Molecular cloning and characterization of drimenol synthase from valerian plant (Valeriana officinalis)

Article abstract of DOI:10.1016/j.febslet.2014.10.031

Drimenol, a sesquiterpene alcohol, and its derivatives display diverse bio-activities in nature. However, a drimenol synthase gene has yet to be identified. We identified a new sesquiterpene synthase cDNA (VoTPS3) in valerian plant (Valeriana officinalis). Purification and NMR analyses of the VoTPS3-produced terpene, and characterization of the VoTPS3 enzyme confirmed that VoTPS3 synthesizes (-)-drimenol. In feeding assays, possible reaction intermediates, farnesol and drimenyl diphosphate, could not be converted to drimenol, suggesting that the intermediate remains tightly bound to VoTPS3 during catalysis. A mechanistic consideration of (-)-drimenol synthesis suggests that drimenol synthase is likely to use a protonation-initiated cyclization, which is rare for sesquiterpene synthases. VoTPS3 can be used to produce (-)-drimenol, from which useful drimane-type terpenes can be synthesized.

Full text of DOI:10.1016/j.febslet.2014.10.031

Accepted Manuscript
Molecular cloning and characterization of drimenol synthase from Valerian
plant (Valeriana officinalis)
Moonhyuk Kwon, Stephen A. Cochrane, John C. Vederas, Dae-Kyun Ro
PII:
S0014-5793(14)00778-9
DOI:
http://dx.doi.org/10.1016/j.febslet.2014.10.031
FEBS 36888
Reference:
To appear in:
FEBS Letters
Received Date:
Revised Date:
Accepted Date:
7 August 2014
25 October 2014
27 October 2014
Please cite this article as: Kwon, M., Cochrane, S.A., Vederas, J.C., Ro, D-K., Molecular cloning and
characterization of drimenol synthase from Valerian plant (Valeriana officinalis), FEBS Letters (2014), doi: http://
dx.doi.org/10.1016/j.febslet.2014.10.031
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1
. Introduction
Terpenoids (or isoprenoids) are one of the major classes of natural products with 55,000 known
structures [1]. Terpenoids in animals serve as primary physiological components of membrane
constituents (e.g., cholesterol) and the electron transport chain (e.g., ubiquinone), while in plants
and microbes, they occur as diverse secondary metabolites which have benefited human lives as
aromas, flavors, and pharmaceutical substances [2,3]. Terpenoids of specific hydrocarbon
backbones are synthesized by specific terpene synthases (TPSs), which can fold prenyl
diphosphate precursors, such as geranyl (C ), farnesyl (C ), and geranyl geranyl (C )
1
0
15
20
diphosphate, with subsequent carbocation formation to produce a large variety of different
terpenoids [4-6]. Hence, at the entry point of the terpenoid metabolism, the TPS enzyme dictates
terpene skeletal types. In nature, some terpene skeletons (e.g., farnesene, caryophyllene, and
germacrene) are commonly found in plants, but others are associated with specific lineages or
species of plants through evolutionary adaptations (e.g., amorphadiene from Artemisia annua
and (+)-epi-α-bisabolol from Lippia dulcis) [7,8]. Therefore, it is important to characterize TPSs
from diverse taxa.
Drimane is one of the unique C sesquiterpene backbones (Figure 1A), of which the
1
5
biosynthetic gene has not been identified [9]. Hundreds of drimane-type sesquiterpenoids have
been isolated from plants (primarily Canellaceae and Polygonum families), liverworts, fungi, and
sponges [9-16]. Intriguingly, many of them show strong biological activities, such as antifungal,
plant growth regulatory and antifeedant effects [9,13,17-19]. Hence, it is apparent that drimane
derivatives are widespread in nature and deliver important eco-physiological roles to the
synthesizers. However, it should be noted that drimane is rarely isolated as a natural product and
that the simplest and closest drimane-type sesquiterpene found in nature is drimenol (Figure 1B),
which implies that drimenol or perhaps drimenyl pyrophosphate is the biosynthetic precursor to
the various drimane sesquiterpenoids. A native sesquiterpene synthase (sesqui-TPS), that can
2
+
synthesize drimenol from (E,E)-farnesyl diphosphate (FPP) in a Mg -dependent manner, has
been purified from the Polygonum hydropiper [20]. This plant produces a drimane
sesquiterpenoid, polygodial (Figure 1C), which possesses potent antifeedant activity against
insects [9,17,21]. These observations support that drimenol is the first skeletal backbone
synthesized from FPP, and that drimenol serves as the central precursor of naturally occurring
1

