Bifunctional cis-abienol synthase from Abies balsamea discovered by transcriptome sequencing and its implications for diterpenoid fragrance production

Article abstract of DOI:10.1074/jbc.M111.317669

The labdanoid diterpene alcohol cis-abienol is a major component of the aromatic oleoresin of balsam fir (Abies balsamea) and serves as a valuable bioproduct material for the fragrance industry. Using high-throughput 454 transcriptome sequencing and metabolite profiling of balsam fir bark tissue, we identified candidate diterpene synthase sequences for full-length cDNA cloning and functional characterization. We discovered a bifunctional class I/II cis-abienol synthase (AbCAS), along with the paralogous levopimaradiene/ abietadiene synthase and isopimaradiene synthase, all of which are members of the gymnosperm- specific TPS-d subfamily. The AbCAS-catalyzed formation of cis-abienol proceeds via cyclization and hydroxylation at carbon C-8 of a postulated carbocation intermediate in the class II active site, followed by cleavage of the diphosphate group and termination of the reaction sequence without further cyclization in the class I active site. This reaction mechanism is distinct from that of synthases of the isopimaradiene- or levopimaradiene/ abietadiene synthase type, which employ deprotonation reactions in the class II active site and secondary cyclizations in the class I active site, leading to tricyclic diterpenes. Comparative homology modeling suggested the active site residues Asp-348, Leu-617, Phe-696, and Gly-723 as potentially important for the specificity of AbCAS. As a class I/II bifunctional enzyme, AbCAS is a promising target for metabolic engineering of cis-abienol production.

Full text of DOI:10.1074/jbc.M111.317669

Plant Biology:
Bifunctional cis-Abienol Synthase from
Abies balsamea Discovered by
Transcriptome Sequencing and Its
Implications for Diterpenoid Fragrance
Production
Philipp Zerbe, Angela Chiang, Macaire Yuen,
Björn Hamberger, Britta Hamberger, Jason A.
Draper, Robert Britton and Jörg Bohlmann
J. Biol. Chem. 2012, 287:12121-12131.
doi: 10.1074/jbc.M111.317669 originally published online February 15, 2012
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THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 287, NO. 15, pp. 12121–12131, April 6, 2012
© 2012 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Bifunctional cis-Abienol Synthase from Abies balsamea
Discovered by Transcriptome Sequencing and Its
□
S
Implications for Diterpenoid Fragrance Production*
Received for publication, October 26, 2011, and in revised form, February 14, 2012 Published, JBC Papers in Press, February 15, 2012, DOI 10.1074/jbc.M111.317669
Philipp Zerbe‡, Angela Chiang‡, Macaire Yuen‡, Björn Hamberger‡1, Britta Hamberger‡1, Jason A. Draper§,
Robert Britton§, and Jörg Bohlmann‡1,2
From the ‡Michael Smith Laboratories, University of British Columbia, 301-2185 East Mall, Vancouver, British Columbia V6T 1Z4,
Canada and the §Department of Chemistry, Simon Fraser University, Burnaby, British Columbia V5A 1S6, Canada
Background: Balsam fir produces cis-abienol, a natural product of value to the fragrance industry.
Results: We describe the genomics-based discovery of balsam fir cis-abienol synthase.
Conclusion: cis-Abienol synthase is a bifunctional diterpene synthase that produces a bicyclic diterpenol in the class II active
site.
Significance: cis-Abienol synthase is a new enzyme for metabolic engineering of plants or microorganisms to produce high
value fragrance compounds.
The labdanoid diterpene alcohol cis-abienol is a major com- tional enzyme, AbCAS is a promising target for metabolic engi-
ponent of the aromatic oleoresin of balsam fir (Abies balsamea) neering of cis-abienol production.
and serves as a valuable bioproduct material for the fragrance
industry. Using high-throughput 454 transcriptome sequencing
and metabolite profiling of balsam fir bark tissue, we identified
candidate diterpene synthase sequences for full-length cDNA
cloning and functional characterization. We discovered a
bifunctional class I/II cis-abienol synthase (AbCAS), along with
the paralogous levopimaradiene/abietadiene synthase and
isopimaradiene synthase, all of which are members of the gym-
nosperm-specific TPS-d subfamily. The AbCAS-catalyzed for-
mation of cis-abienol proceeds via cyclization and hydroxyla-
tion at carbon C-8 of a postulated carbocation intermediate in
the class II active site, followed by cleavage of the diphosphate
group and termination of the reaction sequence without further
cyclization in the class I active site. This reaction mechanism is
distinct from that of synthases of the isopimaradiene- or levopi-
maradiene/abietadiene synthase type, which employ deproto-
nation reactions in the class II active site and secondary cycliza-
tions in the class I active site, leading to tricyclic diterpenes.
Comparative homology modeling suggested the active site resi-
dues Asp-348, Leu-617, Phe-696, and Gly-723 as potentially
important for the specificity of AbCAS. As a class I/II bifunc-
Conifers produce a diverse array of diterpenoids as major
oleoresin components that play a key role in the chemical
defense against herbivores and pathogens, such as bark beetles
and their associated fungi (1–3). Oleoresin diterpenoids are
also used as large volume, renewable raw material for the pro-
duction of a suite of industrial resins and coatings and other
bioproducts (4, 5). Although tricyclic diterpene resin acids are
ubiquitously abundant in the pine family (Pinaceae), the oleo-
resin of balsam fir (Abies balsamea (L.) Mill.) contains cis-abi-
enol 4 (see Fig. 1), a bicyclic, tertiary diterpene alcohol, as the
major diterpenoid (6). Among other applications, cis-abienol 4
and other oxygen-containing diterpenoids of plant origin (e.g.
sclareol and manool) are important in the fragrance industry to
produce Ambroxꢀ, which serves as a sustainable replacement
for the use of ambergris in high end perfume formulations (7).
Whereas Ambroxꢀ is produced from plant terpenoids, amber-
gris is an animal product secreted from the intestines of sperm
whales, which are listed as an endangered species.
In addition to balsam fir, a gymnosperm tree, only a few
angiosperm plant sources, such as tobacco (Nicotiana taba-
cum; family Solanaceae) trichomes (8, 9) or the tuberous roots
of Bolivian sunroot (Polymnia sonchifolia; family Asteraceae)
(10) are known to produce cis-abienol 4 in amounts that are
relevant for industrial scale extraction. The importance of cis-
abienol 4 as a plant-derived precursor for the fragrance and
bioproducts industry has spawned an interest in the discovery
of the relevant biosynthetic genes and enzymes and their future
application in metabolically engineered microbial or plant pro-
duction systems.
* ThisresearchwassupportedbyGenomeAlberta, GenomeBritishColumbia,
Genome Quebec, and the Government of Canada through Genome Can-
adainsupport of thePhytoMetaSynProjectandbygrantsfromtheNatural
Sciences and Engineering Research Council of Canada (to J. B.).
Thenucleotidesequencesreportedinthispaperhavebeensubmittedtotheshort
read archive at NCBI with the accession number SRX039631; cDNA sequences
described in this paper have been submitted to the GenBankTM/EBI Data Bank
with accession numbers JN254805 (AbdiTPS1), JN254806 (AbdiTPS2),
JN254807 (AbdiTPS3), and JN254808 (AbdiTPS4).
