; Uomori, A.; Takeda, K.; Seto, H.; Ebizuka,
Y.; Sankawa, U. J. Am. Chem. Soc. 1988, 110, 1740–1745.
J. Org. Chem. Vol. 74, No. 4, 2009 1545

Toyomasu et al.
TABLE 1. 1H and 13C NMR Assignments of Phomopsene (1) and Methyl Phomopsenonate (2)
phomopsene (1)
methyl phomopsenonate (2)
δH (multiplicity, J in Hz)
δC
δH (multiplicity, J in Hz)
δC
1
43.08
1.51-1.42 (R-H)
1.17 (d, 12.8, ꢀ-H)
49.66
1.77 (d, 13.6, ꢀ-H)
1.51 (d, 13.6, R-H)
2
3
4
54.63
39.45
30.14
56.37
46.50
23.46
1.51-1.42
2.65 (dd, 8.8, 10.6)
1.72 (m)
2.30 (dddd, 5.6, 10.6, 12.5, 13.4, ꢀ-H)
1.95 (dddd, 4.2, 8.8, 9.2, 13.4, R-H)
2.54 (br dd, 12.5, 16.4, ꢀ-H)
1.14 (m)
2.25 (ddd, 6.0, 9.5, 13.8)
2.14-2.08
5
24.28
25.01
2.45 (ddd, 5.6, 9.2, 16.4, R-H)
6
7
8
140.73
123.13
26.19
162.84
129.56
199.22
2.14-2.08
1.60 (br)
9
24.50
1.36-1.32
39.57
2.39 (dd, 5.0, 15.2, R-H)
2.35 (dd, 2.7, 15.2, ꢀ-H)
2.74 (ddd, 2.7, 5.0, 8.3)
1.33 (d, 8.3)
1.27 (br tt, 4.4, 12.6)
2.03 (dt, 6.7, 3.4)
1.51-1.42
10
11
12
13
39.61
64.28
42.18
40.81
37.25
66.17
42.23
39.52
1.51-1.42
1.36-1.32
1.51-1.42
1.41-1.35
1.33-1.30
1.46 (ddd, 6.2, 13.8, 14.2, ꢀ-H)
1.41-1.35 (R-H)
14
40.63
48.61
40.23
15
48.23
16
172.85
16-Me
17-Me
18-Me
19-Me
20-Me
OMe
14.21
20.13
30.37
24.98
30.83
0.88 (d, 6.7)
1.58 (s)
1.02 (s)
1.01 (s)
0.96 (s)
12.01
30.37
24.49
29.64
51.70
1.74 (d, 1.4)
0.97 (s)
1.03 (s)
0.93 (s)
3.74 (s)
SCHEME 2
described in this report could become an alternative approach
to identify novel secondary metabolites. When combined with
a suitable heterologous expression system, this approach
provides a powerful method to produce natural bioactive
diterpenes that are obtained in small quantities or even not
produced under normal fermentation conditions. According to
a database search of fungal genomes, we found many GGS
homologues in the Aspergillus fungi. Therefore, our reverse
genetic approach would also be useful for genome mining of
novel diterpenes and sesterterpenes.
Experimental Section
General. All commercially supplied reagents were used as
received. 1H and 13C chemical shifts were referenced to the solvent
signals (CDCl3): 7.26 and 77.0 ppm. IPP, DMAPP, GPP, FPP, and
GGPP were purchased from Sigma-Aldrich. [1-14C]IPP (CFA476,
2.15 GBq/mmol) was obtained from Amersham Pharmacia.
Genome Walking from GGS. Genome walking was carried out
using a Universal Genome Walker kit (Clontech). Genomic DNA
was extracted from the 3-day-cultured mycelia of P. amygdali N2
by a Nucleon PhytoPure Kit (Amersham Pharmacia). A genome
walker library was constructed with the genomic DNA and was
used for nested PCR according to the manufacture’s protocol.