drimane-type sesquiterpenoids. However, the respective gene for drimenol biosynthesis had not
been identified until now.
2
. Materials and Methods
2
.1. Plasmid construct
Total RNA was isolated from the roots of Valeriana officinalis with 1 % of cetyl trimethyl
ammonium bromide (0.5 M Tris HCl, pH. 8.0, 0.25 M ethylene diaminetetraacetic acid (EDTA),
4
% NaCl, 0.5 % polyvinylpyrrolidone). The cDNA synthesized from total RNA was used as
template for PCR. The PCR products were amplified with primers (Table S1) and cloned into
pESC-Leu2d for yeast expression, pBluescript II for D307A sub-cloning, and pH9GW or
pDEST17 for E. coli expression.
2
.2. Production and purification of drimenol in yeast
VoTPS3:pESC-Leu2d or the empty vector was transformed into the yeast strain, EPY300, and
the terpene products from the transgenic yeast analyzed, according to published methods [22].
To purify the VoTPS3-product, the VoTPS3-expressing yeasts were cultured at 30 ºC for 6 days
in a 1-L cultivation scale. The yeast culture was extracted with diethyl ether (3 x 500 mL) and
the organic extracts combined, concentrated and purified by flash column chromatography (silica,
4
5 g; 23 cm height; eluent = 95:5 hexanes:ethyl acetate; 180~250 mL fractions). Drimenol-
containing fractions were pooled, concentrated, and fractionated again using flash column
chromatography (silica, 15 g; 23 cm height; eluent = dichloromethane; 80~180 mL fractions).
2
.3. NMR Spectroscopy
The purified product was dried with N gas, dissolved in CD OD (600 μL) and analyzed by
2
3
NMR. NMR spectra were recorded on a Varian Inova 600 MHz or 700 MHz spectrometer
1
3
1
equipped with a C/ H dual cold probe. δ values were referenced to CD OD (3.31 ppm) or D O
3
2
1
13
(
4.90 ppm) for H-NMR and to CD OD (49.0 ppm) for C-NMR. Details of 1D and 2D NMR
3
analyses are given in the Supplementary Materials.
2

