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Synthesis, characterization and in vitro biological activities of ruthenium(II) polypyridyl complexes
Nat. Prod. Bioprospect. (2016) 6:167–171
DOI 10.1007/s13659-016-0096-4
ORIGINAL ARTICLE
Identification and Characterization of a d-Cadinol Synthase
Potentially Involved in the Formation of Boreovibrins
in Boreostereum vibrans of Basidiomycota
Hui Zhou . Yan-Long Yang . Jun Zeng .
Ling Zhang . Zhi-Hui Ding . Ying Zeng
Received: 3 March 2016 / Accepted: 24 March 2016 / Published online: 1 April 2016
The Author(s) 2016. This article is published with open access at Springerlink.com
Abstract Sesquiterpenoids are very common among natural products. A large number of sesquiterpene synthase genes
have been cloned and functionally characterized. However, until now there is no report about the d-cadinol synthase
predominantly forming d-cadinol (syn. torreyol) from farnesyl diphosphate. Sesquiterpenoids boreovibrins structurally
similar to d-cadinol were previously isolated from culture broths of the basidiomycete fungus Boreostereum vibrans. This
led us to expect a corresponding gene coding for a d-cadinol synthase that may be involved in the biosynthesis of
boreovibrins in B. vibrans. Here we report the cloning and heterologous expression of a new sesquiterpene synthase gene
from B. vibrans. The crude and purified recombinant enzymes, when incubating with farnesyl diphosphate as substrate,
gave d-cadinol as its principal product and thereby identified as a d-cadinol synthase.
Graphical Abstract A new sesquiterpene synthase gene was cloned from the basidiomycete fungus Boreostereum vibrans
and heterologously expressed in E. coli. The purified recombinant enzyme gave d-cadinol as its principal product from
farnesyl diphosphate and thereby identified as a d-cadinol synthase (BvCS).
Keywords
Delta-cadinol Sesquiterpene synthase Biosynthesis GC–MS Fungi
Electronic supplementary material The online version of this
article (doi:10.1007/s13659-016-0096-4) contains supplementary
material, which is available to authorized users.
H. Zhou Y.-L. Yang J. Zeng L. Zhang Z.-H. Ding
Y. Zeng (&)
State Key Laboratory of Phytochemistry and Plant Resources in
West China, Kunming Institute of Botany, Chinese Academy of
Sciences, 132 Lanhei Road, Kunming 650201, China
e-mail: biochem@mail.kib.ac.cn
H. Zhou Y.-L. Yang J. Zeng
University of Chinese Academy of Sciences, Beijing 100049,
China
1 Introduction
Higher fungi (Basidiomycota), among the many diverse
organisms, are a major source of biologically active natural
products, since they harbor a huge reservoir of active
secondary metabolites [1–4]. From culture broths of the
basidiomycete fungus Boreostereum vibrans (syn. Stereum
vibrans), vibralactones with distinctly skeletons were
identified [5–9] and generated by unusual and divergent
biosynthetic pathways that we have recently established by
precursor feedings in combination with genome mining
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[10, 11]. Further analyses of our B. vibrans genome draft
assembly revealed several sequences to encode putative
sesquiterpene synthases (STS). Interestingly, cadinane
sesquiterpenoids boreovibrins A–G were isolated from B.