□
S
This article contains supplemental Tables 1–4 and Figs. 1 and 2.
1 Present address: Dept. of Plant Biology and Biotechnology, University of
Copenhagen, Thorvaldsensvej 40, 1871 Frederiksberg C, Copenhagen,
Denmark.
Based on knowledge of diterpenoid biosynthesis in other
conifer species (11–18), we explored balsam fir as a target for a
genomics-enabled discovery strategy of cis-abienol 4 biosyn-
thesis. In general, biosynthesis of oleoresin diterpenoids in
2 Supported in part by the University of British Columbia Distinguished Uni-
versity Scholars program. To whom correspondence should be addressed.
Tel.: 604-822-0282; Fax: 604-822-2114; E-mail: bohlmann@msl.ubc.ca.
APRIL 6, 2012•VOLUME 287•NUMBER 15
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Bifunctional cis-Abienol Synthase
conifers involves the sequential cycloisomerization of (E,E,E)-
geranylgeranyl diphosphate (GGPP)3 1, catalyzed by bifunc-
tional class I/II diterpene synthases (diTPSs) of the TPS-d sub-
family (Fig. 1) (19). Oxygen functionality of conifer oleoresin
diterpenes can be introduced by diTPSs (18) and/or by separate
activity of cytochrome P450-dependent monooxygenases of
the CYP720B subfamily (20–22). Previously characterized con-
ifer diTPSs (11–18) produce either isopimaradiene 7 and minor
amounts of sandaracopimaradiene (Iso-type diTPSs) or epi-
meric forms of 13-hydroxy-8(14)-abietene 9/10 (LAS-type
diTPSs) (Fig. 1). In vitro and perhaps in vivo, 13-hydroxy-8(14)-
abietene 9/10 is readily dehydrated, resulting in a mixture of
abietadiene 11, palustradiene 12, levopimaradiene 13, and
neoabietadiene 14 (18). The reaction mechanism of the Iso-
and LAS-type diTPSs involves the initial protonation-initiated
bicyclization of GGPP 1 at the class II active site, resulting in
copalyl diphosphate (CPP) 5 of (9S,10S)-configuration (i.e. nor-
mal or (ϩ)-configuration). (9S,10S)-CP-P 5 then translocates to
the class I active site, where it undergoes a secondary ioniza-
tion-dependent cyclization and enzyme-specific rearrange-
ment of intermediate carbocations. In Iso-type diTPSs, direct
deprotonation of the sandaracopimarenyl cation 6 results in
the formation of isopimaradiene 7, whereas LAS-type diTPSs
catalyze additional rearrangement and water capture at C-13,
resulting in 13-hydroxy-8(14)-abietene 9/10 as the initial prod-
uct (18).
In contrast to the well characterized biosynthesis of tricyclic
diterpenes by conifer diTPSs of the Iso- and LAS-type, a conifer
gene or enzyme for the biosynthesis of cis-abienol 4 has not yet
been reported. A plausible reaction sequence of cis-abienol 4
formation, catalyzed by a bifunctional conifer class I/II diTPS
could proceed via water capture of a carbocation intermediate
at carbon C-8 and subsequent ionization of the allylic diphos-
phate group without further cycloisomerization (Fig. 1).
Recently, a monofunctional angiosperm diTPS catalyzing the
formation of a bicyclic oxygen-containing diterpenoid, copal-
8-ol diphosphate synthase from Cistus creticus (Cistaceae), has
been reported (23). This enzyme represents a class II diTPS,
which catalyzes the protonation-initiated cyclization of GGPP
1 to form the hydroxylated CPP compound. In addition, Mafu
et al. (24) described a bifunctional class I/II diTPS,
SmCPSKSL1, from the lycophyte Sellaginella moellendorffii for
the formation of labda-7,13-dien-15-ol, where the primary
hydroxyl group is introduced during the reaction of the class I
active site.
FIGURE 1. Schematic of the proposed biosynthesis of diterpene olefins and
alcoholsbyclassI/IIbifunctionaldiTPSsinconifers.TheknownactivityofLAS-
and Iso-type diTPSs in the formation of tricyclic diterpenes involves the stepwise
cyclization of GGPP 1 via (9S,10S)-CPP 5 (i.e. CPP of normal or (ϩ)-stereochemis-
try). Protonation-initiated cyclization of GGPP 1 to CPP 5 is catalyzed by the class
II active site of LAS- and Iso-type diTPSs. At the class I active site of Iso enzymes,
ionization-dependent secondary cyclization of CPP 5 and deprotonation of the
resulting sandaracpoimaren-8-yl cation 6 lead to the formation of isopimara-
diene 7. Alternatively, in the class I active site of LAS enzymes, additional proton
transfer and methyl migration afford the tertiary abietenyl cation 8, and further
deprotonation and hydroxylation (i.e. water capture) result in the formation of
the instable 13-hydroxy-8(14)-abietene 9/10 product of the LAS activity. Dehy-
drationofthistertiaryditerpenealcoholyieldsseveraldifferentditerpeneolefins,
includingabietadiene11, palustradiene12, levopimaradiene13, andneoabieta-
diene 14. A different reaction sequence is proposed for the formation of the
bicyclic diterpene alcohol cis-abienol 4 by the class I/II bifunctional AbCAS
We describe here the development of a 454 transcriptome
resource for balsam fir, which, in conjunction with terpenoid
metabolite profiling, was used for the identification and func-
tional characterization of a class I/II gymnosperm cis-abienol
synthase (CAS). The same sequence resource also revealed two
additional bifunctional class I/II diTPSs, representing the bal-
sam fir Iso and LAS enzymes. Comparative homology modeling
3 The abbreviations used are: GGPP, geranylgeranyl diphosphate; CAS, cis-
abienol synthase; CPP, copalyl diphosphate; diTPS, diterpene synthase; enzyme. The class II activity of AbCAS converts GGPP 1 to labda-13-en-8-ol
diphosphate 3 via water capture at the C-8 carbon of the labda-13-en-8-ylϩ cat-
LAS, levopimaradiene/abietadiene synthase; Iso, isopimaradiene syn-
ion 2. Subsequent ionization of the allylic diphosphate at the class I active site,
without additional cyclization, yields cis-abienol 4.
thase; TPS, terpene synthase; FL, full-length; RACE, rapid amplification of
cDNA ends; APCI, atmospheric pressure chemical ionization.