2
.4. Overexpression and purification of recombinant VoTPS3
An E. coli strain, Rosetta pLysS, harboring VoTPS3-pH9GW or D307A-pDEST17 was cultured
in 500 mL of LB medium at 37 ºC for 2 hr and induced with 0.1 mM isopropyl β-D-1-
thiogalactopyranoside at 22 ºC overnight. The cells were pelleted at 6,000 rpm for 5 min at 4 ºC
and resuspended in lysis buffer (50 mM Na PO , pH. 7.5, 500 mM NaCl, 20 mM Imidazole).
3
4
The cells were lysed by sonication and then centrifuged at 4,000 rpm for 30 min at 4 ºC. The
soluble fraction was bound with Nickel-Nitrilotriacetic acid resin (Qiagen, Venlo, Netherlands)
for 1 hr. Proteins were eluted with 2 mL of elution buffer (50 mM Na PO , pH. 7.5, 500 mM
3
4
NaCl, 50 mM Imidazole), followed by concentration and desalting using a membrane filter
Millipore, Massachusetts, USA).
(
2
.5. Enzyme Characterization
For the in vitro enzyme assay, 20 μg of purified enzyme was mixed with 50 mM Tris (pH 7.5),
0 mM MgCl and 100 μM of (E,E)-FPP (Echelon Biosciences) or 1 mM (E,E)-farnesol (Sigma)
1
2
or 100 μM of drimenyl diphosphate. The reaction mixture (500 μL) was overlaid with hexane
500 μL) and incubated at 30 ºC for 2 hr. The phases were separated and the aqueous layer
(
extracted with hexane (2 x500 μL). The combined hexane extracts were analyzed by GC-MS. To
convert FPP to farnesol or drimenyl diphosphate to drimenol, the reaction mixture was incubated
with 2 units of calf intestine phosphatase (CIP) (NEB). For the assay using heavy water, the
1
8
reaction mixture was incubated with 79 % of H O and 19 % of H O (Sigma). For kinetic
2
2
3
-1
analysis, cold FPP substrates were spiked with [1- H]-FPP (Perkin Elmer, 23 Ci mmol ) at 1%
of the final concentration. A 100 μL mixture (3.2 μg of enzyme, 25 mM HEPES, pH. 7.4, 10 mM
MgCl , 0.5-10 μM FPP) was incubated for 5 min at 30 ºC. The reaction was terminated by
2
adding 4 M NaOH and 1 M EDTA. The products were mixed with pentane (900 μL) and 400 μL
of the upper layer was collected. The pentane extracts were mixed with 3.5 mL of liquid
scintillation cocktail. The radioactivity of products was monitored by a liquid scintillation
counter (Beckman LS6500). The kinetic parameters were calculated with the Enzyme Kinetics
module in Sigmaplot 12.
3

2
.6. GC-MS analysis
GC-MS analysis was performed with an Agilent 6890N gas chromatography system, Agilent
975B mass spectrometer and DB1-MS column (30 m × 250 μm × 0.25 μm). 2 μL of sample
5
-1
was injected at 250 ºC, and the temperature was increased at a rate of 10 ºC min from 40 ºC to
-1
2
50 ºC. Helium (1 mL min ) was used as a carrier gas. The National Institute of Standards and
TM
Technology (NIST) Standard Reference Database (version 5) and the Wiley Registry of Mass
Spectral Data (version 10) were used to identify drimenol.
2
.7. Quantitative read mapping
Illumina read mapping: For quantitative analysis, CLC Genomics Workbench software (version
5
2
.5.1) was employed using a single read Illumina data. The parameters used were: mismatch cost,
; insertion cost, 1; deletion cost, 1; length fraction, 0.8; similarity fraction, 0.98.
3
. Results and Discussion
3
.1. Isolation of new sesqui-TPS from V. officinalis
Drimenol was reported in the valerian plant (V. officinalis) root as a minor constituent [23-26],
and we searched for the valerian transcriptome data as a source of drimenol synthase gene
isolation. From the valerian root transcriptome database, generated by 454 sequencing,
valerenadiene synthase (VoTPS1) and germacrene C/D synthase (VoTPS2) were previously
identified and characterized [27,28]. To acquire deeper transcript sequencing, Illumina
sequencing was additionally performed, and the resulting reads were assembled by a Trinity
algorithm [29]. The BLAST-searchable databases of V. officinalis 454 and Illumina
transcriptomes
www.phytometasyn.ca) [30,31].
From this transcriptome database, the third full-length sesqui-TPS cDNA was identified,
are
publicly
accessible
through
the
PhytoMetaSyn
website
(
and its open reading frame (ORF) was PCR-amplified using primers designed at the start and
stop codon of the assembled transcript. The cloned cDNA encodes a polypeptide of 556 amino
acids with M of 64.8 kDa. The newly identified sesqui-TPS was named VoTPS3 (GenBank
w
number: JQ437841). VoTPS3 was quite divergent from other sesqui-TPSs as it displayed only
4