vibrans [12]; most of them are structurally similar to dcadinol (syn. torreyol). Moreover, production of (?)-dcadinol was observed in mycelial of Stereum hirsutum
which falls into the same genus with B. vibrans [13]. Thus
we speculate that a sesquiterpene synthase catalyzing the
conversion of farnesyl diphosphate (FPP, 1) into d-cadinol
(6) is probably responsible for the production of boreovibrins (Fig. 1). Like other terpene synthases, STS catalyze the release of diphosphate from FPP and then guide
migration of the reactive carbocation along the prenyl
chain, thereby inducing a series of cyclization and rearrangement reactions, until final carbocation quenching by
deprotonation or water [14]. The reaction with the nucleophile water can afford terpene alcohols as direct products
instead of hydrocarbon terpenes. Many sesquiterpene
alcohols have been detected in enzymatic products of STS
from plants and bacteria, but major products similar to 6
are only found for s-cadinol synthase (LaCADS) from the
plant Lavandula angustifolia [15], and T-muurolol synthases from the bacteria Streptomyces clavuligerus [16]
and Roseiflexus castenholzii [17, 18]. With 6 as a minor
product, the sesquiterpene synthase Mg25 from the plant
Magnolia grandiflora was shown to mainly produce bcubebene from 1, hence named as a b-cubebene synthase
[19]. Even now there is no report about the d-cadinol
H. Zhou et al.
synthase predominantly forming d-cadinol from FPP.
Basidiomycete fungi are known to produce numerous
bioactive sesquiterpenoid metabolites, yet only a few STS
have been cloned and functionally characterized. For
examples, the protoilludene synthase in Armillaria gallica
was observed for exclusive production of D6-protoilludene
and involved in the biosynthesis of melleolides [20]. Six
STS from Coprinus cinereus were identified to produce
germacrene A, a-muurolene (4), germacrene D (3), cubebol, and a-cuprenene as major products, respectively [21,
22]; nine STS from Omphalotus olearius to produce amuurolene (4), b-elemene, d-cadinene, c-cadinene,
D6-protoilludene, a-barbatene, trans-dauca-4(11),8-diene
as major products, respectively [23]; and five STS from
Stereum hirsutum to produce d-cadinene, b-barbatene,
D6-protoilludene as major products, respectively [24]. Here
we report the cloning and heterologous expression of a new
sesquiterpene synthase gene from B. vibrans. The purified
recombinant enzyme, when incubating with 1 as a substrate, gave d-cadinol as its principal product and thereby
identified as a d-cadinol synthase.
2 Results and Discussion
Based on the putative sesquiterpene synthase sequence
from B. vibrans genome draft assembly, the full-length
cDNA (designated as BvCS) was recovered by RT-PCR
with specific primers and contained an open reading frame
Fig. 1 Proposed biosynthetic pathway to the sesquiterpenoids formed by the d-cadinol synthase BvCS from B. vibrans
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Identification and Characterization of a d-Cadinol Synthase Potentially Involved in the…
of 1182 nucleotides. The sequence has been submitted to
the GenBank database under accession number KU668561.
The deduced amino acid sequence of BvCS had 49 %
identity to a-muurolene synthase (Cop3, accession no.
A8NE23) from Coprinus cinereus [21] and germacradienol/germacrene D synthase (accession no. KNZ73785)
from Termitomyces sp. J132. Showing 22 % identity,
BvCS was less related to d-cadinene synthase (accession
no. XP_007299839) from Stereum hirsutum [24] (Table S1
in Electronic supplementary material).
Next, functional expression in pET32a?/Escherichia
coli BL21(DE3) was conducted to confirm the catalytic
activity of BvCS. Soluble protein expression was achieved
at 15 C for 22 h with 0.1 mM IPTG (isopropyl-b-D-thiogalactopyranoside), as determined by SDS-PAGE analysis
(Figure S1 in Electronic supplementary material). The
crude enzyme was assayed for sesquiterpene synthase
activity using 1 as a substrate under optional condition as
described in Sect. 3. Based on GC–MS analyses, major
product peak at 18.68 min (retention time) and minor
products were observed for crude BvCS, compared with
the empty vector as control (Electronic supplementary
material). The purified recombinant protein was used for
further characterization. After purification under native
condition on the Ni–NTA Agarose, the analysis of the elute
on SDS-PAGE led to detection of the main band corresponding exactly to the predicted size of the recombinant
BvCS protein of approximately 61 kDa (Fig. 2). When
incubated with FPP, the purified BvCS made d-cadinol (6)
as major product at 18.68 min, and minor products
including germacrene D-4-ol (2) at 17.10 min, a-muurolene (4) at 15.32 min, and c-muurolene (5) at 14.76 min,
compared with the heat-denatured enzyme as control
(Fig. 3, Electronic supplementary material). This is detected and characterized by GC–MS following comparison to
the standards included in the database. The product peak at
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18.68 min generated the dominant mass segments at m/z
161, 119, 204 and 105 perfectly matching d-cadinol (also
torreyol) in mass spectra of the database and the authentic
d-cadinol in publications [13, 25] (Electronic supplementary material).