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Bifunctional cis-Abienol Synthase
and site-directed mutagenesis revealed unique features of cis- was conducted at the McGill University and Génome Québec
abienol 4 biosynthesis and suggest functional divergence in the Innovations Centre (Montreal, Canada). The cDNA library was
evolution of CAS relative to the paralogous Iso- and LAS-type constructed from 200 ng of fragmented mRNA using the cDNA
diTPSs associated with substitution of key residues in the active Rapid Library Preparation kit, GS FLX Titanium series (Roche
sites. A novel feature of CAS is the formation of a tertiary diter- Applied Science) following the manufacturer’s protocols. Yield
penol at the class II active site of a bifunctional class I/II diTPS. of cDNA and the size range of cDNA fragments were assessed
using a Bioanalyzer 2100 Pico Chip (Agilent), and 200 ng of the
EXPERIMENTAL PROCEDURES
cDNA library were subjected to a half-plate reaction of 454
Plant Material—Two-year-old saplings of Abies balsamea pyrosequencing using the Roche GS FLX Titanium technology.
var. phanerolepsis (L.) Mill. were purchased from Arbutus
De Novo Sequence Assembly—The 454 transcriptome
Grove Nursery Ltd. (North Saanich, Canada) and maintained in sequencing generated 797,060 raw reads. After trimming of 5Ј
a greenhouse as described previously (25). Needles, xylem/ adapter sequences and exclusion of reads smaller than 45 bp, de
wood, and phloem/bark were harvested from the upper inter- novo assembly of the bark transcriptome was performed using
whorls and used fresh or flash frozen in liquid N2 for metabolite the GS De Novo Assembler version 2.5p1 (Roche Applied Sci-
and RNA extraction, respectively.
ence). BLASTx search (28) of the resulting isotigs against a
Diterpene Standards—Authentic diterpene resin acid stand- comprehensive in-house curated sequence database of plant
ards were purchased from Orchid Celmark (New Westminster, diTPS sequences was used to identify a subset of candidate
Canada). The corresponding diterpene olefins were synthe- isotigs. Reads from these isotigs were reassembled in PHRAP
sized from the acids at Best West Laboratories Inc. (Salt Lake (29) for closer investigation.
City, UT) as described previously (20).
Isolation of Full-length cDNAs—A full-length (FL) sequence
Metabolite Analysis—Terpenoids were extracted from sam- of AbdiTPS4 was retrieved from the assembled transcriptome
ples of 2 g of pulverized tissue with 1.5 ml of diethyl ether for sequences. Completion of the 3Ј-sequences of AbdiTPS1 and -2
16 h at room temperature. Water was removed by the addition was achieved by 3Ј-RACE using the SMARTer cDNA RACE
of anhydrous Na2SO4, and extracts were passed through cDNA amplification kit (Clontech) and Phusion DNA poly-
0.22-␮m GHP membrane filters (PALL Corp., De Miniac, Can- merase (New England Biolabs, Pickering, Canada) with the fol-
ada). GC-MS analysis was performed on an Agilent 6890N GC lowing PCR program: initial denaturation at 98 °C for 30 s, five
(Agilent Technologies Inc., Mississauga, Canada), 7683B series cycles of denaturation at 98 °C for 20 s and extension at 72 °C
autosampler and a 5975 Inert XL MS Detector at 70 eV and 1 ml for 90 s, five cycles of denaturation at 98 °C for 20 s, annealing at
Ϫ1
min helium as carrier gas using a SGE Solgel-Wax column 70 °C for 20 s and extension at 72 °C for 90 s, 27 cycles of dena-
(polyethylene glycol, 30 m, 250 ␮m inner diameter, 0.25-␮m turation at 98 °C for 20 s, annealing at 65 °C for 20 s and exten-
film) with the following GC temperature program: 40 °C for 5 sion at 72 °C for 90 s, and a final extension at 72 °C for 3 min.
min, 3 °C minϪ1 to 80 °C, 8 °C minϪ1 to 250 °C, 10 °C minϪ1 to The obtained amplicons were gel-purified and ligated into pJET
270 °C, hold 5 min, pulsed splitless injector held at 250 °C. using the CloneJET kit (Clontech). Pseudomature versions of
Diterpene resin acids were extracted from samples of ϳ150 mg the FL cDNAs (starting at (K/N)REFPP) were amplified (initial
of tissue with 1.5 ml of tert-butylmethylether following the denaturation at 98 °C for 30 s followed by 30 cycles of denatur-
method described by Lewinsohn et al. (26) and derivatized with ation at 98 °C for 10 s, annealing at 65–68 °C for 30 s, and
2 M trimethylsilyl diazomethane (Sigma) prior to GC-MS anal- extension at 72 °C for 80 s and final extension at 72 °C for 10
ysis using an Alltech AT-1000 column (polyethylene glycol min), cloned into pJET, and subcloned into the NheI/SalI and
acid-modified, 30 m, 250-␮m inner diameter, 0.25-␮m film) NotI restriction sites of pET28b(ϩ) (EMD Biosciences, San
and GC specifications as follows: 150 °C initial temperature, Diego, CA). All cDNA constructs were sequence-verified at the
Ϫ1
Ϫ1
1.5 °C min to 220 °C, 20 °C min to 240 °C. Compound Nucleic Acid Protein Service Unit (University of British Colum-
identification was achieved by comparison of mass spectra with bia, Vancouver, Canada) prior to expression in Escherichia coli.
those of authentic standards and reference mass spectral data- Oligonucleotides used in PCR procedures are listed in supple-
bases of the National Institute of Standards and Technology MS mental Table 1.
library searches (Wiley W9N08). Quantifications were based
on three independent biological replicates.
Functional Characterization of Diterpene Synthases—Re-
combinant proteins were expressed in E. coli BL21DE3-C41,
RNA Isolation and cDNA Synthesis—Total RNA was isolated Ni2ϩ affinity-purified as described elsewhere (16), and desalted
from samples of 150 mg of bark tissue according to the method into 20 mM HEPES (pH 7.2), 150 mM NaCl, 10% glycerol, 5 mM
of Kolosova et al. (27), and mRNA was purified on Dynabeads DTT using PD MiniTrap G-25 columns (GE Healthcare).
(Invitrogen). RNA integrity and amounts were determined on a Enzyme assays were carried out as described before (16) in 50
Bioanalyzer 2100 using an RNA Pico Chip (Agilent). Synthesis mM HEPES (pH 7.2), 10 ␮M MgCl2, 5% glycerol, 5 mM DTT,
of cDNA as a template for PCR amplification of candidate genes using 100 ␮g of purified protein (100 ␮g each for coupled
was carried out with random hexamer oligonucleotides using assays) and 15 ␮M (E,E,E)-GGPP 1 (Sigma) with incubation for
the SMARTer cDNA rapid amplification of cDNA ends 1 h at 30 °C. After extraction of reaction products with 500 ␮l of
(RACE) amplification kit (Clontech, Mountain View, CA).
pentane, GC-MS analysis was conducted on an Agilent 7890A
cDNA Library Construction and 454 Transcriptome GC, 7683B series autosampler and a 7000A Triple Quad MS
Ϫ1
Sequencing—Construction of a non-normalized bark tissue Detector at 70 eV and 1.2 ml min helium flow using an
cDNA library and subsequent 454 transcriptome sequencing HP5ms column (5% phenyl methyl siloxane, 30 m, 250 ␮m
APRIL 6, 2012•VOLUME 287•NUMBER 15
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Bifunctional cis-Abienol Synthase
inner diameter, 0.25-␮m film) and the following GC tempera- tively, thus allowing for a structural comparison of the active
Ϫ1
ture program: 40 °C for 2 min, 20 °C min to 300 °C, hold 2 sites. Using Molegro Virtual Docker 2010.4.0.0 (36), a semiau-
min, pulsed splitless injection (injector held at 250 °C). For the tomated docking approach was applied to place labda-13-en-
detection of diphosphate intermediates, reaction products 8-ol diphosphate 3 in the class I and class II active site of AbCAS
were dephosphorylated prior to extraction by incubation with (Fig. 6). For this purpose, active site cavities were predicted
10 units of calf intestinal alkaline phosphatase (Invitrogen) for using a probe radius of 1.0 Å and a grid resolution of 0.6 Å with
16 h at 37 °C and analyzed on a Solgel-Wax column as described further manual optimization. Substrate docking was then
above (see “Metabolite Analysis”) with modified GC parame- restricted to the predicted cavities, inclusive of the required
ters: 40 °C for 2 min, 25 °C minϪ1 to 250 °C, hold 5 min, pulsed Mg2ϩ complex. Energy-minimized Protein Data Bank coordi-
splitless injector held at 250 °C. Analysis of reaction products nates of the ligands were generated using the PRODRG server
via LC-MS was performed on an Agilent 1100 series LC/MSD (37).