4
1% and 39% sequence identity to VoTPS1 and VoTPS2, respectively, and its closest homolog in
the public database showed limited amino acid identity (49% identity with vetispiradiene
synthase from Solanum tuberosum; GenBank number, BAA82108). To infer the relative
expression level of VoTPS3, ~30 million raw Illumina reads were computationally mapped to
VoTPS1/2/3 ORF nucleotide sequences. In this in silico analysis, VoTPS1 and VoTPS2 showed a
comparable level of expression to each other, but the read count of VoTPS3 was only 2.4% of
VoTPS1 (Table 1). Therefore, VoTPS3 appears to only marginally contribute to the terpene
constituents of the valerian root, in comparison to the VoTPS1 (valerenadiene synthase) that is
responsible for a major sesquiterpenoid, valerenic acid [27].
3
.2. In vivo functional assessment of sesqui-TPS
To assess the catalytic function of VoTPS3, its ORF was expressed under a galactose-inducible
promoter in the EPY300 strain, a yeast strain engineered to accumulate an elevated level of FPP
[
32,33]. After galactose-induction, volatile terpenes were sequestered by dodecane (2 mL)
overlaid on the yeast culture (20 mL) during the 3-day cultivation. The dodecane layer was then
analyzed by GC-MS. In the GC-MS profile, a new peak (Figure 2, peak 2) was identified from
the yeast expressing VoTPS3, whereas this peak was absent in the control yeast harboring an
empty plasmid. On the other hand, (E,E)-farnesol (Figure 2, peak 1) derived from the
dephosphorylation of FPP was the most abundant compound in the control yeast, but it was
markedly reduced in the yeast expressing VoTPS3. This observation implied that an efficient
conversion of FPP to the new terpene product was occurring in the VoTPS3-expressing yeast. In
the MS spectral database search, the electron-impact (EI) fragmentation pattern of the new
terpene showed a strong spectral match (98%) to that of drimenol with a parental m/z of 222.
This result indicated that VoTPS3 might catalyze the synthesis of the sesquiterpene alcohol,
drimenol, from FPP.
3
.3. Structural elucidation of (-)-drimenol
To unambiguously assign the chemical identity of the VoTPS3-product as drimenol, the
cultivation of the yeast expressing VoTPS3 was scaled up to 1-L, and the whole yeast culture was
extracted with diethyl ether (3 x 500 mL). The diethyl ether fraction was fractionated using flash
5

column chromatography (see section 2.2) to yield 6 mg of the VoTPS3-product with >95%
purity. The purified terpene was fully characterized by one- and two-dimensional NMR analyses.
1
13
1
1
1
13
H-NMR, C-NMR, H- H COSY, H- C HSQC, HMBC, and TOCSY were used to validate
the drimenol skeleton. 1D-NOESY experiments were additionally used to assign the relative
stereo-configuration of the stereo-centers at C5, C9 and C10 (details of structural determination
are given in the Supporting Information). To determine if the isolated drimenol was the RRR or
SSS enantiomer, a specific rotation value was measured, and its ] value compared to a
D
previously reported synthetic SSS standard [34]. Both the isolated drimenol sample and the
synthetic literature compound had comparable negative values (see Supplementary Materials),
which allowed us to assign the absolute configuration as the SSS enantiomer, (-)-drimenol. From
these results, we concluded that VoTPS3 encodes the Valeriana officinalis (-)-drimenol synthase
(VoDMS).
3
.4. Characterization of recombinant drimenol synthase
In order to evaluate the kinetic properties of VoDMS, VoDMS was N-terminally tagged with
nine histidines (9xHis-VoDMS) and was expressed in E. coli. Approximately, 0.5 mg of the
recombinant enzyme with 99% purity, as assessed by SDS-PAGE, was purified by a Nickel-
NTA affinity column (Supplementary Figure 1A). Incubation of purified VoDMS with FPP and
2
+
the cofactor Mg resulted in the in vitro synthesis of drimenol (Supplementary Figure 1B/C).
3
Furthermore, kinetic properties of VoDMS were obtained using H-labeled FPP. The K and k
m
cat
-1
values of VoDMS were determined to be 9.6 ± 2.7 μM and 0.025 ± 0.004 s (mean ± S.D; n=3).
This result assured that VoDMS encodes a catalytically active sesqui-TPS enzyme.
3
.5. Mechanism of Drimenol synthesis by VoDMS
2
+
Terpene synthases are known to utilize two different mechanisms: 1) Mg -dependent
“
ionization-initiated” reaction (class I) by cleaving an allylic diphosphate bond or 2)
protonation-initiated” reaction (class II) by protonating a double-bond at the terminal isoprene
“
unit (Figure 3) [35-37]. The ionization-initiated reaction is mediated by the conserved DDxxD
motif, which binds a divalent metal ion for substrate ionization. On the other hand, the
“protonation-initiated” reaction involves an acidic residue, often conserved in DxDD motif,
6