Primers were first designed on the basis of the cDNA fragment
sequences of PaGGS3.11 Gene prediction was carried out based
on homology search using BLAST (http://www.ncbi.nlm.nih.gov/
BLAST/). The sequence of the identified biosynthetic gene cluster
reported in this paper has been deposited in the DDBJ database
with the accession code AB254159.
3′ RACE. To determine the 3′ sequences of the terpene cyclase-
like gene, 3′ RACE was done using gene specific primers based
on the corresponding genome DNA sequences, according to
FIGURE 8. Biosynthetically related metabolites of methyl phomopse-
nonate (2).
1546 J. Org. Chem. Vol. 74, No. 4, 2009

Biosynthetic Gene-Based NoVel Diterpene Screening
methods described previously.23 A full-length cDNA sequence of
PaPS determined using RACE has been deposited in the DDBJ
database with accession code AB252833.
amygdali N2 was separated by silica gel column chromatography
(hexane/ethyl acetate ) 10:1). The fraction showing characteristic
1H NMR methyl signals like those of phomopsene was purified by
silica gel column chromatography (hexane/acetone ) 20:1), and
recrystallization from hexane/ethyl acetate afforded methyl pho-
mopsenonate (5.0 mg): colorless crystal; mp 151 °C; [R]25D 12° (c
0.50, CHCl3); UV λmax nm (ꢀ): 318 (370), 248 (17000). See Table
1 for 1H and 13C NMR data. IR νmax cm-1: 1731, 1718 (w), 1666;
EI-HR-MS (positive): calcd for C21H30O3 (M) 330.2195, found m/z
330.2199.
Functional Analysis for Cyclase. To amplify the ORF cDNAs
of PaPS, reverse transcriptase-polymerase chain reaction (RT-PCR)
was carried out using a set of primers, 5′-GGATCCGTCCAT-
CAACATTCATTCATCATG-3′ (sense, BamHI site underlined) and
5′-GCGGCCGCACACGACCCGACAGATCAGG-3′ (antisense,
Not I site underlined). For amplification of a cDNA fragment
encoding N347 mutant, another reverse primer was used: 5′-
GCGGCCGCCTAGGTAAGCTTGGTCACTTCG-3′ (antisense,
Not I site underlined and added stop codon in boldface type). Each
PCR product was digested with BamHI and Not I and ligated into
pGEX-4T-3 vector (Amersham Pharmacia) for expression as fusion
protein with GST at the N-terminus. The plasmid was transformed
into E. coli BL21trx. Procedures for growth of the E. coli cells,
induction of the gene expression, extraction and purification of
recombinant enzymes, and measurement of cyclase enzyme activity
were identical with those described previously.24 Twenty micro-
grams of GPP, FPP, or GGPP was used as the substrate with or
without IPP. Hydrocarbon products and dephosphorylated deriva-
tives from allylic diphosphates remained in the reaction mixture
were analyzed by GC-MS.24
Functional Analysis for Prenyltransferase. For amplification
of a cDNA fragment encoding C398 mutant, a set of primers were
used: 5′-GGATCCATGATGGTTGCTCGCATGAA-3′ (sense, Bam-
HI site underlined) and 5′-GTCGACCTAAACTTTGAGTAAGCT-
GA (antisense, SalI site underlined). PCR product was digested
with BamHI and SalI and subcloned into pQE30 vector (QIAGEN),
and the plasmid was transformed into E. coli BL21 (DE3). All
procedures following were the same as those described previously.25
Allylic substrates used were DMAPP, GPP, or FPP. The products
derived from allylic substrates with [14C]IPP were dephosphorylated
and subjected to reverse-phase TLC analysis using LKC-18
developed with acetone/H2O (9:1).