Trap XCT Plus MS with atmospheric pressure chemical ioni-
RESULTS
zation (APCI) in positive mode on an Agilent Zorbax RX-Sil
silica column (4.6 mm inner diameter ϫ 150 mm ϫ 5 ␮m) as
Terpenoid Metabolite Profiling Confirms cis-Abienol as
reported previously (18).
Major Diterpene in Bark Tissue of Balsam Fir—To identify a
Nuclear Magnetic Resonance (NMR) Analysis—cis-Abienol 4 suitable tissue source for transcriptome mining of genes of cis-
was prepared from a pool of 10 individual enzyme assays as abienol 4 biosynthesis, we established diterpenoid metabolite
described above using 20 ␮M GGPP and an incubation time of profiles for wood and xylem, bark and phloem, and needles of
2 h to maximize product formation. To enhance product purity, balsam fir sapling trees (supplemental Table 2). As supporting
pentane was purified on alumina prior to use, and assays were information, we also measured mono- and sesquiterpenoids.
performed in buffer pre-extracted with alumina-purified pen- Although only trace amounts of these terpenoids were detected
tane. Nuclear magnetic resonance (NMR) spectra were in wood tissue (data not shown), bark/phloem and needle sam-
recorded on a Bruker Avance 600 spectrometer equipped with ples had similar total amounts of terpenoids, consisting mostly
a QNP or TCI cryoprobe (600 MHz) using deuterochloroform of diterpenoids, with lesser amounts of monoterpenoids and
(CDCl3) as the solvent, which was neutralized by distillation relatively minor amounts of sesquiterpenoids (supplemental
and filtration through potassium carbonate prior to prepara- Table 2). Both the bark/phloem and the needle samples con-
tion of the sample. Signal positions (␦) were calculated in parts tained the diterpene resin acids, abietic acid, isopimaric acid,
per million (ppm) as compared with tetramethylsilane (␦ 0) and dehydroabietic acid, and palustric acid, as major diterpenoids.
were measured relative to the signal of the solvent (CDCl3: ␦ These are all commonly abundant in conifers. cis-Abienol 4 was
7.26, 1H NMR; ␦ 77.0, 13C NMR).
found almost exclusively in the bark/phloem tissue, where it
Generation of Site-directed Protein Variants of AbdiTPS4— was the most abundant terpenoid metabolite, accounting for
Alanine substitutions of Asp-402, Asp-404, or Asp-621 of more than 25% of the total diterpenoid content of balsam fir
AbdiTPS4 were generated following the QuikChange site-di- stem tissue (supplemental Table 2).
rected mutagenesis protocol (Stratagene, Mississauga, Canada)
Discovery of diTPS Genes via Transcriptome Sequencing and
with the cDNA in pET28b(ϩ) as template and the following de Novo Sequence Assembly—Based on the high abundance of
PCR program: initial denaturation at 98 °C for 90 s followed by cis-abienol 4 in bark/phloem of balsam fir sapling stems, we
29 cycles of denaturation at 98 °C for 50 s, annealing at 60 °C for used this tissue source for the preparation of a non-normalized
30 s and extension at 72 °C for 4 min, and final extension at cDNA library and subsequent transcriptome sequencing. A
72 °C for 10 min. Oligonucleotides used for mutagenesis are half-plate reaction of Roche 454 sequencing generated a total of
listed in supplemental Table 1.
797,060 sequence reads with an average GC content of 46% and
Phylogenetic Analysis—Multiple protein sequence align- an average read length of 359 bp (supplemental Table 3). After
ments were performed using the CLC bio Main Workbench adapter trimming, the remaining high quality reads were sub-
5.7.1 (CLC bio, Århus, Denmark). Phylogenetic analyses were jected to a de novo assembly in GS De Novo Assembler 2.5p1
conducted on the basis of the maximum likelihood algorithm with a size exclusion of 45 bp. The assembly contained 85% of
using PhyML 3.0 (30) with four rate substitution categories, LG all input reads, yielding 14,699 isogroups from 17,122 isotigs
substitution model, BIONJ starting tree and 100 bootstrap rep- with an average size of 1,114 nucleotides. For the discovery of
etitions, and displayed as phylogram using treeview32 1.6.6.
TPS genes, these isotigs were searched against an in-house
Computational Structure Analysis—Homology models of curated database of 146 known plant TPSs, resulting in a subset
AbLAS, AbIso, and AbCAS were built using the CPHmodels 3.0 of isotigs that represented candidate genes for nine different
server (31) based on the tertiary structure of TbTXS (32) (PDB putative mono- and sesqui-TPSs and five putative diTPSs (sup-
code 3p5pA, chain A) and certified as high quality exceeding plemental Table 4). Of the diTPSs, four candidate isotigs
91% residues assigned to most favored regions in Ramachan- (AbdiTPS1, AbdiTPS2, AbdiTPS3, and AbdiTPS4) resembled
dran plot statistics using PROCHECK (33). Lack of structural bifunctional class I/II gymnosperm diTPSs of the TPS-d group
errors in the models was validated using the ProSA-web server (14, 17, 19) containing the characteristic DXDD, DDXXD, and
(34). Pairwise comparison of these modeled structures with the NSE/DTE motifs (38–40). To optimize and validate the
DaliLite server (35) demonstrated a high structural similarity of sequence assemblies, reads corresponding to these four isotigs
AbLAS, AbIso, AbCAS, and TbTXS with root mean square were reassembled in PHRAP (29), from which three unique
deviations of Յ1 Å for the ␤␥ domain and the ␣ domain, respec- partial diTPS cDNA sequences (AbdiTPS1, AbdiTPS2, and
12124 JOURNAL OF BIOLOGICAL CHEMISTRY
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Bifunctional cis-Abienol Synthase
AbdiTPS3) and one FL cDNA sequence (AbdiTPS4) were
obtained. The encoded proteins of AbdiTPS1, AbdiTPS2, and
AbdiTPS3 showed highest similarity with grand fir (Abies gran-
dis) abietadiene synthase (AgAS) (11, 12), with more than 90%
protein sequence identity. In contrast, the deduced protein
sequence AbdiTPS4 showed highest similarity with Norway
spruce (Picea abies) isopimaradiene synthase (PaIso) (14),
although at a substantially lower level of sequence identity of
only 75%, highlighting AbdiTPS4 as a unique candidate diTPS
sequence (supplemental Fig. 1).