which donates a proton to the double-bond at a terminal isoprene unit. Both reactions produce
reactive carbocation intermediates which undergo carbocation cascade reactions to yield various
isoprene products. A large majority of sesqui-TPS’ use the class I “ionization-initiated”
mechanism, whereas the class II “protonation-initiated” reaction is a typical terpene-forming
reaction for triterpene synthases.
As expected, VoTPS sequences align seamlessly with those of other sesqui-TPSs and
possess the conserved DDxxD motif for FPP ionization (Supplementary Figure 2). Therefore, we
considered the biosynthesis of drimenol using an “ionization-initiated” carbocation formation at
C11. However, a simple mechanism for drimenol formation by this typical ionization (class I)
mechanism is not readily apparent. Rather, the drimenol formation from FPP can be postulated
without difficulty by employing the class II reaction by protonating at C3 (Figure 4A). In this
mechanism, the reaction proceeds with a protonation at C3, followed by two sequential double-
bond rearrangements and a deprotonation. Then the cleavage of diphosphate by DDxxD and
2
+
Mg could generate a primary carbocation which can be quenched by a water molecule. In this
model, both class I and II mechanisms contribute to the formation of drimenol.
We recognized, however, the occurrence of an unstable primary carbocation in this
mechanism and hence reasoned that drimenol could be synthesized by an enzymatic hydrolysis
of the diphosphate. To probe the biosynthetic nature of the alcohol moiety at C11, VoDMS
18
2+ 18
enzyme was incubated with H O. If C11 is ionized by the DDxxD motif and Mg , O will be
2
incorporated into drimenol. On the other hand, if a hydrolysis of diphosphate is responsible for
1
8
18
2
the reaction, O from the heavy water will not be incorporated into the drimenol. With H O as
1
9% of total H O in the enzyme assay, comparable amount (20.3%) of drimenol parental ions
2
with two mass increase (m/z = 224) was detected by GC-MS analysis (Figure 5). This result
proved that the oxygen atom from water is incorporated into drimenol, supporting the class I
ionization reaction by VoDMS. The proposed homoallylic cation could be stabilized by its
resonance structure, cyclopropylcarbinyl and cyclobutyl cation (Figure 4A). In this proposed
catalysis, drimenyl diphosphate is a key intermediate, and thus VoDMS could utilize drimenol
diphosphate as a substrate. To test this possibility, drimenyl diphosphate was synthesized from
the purified drimenol and incubated with VoDMS, but no evidence of drimenol formation from
drimenyl diphosphate was obtained (Supplementary Figure 3). From this result, we can deduce
7