For the biosynthesis of cadalane sesquiterpenes, three
alternative mechanisms were proposed and elucidated in
bacteria [16, 17] and plants [25, 26]. The intermediacy of
nerolidyl diphosphate (NPP) was generally accepted.
Recently, however, the pathway via the protonation of the
neutral intermediate 3 was demonstrated through labeling
experiments to account for production of 92 % of cadalane
sesquiterpenes by MtTPS5 from the plant Medicago truncatulathe, which can give 6 from 1 as its minor product
[25]. Furthermore, the germacrene D (3) pathway was
proposed for the bacterial STS from Chitinophaga pinensis
DSM 2588 that can afford both d-cadinene and germacrene
D-4-ol (2), although no germacrene D was present in the
enzymatic products [17]. As shown in this study, BvCS can
form 6 as major product and 2 as minor product (Fig. 3).
Therefore it is likely for the enzyme to follow the germacrene D pathway as that from C. pinensis DSM 2588 [17]
(Fig. 1, Electronic supplementary material), while alternative mechanisms with the intermediacy of NPP could
also be possible.
In conclusion, we cloned the full-length cDNA of a new
sesquiterpene synthase gene from the basidiomycete
Boreostereum vibrans and expressed it in E. coli for
functional characterization. Based on GC–MS analyses, the
recombinant enzyme was demonstrated to mainly produce
d-cadinol from farnesyl diphosphate and thereby identified
as a d-cadinol synthase.
3 Experimental Section
3.1 Gene Cloning
Fig. 2 Expression of BvCS/pET32a? in E. coli and purification of
recombinant fusion proteins. 0 Empty vector; 1 whole proteins; 2
soluble proteins; 3 unbinding proteins; 4 washing; 5 elute (the purified
enzyme); M protein size marker
Mycelia of the fungus Boreostereum vibrans was inoculated in 0.5 L modified PDB medium (potato 200.0 g,
glucose 20.0 g, KH2PO4 3.0 g, MgSO4 1.5 g, citric acid
0.1 g, and thiamin hydrochloride 10 mg in 1 L of deionized
water, pH 6.5), cultured at 25 C on a rotary shaker at
140 rpm. Mycelia were activated on PDB agar plates
before inoculation in liquid PDB. Total RNA was isolated
from the mycelia on day 20 using the Plant RNA Mini Kit
(Qiagen). The first-strand cDNA was synthesized with
SuperscriptTM III First-strand Synthesis System (Invitrogen). The full length cDNA was obtained with specific
primer pairs 50 -CCCGACCTTCTCACCATCTGT-30 (forward) and 50 -CGCGAGGTATAGAGCACCTGT-30 (reverse) according to the predicted gene sequence in our B.
vibrans genome draft assembly. For PCR, 30 cycles of
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H. Zhou et al.
Fig. 3 GC–MS analyses of products formed by the recombinant
BvCS and the heat-denatured enzyme as control, respectively, with
FPP as substrate. Total ion chromatograms (left) and the
corresponding mass spectra (right) illustrate the product peaks at
17.10 and 18.68 min matching germacrene D-4-ol and d-cadinol,
respectively, in mass fragmentation patterns included in the database
reactions were performed at the condition (95 C, 30 s;
58 C, 30 s; 72 C, 100 s) with final extension at 72 C,
10 min. The amplicons were cloned and sequenced to
verify the encoding region. The sequence, designated as
BvCS, has been submitted to the GenBank database under
accession number KU668561.