Functional Characterization of Balsam Fir diTPSs and Dis-
covery of Novel cis-Abienol Synthase—The FL cDNA clone for
AbdiTPS4 was amplified based on the in silico assembled FL
sequence. FL or nearly FL cDNAs for AbdiTPS1 and AbdiTPS2,
respectively, were recovered by 3Ј-RACE, with AbdiTPS2 lack-
ing ϳ40–50 bp of the plastidial transit peptide. Despite using a
number of different primer combinations and PCR conditions,
we could not amplify a cDNA clone corresponding to the
AbdiTPS3 sequence assembly. For functional characterization
of AbdiTPS1, AbdiTPS2, and AbdiTPS4, we cloned pseudoma-
ture versions of these diTPSs starting at the conserved
(N/K)RX6W motif and lacking the putative plastidial transit
peptide (16). Recombinant proteins were expressed in E. coli
and Ni2ϩ affinity-purified, resulting in soluble proteins of the
expected molecular mass of 90–91 kDa (supplemental Fig. 2).
Enzyme assays with GGPP 1 as substrate followed by GC-MS or
LC-MS analysis of reaction products against proper controls
and, where available, authentic standards identified functions
for AbdiTPS1, AbdiTPS2, and AbdiTPS4 as described below.
The product profile of AbdiTPS1 was identified by GC-MS to
consist of four peaks corresponding to abietadiene 11, palustra-
diene 12, levopimaradiene 13, and neoabietadiene 14 in a ratio
of ϳ4:3:2:1 (Fig. 2). This profile closely matches that previously
identified for grand fir AgAS (12), a diTPS that is 99% identical
with AbdiTPS1 on the amino acid level. According to these
patterns, AbdiTPS1 can be classified as an LAS-type bifunc-
tional class I/II diTPS, which appears to be orthologous with
AgAS. Following the recent discovery of epimers of a tertiary
alcohol, 13-hydroxy-8(14)-abietene 9/10, as the initial enzyme
products of Norway spruce PaLAS (18), we also investigated
the product of AbdiTPS1 by LC-MS, which confirmed the
formation of 13-hydroxy-8(14)-abietene 9/10 (Fig. 3). The
detected mass fragment of m/z 273 in the LC-MS analysis (Fig.
3) corresponds to dehydration product of 13-hydroxy-8(14)-
abietene 9/10 as detailed by Keeling et al. (18).
FIGURE 2. Activity of balsam fir diTPSs AbdiTPS1 (AbLAS), AbdiTPS2
(AbIso), and AbdiTPS4 (AbCAS). A, total ion chromatograms (TIC) of reaction
products from in vitro assays with purified recombinant enzymes using GGPP
1 as a substrate. GC-MS analysis was performed on an Agilent HP5ms column
with electronic ionization at 70 eV. Enzymatic activity assays were confirmed
with three independent experiments. Peak IS, internal standard 1.6 ␮M 1-ei-
cosene; peak a, palustradiene 12; peak b, levopimaradiene 13; peak c, abieta-
diene 11; peak d, neoabietadiene 14; peaks e and f, epimers of 13-hydroxy-
8(14)-abietene 9/10; peak g, isopimaradiene 7; peak h, cis-abienol 4; the
authentic standard of abietadiene 11 contained an unknown contamination;
the authentic standard of levopimaradiene 13 contains other diterpene iso-
mers, which could not be removed. B, mass spectrum of cis-abienol 4, pro-
duced by recombinant AbdiTPS4 with GGPP 1 as substrate and comparison
with the reference mass spectrum of cis-abienol 4 as described by Vlad et al.
(41) and obtained from the National Institute of Standards and Technology
MS library searches (Wiley W9N08). Authentic cis-abienol 4 standard is not
commercially available.
AbdiTPS2 formed exclusively isopimaradiene 7 (Fig. 2), sim-
ilar to the previously characterized isopimaradiene synthases
from Norway spruce (PaIso) and Sitka spruce (PsIso) (14, 16,
17). AbdiTPS2 was thereby identified as an Iso-type bifunc-
tional class I/II diTPS.
AbdiTPS4 showed a unique single-peak product profile (Figs.
2 and 3), which corresponded to cis-abienol 4 according to
retention time consistent with the compound extracted from
plant tissue and shared characteristic mass fragments (e.g. m/z
ϩ
ϩ
290 [M ], m/z 272 [M -H2O], and m/z 134) in comparison olution of the shape of the cis-abienol 4 peak (Fig. 2), relative to
with reference mass spectra (41).
the peak shape of diterpene olefins, and the occurrence of trace
When the product of AbdiTPS4 was analyzed by GC-MS, amounts of two additional compounds. This profile is probably
under a number of different conditions, we observed poor res- due to degradation of cis-abienol 4 during GC-MS as reported
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Bifunctional cis-Abienol Synthase
FIGURE3.LC-MSanalysisofditerpenolproductsofAbdiTPS1(AbLAS)and
AbdiTPS4 (AbCAS). Assay products were analyzed by normal phase
LC-APCI-MS in positive mode and are shown as extracted ion chromatograms
(EIC) of the base peak m/z 273, which is indicative of dehydration of the unsta-
ble diterpene alcohol compounds in the APCI interface. Comparison with
abietadiene 11 standard showed separation of olefin compounds, such as
abietadiene 11 (peak c) and the more polar hydroxylated 13-hydroxy-8(14)-
abietene epimers 9/10 (peaks e and f) and cis-abienol 4 (peak h).
previously (42–44). However, LC-MS analysis confirmed cis-
abienol 4 as a single product of AbdiTPS4, with m/z 273 in the
LC-MS analysis corresponding to the predicted dehydration
product of the diterpene alcohol (Fig. 3). Additional structural
and stereochemical analysis by proton and carbon NMR and
comparison with previously reported analyses (45, 46) con-
firmed the identity of the AbdiTPS4 product as cis-abienol 4.