X-ray Analysis of Methyl Phomopsenonate (2).26The 0.33 ×
0.33 × 0.26 mm3 crystal was orthorhombic (P212121), a )
8.9881(7), b ) 13.7561(10), c ) 14.6616(11) Å, V ) 1812.8(2)
Å3, Z ) 4, and Dc ) 1.211 g/cm3. All diffraction intensities
with 2θ < 136.5° were collected at -180 °C in the ω-2θ scan
mode by a Rigaku RAXIS RAPID imaging plate area detector
with graphite monochromated Cu KR radiation (λ ) 1.54187
Å). Of the 8191 reflections collected, 1117 were unique, and
the structure was solved by direct methods (SIR92).27 The non-
hydrogen atoms were refined anisotropically. Hydrogen atoms
were refined isotropically. Full-matrix least-squares refinement
on F converged to a final Rw factor of 0.0280 and an R factor of
0.0245 for the 18,222 observed reflections (I > 2.00σ(I)).
(R)-r-Methoxy-r-trifluoromethylphenylacetate 4a. To a solu-
tion of methyl phomopsenonate (2, 20 mg, 0.061 mmol) and
CeCl3 · 7H2O (38 mg, 0.10 mmol) in methanol (0.2 mL) was
added NaBH4 (100 mg, 2.64 mmol) by portions at 0 °C. Acetone
(0.5 mL) was added, and the mixture was extracted with ethyl
acetate. The organic layers were dried over anhydrous Na2SO4
and concentrated in vacuo. The residue was purified by prepara-
tive TLC (hexane/ethyl acetate ) 8:1) giving the allylic alcohol
3. The product was unstable and was immediately used for the
next reaction. To a solution of 3 and (R)-MTPA (18 mg, 0.077
mmol) in CH2Cl2 (0.5 mL) were added dicyclohexylcarbodiimide
(62 mg, 0.30 mmol) and 4-(dimethylamino)pyridine (22 mg, 0.18
mmol). After standing at 25 °C for 12 h, the reaction mixture
was filtered through Celite and concentrated in vacuo. The
residue was purified by preparative TLC (hexane/ethyl acetate
) 6:1) affording the (R)-MTPA ester 4a (5 mg, 15% in 2 steps):
Extraction and Partial Purification of the Hydrocarbon
Fraction. The n-hexane extract was prepared in the usual way
from the samples of P. amygdali mycelia or E. coli cells. This
extract was loaded into a column packed with silica gel (BW-
820MH, Fuji Silysia Chemical). The column was eluted by
n-hexane, and the eluate was evaporated and subjected to
GC-MS analysis.
1
colorless oil; H NMR (400 MHz, CDCl3) δ 7.55 (2H, m), 7.42
Isolation of Phomopsene. Fermentation of P. amygdali N2 was
carried out at 25 °C on a reciprocal shaker for 3 days in two hundred
500-mL Sakaguchi flasks each containing 120 mL of medium of
5.0% glucose, 1.0% Pharmamedia (Southern Cotton Oil Inc.), 0.5%
KH2PO4, and 0.1% MgSO4. The mycelia were collected by filtration
and extracted with acetone. The acetone layer was concentrated in
vacuo, and the residual aqueous layer was adjusted to pH 9.5 with
5% aqueous Na2CO3. The resulting aqueous layer was extracted
with ethyl acetate, and the organic layers were concentrated in
vacuo. The ethyl acetate extract (11 g) was partitioned between
hexane and acetonitrile. The hexane layer was concentrated in
vacuo, and the residue (8 g) was purified by silica gel column
chromatography (hexane) twice giving the mixture of hydrocarbon
products (8 mg). This mixture was further separated by reverse-
phase HPLC (Develosil ODS UG-5, φ 10 × 250 mm, acetonitrile)
affording 1 (4 mg): colorless oil; [R]25D -97° (c 0.27, CHCl3). See
Table 1 for 1H and 13C NMR data. EI-HR-MS (positive): calcd for
C20H32 (M) 272.2504, found m/z 272.2505.