3.3 Enzyme Assays and GC–MS Analyses
3.2 Expression in E. coli and Enzyme Purification
The ORF of BvCS was cloned into the expression vector
pET32a? (Novagen) which was subsequently transformed
into E. coli BL21(DE3) (Novagen) for a fusion expression,
using the original pET32a? as negative control. Protein
expression at 37 or 15 C with 0.1 or 0.5 mM IPTG was
determined by SDS-PAGE analysis. Cells induced with
0.1 mM IPTG at 15 C for 22 h were collected by centrifugation (8000 rpm, 4 C, 5 min), washed by deionized
water and suspended in 50 mM Tris–HCl buffer (pH 7.5)
for crude enzyme preparation or in the binding buffer
(20 mM Tris–HCl pH 7.5, 0.3 M NaCl, 5 mM imidazole)
for enzyme purification. The cell lysate obtained by sonication on ice was then centrifuged at 12000 rpm for 10 min
at 4 C and the supernatant, containing the soluble
recombinant enzyme was used for crude enzyme assay or
enzyme purification. Purification of His-tagged enzymes
was performed according to the Ni–NTA Agarose protocol
(Qiagen) with a wash buffer (20 mM Tris–HCl pH 7.5,
0.5 M NaCl, 150 mM imidazole) and an elution buffer
(20 mM Tris–HCl pH 7.5, 0.5 M NaCl, 300 mM imidazole). The elute containing the purified enzyme was
immediately desalted with 50 mM Tris–HCl buffer
(pH 7.5), concentrated by centrifugation at 4 C (Amicon
Ultra-15, Merck Millipore), and immediately used for
activity assays. All fractions were analyzed by SDS-PAGE
on 12 % polyacrylamide gel under non-reduced condition
at 140 V for 1.5 h.
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Crude enzyme was assayed for sesquiterpene synthase
activity using FPP (Sigma) of 10 lg in assay buffer
(50 mM Tris–HCl pH 7.5, 10 % glycerol, 10 mM MgCl2,
1 mM dithiothreitol, 100 mM NaCl), incubating for 4 h at
30 C, using the empty vector as control. After extraction
with 2 mL hexane, the hexane phase was collected by
centrifugation, dehydrated over anhydrous sodium sulfate
and concentrated under a stream of air for GC–MS analysis. The purified enzyme activity was detected as described above with FPP of 5 lg, using heat-denatured enzyme
as control.
GC–MS
[Agilent
HP6890/5973,
column:
0.25 mm 9 30 mm, 0.25 lm (HP-5MS)] was conducted
under electron-impact (EI) mode (70 eV). The flow rate of
helium carrier gas was set at 1.0 mL/min. Samples (2 lL)
were injected at 80 C. After holding the samples for 5 min
at 80 C, the column temperature was increased at 5 C/min
to 280 C and hold for 30 min. The MS date was collected
from 35 to 500 m/z. The identification of the compounds
was achieved by comparing the retention time and the mass
spectra with those of the standards included in the library
(wiley7n.1) and the authentic compounds in publications.
Acknowledgments This work was supported by a grant from the
National Natural Science Foundation of China (21572237). We are
grateful to Professor Jian-Xin Pu (Kunming Institute of Botany) for
his helpful suggestion.
Compliance with Ethical Standards
Conflict of interest
The authors declare no conflict of interest.
Open Access This article is distributed under the terms of the
Creative Commons Attribution 4.0 International License (http://
creativecommons.org/licenses/by/4.0/), which permits unrestricted
use, distribution, and reproduction in any medium, provided you give
appropriate credit to the original author(s) and the source, provide a
Identification and Characterization of a d-Cadinol Synthase Potentially Involved in the…
link to the Creative Commons license, and indicate if changes were
made.
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