Based on functional characterization, AbdiTPS1, AbdiTPS2,
and AbdiTPS4 will be referred to as AbLAS, AbIso, and AbCAS,
respectively. AbCAS represents a new type of conifer diTPS,
producing a bicyclic, tertiary diterpene alcohol, as opposed to
tricyclic products of the LAS- and Iso-type diTPSs. With the
present study, LAS- or Iso-type diTPSs have now been identi-
fied in three different genera of the pine family, namely in true
firs (Abies), spruce (Picea), and pine (Pinus), which allows for
analysis of gene orthology within this family (Fig. 4). It appears
that gene duplications and neofunctionalization leading to
paralogous pairs of LAS and Iso genes occurred independently
in Abies and Picea, after the separation of these genera. Within
the spruce genus, the dichotomy of LAS and Iso genes happened
FIGURE 4. Phylogeny of balsam fir diTPSs of the LAS-, Iso-, and CAS-type
inthecontextoftheTPS-d3subfamilyofconiferdiTPSs. Thephylogenetic
tree was generated by sequence alignment in CLC bio, phylogenetic analysis
with PhyML (four rate substitution categories, LG substitution model, BIONJ
startingtree, 100bootstraprepetitions), andvisualizationintreeviewbyroot-
ing with the outgroup copalyl diphosphate synthase/kaurene synthase from
the moss P. patens (PpCPS/KS). TbrTS, Taxus brevifolia taxadiene synthase
(NCBI accession number AAC49310); TcuTS, Taxus cuspidata taxadiene syn-
thase (NCBI accession number ABW82997); TxmTS, Taxus x media taxadiene
synthase (NCBI accession number AAS18603); PaIso, P. abies isopimaradiene
synthase (NCBI accession number AAS47690); PsIso, Picea sitchensis isopima-
radiene synthase (NCBI accession number ADZ45512); PaLAS, P. abies (NCBI
accession number AAS47691); PsLAS, P. sitchensis levopimaradiene/abieta-
diene synthase (NCBI accession number ADZ45517); PtLAS, P. taeda levopi-
maradiene synthase (NCBI accession number AY779541); AgAS, A. grandis
abietadiene synthase (NCBI accession number AAK83563); GbLS, G. biloba
levopimaradiene synthase (NCBI accession number AAL09965); PpCPS/KS,
P. patens ent-copalyl diphosphate/ent-kaurene synthase (NCBI accession
number BAF61135). Asterisks indicate nodes supported by Ͼ90% bootstrap
values.
apparently prior to the speciation of Norway spruce and Sitka principle, hydroxylation can occur during reactions at the class
spruce. II active site or at the class I active site. To delineate which of the
The bifunctional class I/II AbCAS described here has only two active sites of bifunctional AbCAS catalyzed the formation
been cloned from balsam fir, matching the major diterpenoid of the tertiary alcohol, we generated a set of alanine substitu-
produced by this species (supplemental Table 2). Within the tions of the DXDD (class II active site) and DDXXD (class I
group of conifer class I/II diTPSs, the AbCAS gene is separate active site) motifs to obtain monofunctional AbCAS variants
from and appears basal to a clade of LAS and Iso genes from firs, that contain either a non-functional class II (AbCAS:D402A/
spruces, and pine (Fig. 4).
D404A) or a non-functional class I (AbCAS:D621A) active site.
AbCAS:D621A converted GGPP 1 (peak i in Fig. 5) into trace
Site-directed Mutagenesis of DXDD (Class II Active Site) and
DDXXD Motifs (Class I Active Site) of AbCAS Demonstrate amounts of CPP 5 (peak j) and a product with a significantly
That Formation of the Tertiary Alcohol and Cyclization Occur longer retention time, indicative of a hydroxylated diphosphate
during the Class II Reaction—A plausible mechanism for the (peak k). This compound was identified, upon cleavage of the
formation of cis-abienol 4 requires hydroxylation via water diphosphate group, as labda-13-en-8,15-diol based on charac-
ϩ
quenching of a labda-13-en-8-yl carbocation 2 at C-8 (Fig. 1). In teristic mass fragments (m/z 290 [M ], m/z 275, m/z 257, m/z
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Bifunctional cis-Abienol Synthase
phorylated labda-13-en-8-ol diphosphate 3 under GC-MS con-
ditions because they were not seen in the wild-type enzyme
product profile and did not occur under different GC condi-
tions. Results from enzyme assays with AbCAS:D621A sug-
gested that the class II active site of AbCAS catalyzes protona-
tion-initiated formation of bicyclic labda-13-en-8-ol
diphosphate 3 via water quenching of the parental carbocation.
Alanine substitution of Asp-402 and Asp-404 (AbCAS:
D402A/D404A) in the class II active site resulted in complete
loss of enzymatic activity with GGPP 1 as substrate. Activity of
AbCAS:D402A/D404A could not be restored by combining this
mutant with a protein variant of PaLAS (PaLAS:D611A), which
contains an inactive class I active site and accumulates (9S,10S)-
CPP 5 (peak j in Fig. 5). This result suggested that, unlike the
LAS and Iso-type enzymes, the class I active site of AbCAS is
not active with (9S,10S)-CPP 5 as an intermediate substrate.
Formation of cis-abienol 4 (peak h in Fig. 5) from GGPP 1 was
restored in coupled assays of the two monofunctional AbCAS
class I and class II protein variants, AbCAS:D402A/D404A and
AbCAS:D621A, confirming that the non-mutated active sites
remained functional in the two mutated proteins.
Together, the mutational analysis of class I and class II active
sites of AbCAS suggested a reaction sequence of the bifunc-
tional class I/II enzyme in which both the bicyclization and
water capture occur in the class II active site, resulting in an
intermediary labda-13-en-8-ol diphosphate 3, which under-
goes cleavage of the diphosphate group and final deprotonation
at the class I active site (Fig. 1).
DISCUSSION
Variations on a common theme of diTPS-catalyzed
cycloisomerization of GGPP 1 contribute substantially to the
chemical diversity of diterpene metabolites found in the oleo-
resin of conifers and in nature in general. Introduction of
hydroxy functions further increases the diversity of naturally
occurring diTPS products. Hydroxylation of conifer diterpenes
can result from activity of P450s acting on products of diTPSs
(20, 22) or can result from capture of water by carbocation
intermediates during the diTPS reaction (18). For the fragrance
industry, bicyclic hydroxylated diterpenes, such as cis-abienol 4
and sclareol, are of particular value as plant-derived precursors
for the sustainable production of Ambroxꢀ, which replaces the
controversial use of animal-derived ambergris in perfume
formulations.
FIGURE 5. Characterization of AbCAS protein variants. Activity analysis of
protein variants of AbCAS was conducted with 20 ␮M GGPP 1 as substrate and
dephosphorylation of the reaction products prior to GC-MS analysis (AbCAS:
D402A/D404A)orintheformofcoupledassays, combiningtheAbCAS:D621A
variant with a point mutant of PaLAS (PaLAS:D611A), containing a non-func-
tional class I active site (C. I. Keeling and J. Bohlmann, unpublished results),
and direct GC-MS analysis of the reaction products. A, total ion chromato-
grams (TIC) of reactions products analyzed on an Agilent Solgel-Wax column
with electronic ionization at 70 eV. Peak IS, internal standard 1.6 ␮M 1-ei-
cosene; peak PC, plasticizer contamination; peak h, cis-abienol 4; peak i, gera-
nylgeraniol; peak j, (9S,10S)-copalol; peak l, epi-manoyl oxide; peak m, manoyl
oxide; peak k, labd-13-en-8,15-diol. B, characteristic mass spectra of (9S,10S)-
copalol (top) and labd-13-en-8,15-diol (bottom), produced by PaLAS:D611A
and AbCAS:D621A, respectively.