(3H, m), 5.66 (1H, m, H-8), 3.69 (3H, s, COOMe), 3.65 (3H, s,
OMe), 2.695 (1H, m, H-10), 2.443 (1H, dd, J ) 8.7, 10.7 Hz,
H-3), 2.288 (2H, broad, t, J ) 7.4 Hz, H-5), 2.10 (1H, m, H-4),
1.992 (1H, ddd, J ) 3.2, 4.9, 12.2 Hz, H-9b), 1.82 (1H, m, H-4),
1.594 (3H, s, H-17) 1.55 (1H, d, J ) 7.2 Hz, H-11), 1.47-1.35
(7H, m), 1.024 (3H, s), 1.016 (3H, s), 1.010 (3H, s); ESI-HR-
MS (positive): calcd for C31H39F3NaO5 (M + Na), 571.2647,
found m/z 571.2691.
(S)-r-Methoxy-r-trifluoromethylphenylacetate (4b). The (S)-
MTPA ester 4b was obtained with (S)-MTPA by using the
procedure just described for 4a. 4b: colorless oil; 1H NMR (400
MHz, CDCl3) δ 7.58 (2H, m), 7.41 (3H, m), 5.69 (1H, m, H-8),
3.70 (3H, s, COOMe), 3.61 (3H, s, OMe), 2.723 (1H, m, H-10),
2.473 (1H, dd, J ) 8.7, 10.5 Hz, H-3), 2.255 (2H, broad, t, J )
7.2 Hz, H-5), 2.10 (1H, m, H-4), 2.020 (1H, ddd, J ) 3.3, 4.9,
12.1 Hz, H-9b), 1.82 (1H, m, H-4), 1.553 (3H, s, H-17) 1.55
(1H, d, J ) 7.2 Hz, H-11), 1.47-1.33 (7H, m), 1.018 (3H, s),
1.013 (6H, s); ESI-HR-MS (positive): calcd for C31H39F3NaO5
(M + Na), 571.2647, found m/z 571.2661.
Isolation of Methyl Phomopsenonate (2). The mycelial ethyl
acetate extract (1.6 g) from 3.5 L of 6-day cultural broth of P.
(23) Toyomasu, T.; Kawaide, H.; Mitsuhashi, W.; Inoue, Y.; Kamiya, Y.
Plant Physiol. 1998, 118, 1517–1523.
(24) Otomo, K.; Kenmoku, H.; Oikawa, H.; Konig, W. A.; Toshima, H.;
Mitsuhashi, W.; Yamane, H.; Sassa, T.; Toyomasu, T. Plant J. 2004, 39, 886–
893.
(25) Kawasaki, T.; Hamano, Y.; Kuzuyama, T.; Itoh, N.; Seto, H.; Dairi, T.
J. Biochem. 2003, 133, 83–91.
(26) CCDC 703562 contains the supplementary crystallographic data for
this paper. These data can be obtained free of charge via www.ccdc.cam.ac.uk/
data_request/cif, by emailing, or by contacting The Cambridge Crystal-
lographic Data Centre, 12, Union Road, Cambridge CB2 1EZ, U.K.; fax +44
1223 336033.
J. Org. Chem. Vol. 74, No. 4, 2009 1547

Toyomasu et al.
Acknowledgment. We are grateful to Dr. S. Kanematsu
(National Institute of Fruit Tree Science, Japan) for providing
the fungus, P. amygdali N2, and to Prof. M. Yotsu-Yamashita
(Tohoku University, Japan) for HR-EIMS measurements. This
work was supported in part by a grant-in-aid for Science
Research (A) (nos. 13306009 and 16208012) from the Japanese
Society for the Promotion of Science.
Supporting Information Available: Data on PaPS, 1H and
13C NMR spectra for compounds, and CIF file for methyl
phomopsenonate. This material is available free of charge via
the Internet at http://pubs.acs.org.
(27) Altomare, A.; Cascarano, G.; Giacovazzo, C.; Guagliardi, A. J. Appl.
Crystallogr. 1993, 26, 343–350.
JO802319E
1548 J. Org. Chem. Vol. 74, No. 4, 2009