two possible explanations: 1) drimenyl diphosphate is not an intermediate in VoDMS reaction or
) drimenyl diphosphate remains tightly bound to the active site of VoDMS during catalysis.
2
Even though the drimenyl diphosphate is an intermediate in VoDMS reaction, it is apparent that
VoDMS does not have a separate active site for the intermediate (i.e., drimenyl diphosphate) as
shown in the bifunctional abietadiene synthase that encodes two distinct active sites in a single
polypeptide [38].
A triterpene synthase-class enzyme, squalene-hopene cyclase, has been shown to catalyze
an enzymatic conversion of (E,E)-farnesol to drimenol by its promiscuous protonation activity
on the C3 of farnesol [35,39,40]. Based on this observation, we investigated if farnesol could be
an intermediate of the VoDMS reaction as shown in Figure 4B. In this model, ionization occurs
at C11, and the C11 carbocation is immediately quenched by a water molecule, yielding a
farnesol intermediate. Farnesol can undergo C3 protonation, double-bond rearrangements, and
deprotonation to produce drimenol. To test this possible mechanism, we incubated VoDMS with
up to 10 mM of the farnesol substrate. However, no evidence of drimenol formation was
obtained from this assay (Supplementary Figure 4), suggesting that farnesol is not likely to be an
intermediate in the VoDMS reaction. Despite this result, we cannot exclude the possibility that
the enzyme initially binds only FPP, which may induce a conformational change so that
exogenous farnesol cannot access the active site despite the possible transient existence of that
species during the reaction.
It is unprecedented that a sesqui-TPS possess both class I and class II reactivity in a
single polypeptide. We attempted to decouple the class I (ionization) and class II (protonation)
reaction by mutating the DDxxD motif in VoDMS. The first Asp residue of the DDxxD motif,
2
+
known to ionize the substrate in concert with Mg , was mutated to Ala to generate an ADxxD
motif (D307A). This mutant enzyme, possessing the weakened metal-binding motif, was
incubated with FPP, and the product was dephosphorylated by phosphatase and analyzed by GC-
MS. From this experiment, we aimed to examine the possibility that the substrate FPP binds,
although weakly, to the active site, but is not ionized due to the D307A point-mutation in the
conserved DDxxD motif. While FPP is being bound to the mutant enzyme, C3-protonation could
still occur to produce drimane-diphosphate, which can be converted to drimenol by phosphatase
8

treatment. However, in assays using the mutant VoDMS, drimenol was not detected by GC-MS
analysis (Supplementary Figure 5).
Taken together, a specific reaction mechanism could not be inferred either by feeding
farnesol or drimenyl diphosphate to the enzyme or by using the weakened metal-biding motif
(ADxxD; D307A). However, these results need to be cautiously interpreted. During the reaction,
the reaction intermediates (farnesol or drimenyl diphosphate) could be deeply buried in the
substrate-binding site of VoDMS by the conformational change of the enzyme, and hence
exogenous intermediates may not be accessible to the active site. Also, FPP may not be able to
bind strongly to the active site of the mutant VoDMS (D309A). Therefore, the lack of evidence
for C3-protonation from these experiments cannot exclude a class II reaction in VoDMS. We
predict that an unrevealed acidic residue in VoDMS may play a critical role in protonation.
4
. Conclusion
In summary, a new sesqui-TPS, identified from the valerian root transcriptomic analysis, was
comprehensively characterized and demonstrated to encode a (-)-drimenol synthase. Drimenol
can serve as a useful synthon for the semi-synthetic preparations of various bioactive drimane-
type sesquiterpenes. Hence, the VoDMS can be used to biologically synthesize drimenol, as
shown in our yeast system. VoDMS appears to utilize both class I (ionization) and class II
(protonation) mechanisms in a single active site that are unprecedented among the known sesqui-
TPSs.
Acknowledgements
This work was supported by Genome Canada [PP780422], Genome Alberta, Government of
Alberta [PP789067], Genome Quebec, McGill Innovation Centre, MDEIE (Ministére du
Développement économique, Innovation et Exportation) to D.K.R., and Canada Research Chair
program to D.K.R. and J.C.V. This work was also supported by the Next-Generation BioGreen
2
1 Program (SSAC grant PJ009549032014), Rural Development Administration, Republic of
Korea to M.K.
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1
2