Surprisingly, relatively little is known about diTPSs catalyz-
ing cyclohydration reactions. Reported examples are the bio-
synthesis of copal-8-ol diphosphate by a monofunctional class
II diTPS from Cistus creticus (23), the formation of ent-16␣-
hydroxy-kaurene as a product of the bifunctional class I/II ent-
copalyl diphosphate/ent-kaurene synthase from the non-vas-
cular plants Physcomitrella patens and Jungermannia subulata
(47, 48), and the biosynthesis of labda-7,13-dien-15-ol cata-
lyzed by an ent-copalyl diphosphate/ent-kaurene synthase-like
diTPS from the lycophyte Sellaginella moellendorffii (24). In
192, and m/z 177) as compared with the mass spectrum of the addition, a recent study on the product specificity of Norway
authentic compound (23). epi-Manoyl oxide and manoyl oxide spruce PaLAS (18) demonstrated a tricyclic, tertiary diterpene
(peaks l and m in Fig. 5) were apparently not products of AbCAS alcohol as the initial, but highly unstable, product of this diTPS.
but rather the result from ether formation of the dephos- In the case of PaLAS, the allylic diterpenol product dehydrates
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Bifunctional cis-Abienol Synthase
to a set of diterpene olefins. Against this background of prior tion. A bifunctional cis-abienol synthase has not been previ-
knowledge, the identification of bifunctional class I/II AbCAS ously reported for any plant species. Together, the set of three
adds a unique new catalyst to the known portfolio of diTPSs, types of bifunctional diTPSs, AbIso, AbLAS, and AbCAS,
with possible applications in the bioproducts industry. The account for the majority of diterpene structures found in the
genomics-based discovery of AbCAS, alongside AbLAS and specialized diterpene metabolism of balsam fir bark tissue.
AbIso, also substantially contributes to our understanding of
the molecular, biochemical, and evolutionary underpinnings of stereochemistry of the product of AbdiTPS4 was assigned
conifer diterpenoid diversity. based on comparison of retention times, under several different
In the absence of pure cis- or trans-abienol standards,
The present study highlights the powers of combined metab- GC and LC conditions, with those of the cis-abienol metabolite
olite profiling, tissue-specific deep transcriptome sequencing, in the resin mixture of balsam fir. Previous studies demon-
and functional (i.e. biochemical) genomics for the successful strated that abienol from balsam fir exhibits the cis-configura-
discovery and characterization of new enzymes of natural prod- tion (51). Our proton and carbon NMR analysis further corrob-
uct biosynthesis. The general framework for such an approach orated the stereochemistry of the reaction product of AbdiTPS4 as
has also recently been applied for the discovery of a suite of new cis-abienol.
TPS genes in, for example, tomato (49) and Euphorbia fischeri-
Despite extensive efforts of TPS gene discovery in species of
ana (50). Of fundamental importance for the success of a spruce (14, 17) and in grand fir (52), there is no known gene in
genomics- or transcriptomics-based approach to natural prod- any plant species that is closely related (Ͼ70% protein identity)
uct enzyme discovery is the traditional or new knowledge of the to AbCAS. It is therefore possible that a CAS-type bifunctional
occurrence of specialized metabolites associated with particu- class I/II diTPS is unique to balsam fir, which is the first conifer
lar plant species or particular tissues. Balsam fir was ideally species for which three types of functionally distinct diTPSs are
suited for the discovery of CAS because this species accumu- now known. A phylogenetic position of AbCAS between
lates large amounts of cis-abienol 4 in the oleoresin of bark/ Gingko biloba GbLS and other known conifer diTPSs of special-
phloem tissue (supplemental Table 2). Xylem/wood or needles ized metabolism in spruce, firs, and pine (Fig. 4) may suggest an
of balsam fir do not contain cis-abienol 4 in relevant amounts evolutionary origin of AbCAS by gene duplication prior to spe-
and therefore were not included in the transcriptome sequenc- ciation within the pine family with its possible loss in some or all
ing for CAS discovery.
of the other species of this comparison.
Beyond the selection of species and tissues via metabolite
Bifunctional class I/II diTPSs of conifer specialized metabo-
profiling prior to 454 sequencing, another key element in the lism are members of the TPS-d group (14, 19). Within this
successful discovery strategy of AbCAS was the application group, new genes and enzyme functions appear to have evolved
of a directed BLASTx search of the de novo transcriptome from a common ancestor by repeated gene duplication. Neo- or
assembly. Specifically, we directed the BLASTx search of the sub-functionalization of members of this gene family involved
de novo transcriptome assembly against a comprehensive mutation of specific active site residues (16, 53–55). The muta-
sequence database of characterized TPSs. This strategy tional analysis of AbCAS and comparative structural analysis of
allowed for the curator-optimized annotation of TPSs genes, AbCAS with AbIso and AbLAS identified distinct catalytic
which substantially streamlined the efforts of functional mechanisms and revealed unique residues in the AbCAS class I
gene characterization toward four candidate AbdiTPSs. Of and class II active sites that may determine product specificity.
these four candidates, the corresponding enzymes of The initial bicyclization of GGPP 1, resulting in a proposed
ϩ
AbdiTPS1 (AbLAS), AbdiTPS2 (AbIso), and AbdiTPS4 labda-13-en-8-yl intermediate 2 of (9S,10S) stereochemistry,
(AbCAS) were successfully characterized.
is probably a common feature of the class II reactions of CAS-,
Based on the relatively high protein sequence identity of LAS-, and Iso-type diTPSs (Fig. 1). Previous work showed that
more than 90% with AgAS, a similar catalytic function was pre- naturally occurring cis-abienol 4 is optically pure and repre-
dicted for AbdiTPS1 and AbdiTPS2, whereas the lower sents the (9S,10S) configuration (51). In a unique sequence of
sequence identity of AbdiTPS4 (75%) was indicative of a dis- the class II reaction, AbCAS may then promote water capture at
tinct function. This general prediction was confirmed by func- the C-8 carbon of the carbocation to form labda-13-en-8-ol
tional characterization, which remains a necessary part of TPS diphosphate 3, whereas the LAS- and Iso-type diTPSs form
gene annotation. The functional characterization of AbdiTPS1 (9S,10S)-CPP 5 through deprotonation at the C-8 position (Fig.
(AbLAS) with 13-hydroxy-8(14)-abietene 9/10 as the initial 1). Structural modeling and molecular docking of labda-13-en-
reaction product and multiple olefin compounds occurring 8-ol diphosphate 3 in the class I and class II active sites of
upon dehydration of the alcohol was important to substantiate AbCAS (Fig. 6) revealed only one unique amino acid in the class
a recent discovery of the formation of a tricyclic tertiary C-13 II active site, namely Asp-348, in proximity of the docked inter-
alcohol as the primary product of Norway spruce PaLAS (18). mediate that may control this particular hydroxylation reac-
Likewise, the discovery of AbdiTPS2 (AbIso) was important tion. Located at the posterior of the active site opposite of the
because it was the first Iso-type diTPS to be characterized out- DIDD motif and Trp-358, which have previously been reported
side of the spruce genus (Picea), with phylogenetic patterns to contribute to the AgAS-catalyzed class II reaction (39), the
suggesting that functional divergence of LAS- and Iso-type negatively charged side chain of Asp-348 may stabilize the pos-
diTPS occurred independently in the spruce and fir (Abies) lin- itive charge at C-8 for water quenching to occur in the forma-
eages (Fig. 4). In agreement with predictions based on sequence tion of labda-13-en-8-ol diphosphate 3 (Fig. 6B). This local neg-
divergence, AbdiTPS4 (AbCAS) represents a novel diTPS func- ative charge is not present in any LAS- or Iso-type enzymes,
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Bifunctional cis-Abienol Synthase
FIGURE 6. Unique active site residues of AbCAS relative to AbLAS and AbIso and implication for the formation of cis-abienol. Structural models were
built based on the tertiary structure of T. brevifolia taxadiene synthase (32) (Protein Data Bank code 3p5p, chain A). A, superimposed homology models of
AbCAS, AbLAS, and AbIso resembling the common ␣-helical folding pattern, comprised of the N-terminal ␤ domain (cyan) and ␥ domain (orange) and
C-terminal ␣ domain (green). The position of labda-13-en-8-ol diphosphate 3 (yellow) was estimated in the active sites by molecular docking using Molegro
Virtual Docker 2010.4.0.0 (36), with the diphosphate group bound to the Mg2ϩ cluster (pink), which in return is coordinated by the DDXXD and NDXXTXXXE
motifs (red). B, superimposition of putative catalytic residues in the class II active site of AbLAS (blue), AbIso (green), and AbCAS (light brown). Asp-348 is located
at the posterior of the class II active site, opposite the DIDD motif, with its side chain protruding toward C-8 of labda-13-en-8-ol diphosphate 3. C, Leu-617 and
Phe-696 are located in the class I active site cavity upstream of the DDLYD motif, creating an expansion of the hydrophobic pocket relative to AbLAS and AbIso.
D, Gly-723 and Val-724 contribute to a change in the hinge region between helix G1 and G2, which has previously been shown to be critical for the plasticity
of conifer diTPSs (16, 51, 54).
which contain a conserved histidine in this position (supple- CPP 5, which is the class I active site substrate of LAS-, and
mental Fig. 1).
Iso-type diTPSs (Fig. 5). Unlike LAS- and Iso-enzymes, the class
Mutational analysis and complementation assays suggest I active site of AbCAS catalyzes the ionization of the diphos-
that the class I active site of AbCAS is selective for labda-13-en- phate group without cyclization of a C-ring (Fig. 1). Several
8-ol diphosphate 3 as a substrate but does not convert (9S,10S)- unique residues were found in a radius of 7 Å around the
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Bifunctional cis-Abienol Synthase
Phytochemistry 67, 2415–2423
hydroxy group of labda-13-en-8-ol diphosphate 3 docked
within the class I active site of AbCAS that may control the
AbCAS reaction (Fig. 6, C and D). Among these, Leu-617, Phe-
696, and Gly-723 are of particular interest because they contrib-
ute to the contour of the active site cavity. Leu-617 and Phe-696
are located in the class I active site cavity upstream of the
DDXXD motif on helix D, creating an expansion of the hydro-
phobic pocket relative to AbLAS and AbIso. Gly-723 and Val-
724 account for a change in the hinge region between helix G1
and G2. These residues may contribute to the release of a bicy-
clic product rather than facilitating a secondary cyclization.
Interestingly, residues corresponding to Leu-617, Phe-696, and
Gly-723 have previously been shown to be critical for the cata-
lytic plasticity of conifer diTPSs (16, 53–56) and thus appear to
represent key positions for the functional evolution of these
enzymes.
3. Keeling, C. I., and Bohlmann, J. (2006) Genes, enzymes, and chemicals of
terpenoid diversity in the constitutive and induced defence of conifers
against insects and pathogens. New Phytol. 170, 657–675
4. Bohlmann, J., and Keeling, C. I. (2008) Terpenoid biomaterials. Plant J. 54,
656–669
5. Hillwig, M. L., Mann, F. M., and Peters, R. J. (2011) Diterpenoid biopoly-
mers. New directions for renewable materials engineering. Biopolymers
95, 71–76
6. Gray, P. S., and Mills, J. S. (1964) The isolation of abienol from Canada
Balsam, the oleoresin of Abies balsamea (L.) J. Chem. Soc. 1964,
5822–5825
7. Barrero, A. F., Alvarez-Manzaneda, E. J., Altarejos, J., Salido, S., and
Ramos, J. M. (1993) Synthesis of Ambroxꢀ from (Ϫ)-sclareol and (ϩ)-cis-
abienol. Tetrahedron 49, 10405–10412
8. Guo, Z., Severson R. F., and Wagner, G. J. (1994) Biosynthesis of the
diterpene cis-abienol in cell-free extracts of tobacco trichomes. Arch.
Biochem. Biophys. 308, 103–108
9. Guo, Z., and Wagner, G. J. (1995) Biosynthesis of labdenediol and sclareol
in cell-free extracts from trichomes of Nicotiana glutinosa. Planta 197,
627–632
Plant TPSs are useful enzymes for the metabolic engineering
of bioproducts and biofuels in yeast and E. coli (4, 57). Meta-
bolic engineering of microbial hosts or plants to produce oxy-
genated diterpenoids, such as cis-abienol 4 or sclareol, may pro-
vide a sustainable production platform for these compounds for
the fragrance industry and other applications. The patent liter-
ature (58, 59) suggests that genes for cis-abienol 4 and sclareol
biosynthesis have been cloned from angiosperms. However,
unlike the bifunctional conifer class I/II diTPSs, the known
angiosperm diTPSs are exclusively monofunctional enzymes
(19). Thus, using the angiosperm enzymes for pathway engi-
neering would require the dual expression optimization of sep-
arate class I and class II enzymes. In contrast, the use of a
bifunctional class I/II diTPS, such as AbCAS, would require
only the expression of a single gene. Optimization of protein
expression is also likely to be less complicated for the bifunc-
tional class I/II diTPS (i.e. AbCAS), because the two active sites
required for the conversion of GGPP 1 to cis-abienol 4 will be in
equal amounts as part of the same protein and will be in close
physical proximity of each other. In essence, for applications of
metabolic engineering, the bifunctional class I/II AbCAS offers
a scaffolded arrangement of the two essential active sites. This
scaffold evolved in nature for the high volume production of
cis-abienol 4 in the oleoresin of balsam fir. Because AbCAS is
only very distantly related to angiosperm diTPSs (less than 30%
protein sequence identity), this enzyme would also be a pre-
ferred choice for metabolic engineering of cis-abienol 4 pro-
duction in industrial crops, such as tobacco, because co-sup-
pression effects on endogenous diTPSs are unlikely.
10. Miyazawa, M., and Tamura, N. (2008) J. Essent. Oil Res. 20, 12–14
11. Vogel, B. S., Wildung, M. R., Vogel, G., and Croteau, R. (1996) Abietadiene
synthase from grand fir (Abies grandis). cDNA isolation, characterization,
and bacterial expression of a bifunctional diterpene cyclase involved in
resin acid biosynthesis. J. Biol. Chem. 271, 23262–23268
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Acknowledgments—We thank Karen Reid for excellent laboratory
and project management support, Lina Madilao for support with
GC-MS and LC-MS analysis, and Christopher I. Keeling for helpful
discussions. Transcriptome sequencing was performed at the McGill
University/Genome Quebec Innovation Centre with database
assistance for handling of sequence raw data at the University of
Calgary.
20. Ro, D. K., Arimura, G., Lau, S. Y., Piers, E., and Bohlmann, J. (2005) Lob-
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