Figure legend
Figure 1. Structures of drimane, drimenol, and polygodial.
Figure 2. GC-MS analyses of volatiles from VoTPS3-expressing yeast.
A) Gas chromatographic separation of volatile products from empty plasmid and VoTPS3-
expressing yeast. Peak 1 and 2 are farnesol and VoTPS3-product (drimenol), respectively, and
their structures are shown beside the peaks. B) Mass spectra of VoTPS3-product and its closest
spectral match in the GC-MS library.
Figure 3. Schematic description of class I and class II reaction mechanisms by terpene
synthases.
Figure 4. A proposed biosynthetic mechanism of drimenol catalyzed by drimenol synthase.
See Figure 1 for carbon numbers. A) Protonation at C3 of drimenol occurs first, followed by
double-bond rearrangement and a quench by H O. B) Ionization at C11 occurs first, followed by
2
a quench by a H O and farnesol intermediate formation. Farnesol is further cyclized to drimenol
2
by a protonation at C3. FPP can be converted to farnesol by a phosphatase enzyme reaction, in
which phosphorus in FPP is attacked by a hydroxyl group of serine residue in phosphatase.
1
8
Figure 5. Mass spectra of the drimenol synthesized by VoDMS in the presence of H O
2
Mass spectrum of drimenol synthesized from VoDMS assay with 100% H O (A) and with 81%
2
1
8
16
H O and 19% H O (B). The m/z value of 222 is measured for drimenol parental ions with O.
2
2
1
3

Table 1. Quantitative mapping of three valerian sesqui-TPSs.
Reference
TPS name length (base
pairs)
a
Unique
Relative
read count
abundance
VoTPS1
VoTPS2
VoTPS3
a
1,689
1,692
1,671
24,137
7,674
575
100 %
31.8 %
2.4 %
3
0,362,677 reads generated by Illumina sequencings were mapped to the three reference sequences by
the parameters described in the method.

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Molecular cloning and characterization of drimenol synthase from Valerian plant
(
Valeriana officinalis)
a,b
c
c
a,
Moonhyuk Kwon , Stephen A. Cochrane , John C. Vederas , and Dae-Kyun Ro *
a
Department of Biological Sciences, University of Calgary, 2500 University Dr. NW, Calgary,
T2N 1N4, Canada
b
Department of Agricultural Biotechnology and Research Institute of Agricultural Sciences,
Seoul National University, Seoul 151-921, Korea
c
Department of Chemistry, University of Alberta, Edmonton, T6G 2G2, Canada
*
2
9
Corresponding author. Address: Department of Biological Sciences, University of Calgary,
500 University Dr. NW, Calgary, T2N 1N4, Canada. Tel: +1 403 220 7099, Fax: +1 403 289
311, E-mail address: daekyun.ro@ucalgary.ca (D.K. Ro)

Abstract
Drimenol, a sesquiterpene alcohol, and its derivatives display diverse bio-activities in nature.
However, a drimenol synthase gene has yet to be identified. We identified a new sesquiterpene
synthase cDNA (VoTPS3) in valerian plant (Valeriana officinalis). Purification and NMR
analyses of the VoTPS3-produced terpene, and characterization of the VoTPS3 enzyme
confirmed that VoTPS3 synthesizes (-)-drimenol. In feeding assays, possible reaction
intermediates, farnesol and drimenyl diphosphate, could not be converted to drimenol,
suggesting that the intermediate remains tightly bound to VoTPS3 during catalysis. A
mechanistic consideration of (-)-drimenol synthesis suggests that drimenol synthase is likely to
use a protonation-initiated cyclization, which is rare for a sesquiterpene synthases. VoTPS3 can
be used to produce (-)-drimenol, from which useful drimane-type terpenes can be synthesized.
Keywords: Valeriana officinalis, valerian, terpene synthase, drimenol synthase, (-)-drimenol
Abbreviations: FPP, farnesyl diphosphate; TPS, terpene synthase; sesqui-TPS, sesquiterpene
synthase;

Highlights




Transcriptomics analysis of valerian root identified a drimenol synthase.
Expression of drimenol synthase in yeast allowed de novo synthesis of drimenol.
1D and 2D NMR analyses validated (-)-drimenol structure with SSS configuration.
Drimenol synthase appears to use both protonation and ionization mechanism.