Probing Labdane-Related Diterpenoid Biosynthesis in the Fungal Genus Aspergillus

Article abstract of DOI:10.1021/acs.jnatprod.6b00764

While terpenoid production is generally associated with plants, a variety of fungi contain operons predicted to lead to such biosynthesis. Notably, fungi contain a number of cyclases characteristic of labdane-related diterpenoid metabolism, which have not been much explored. These also are often found near cytochrome P450 (CYP) mono-oxygenases that presumably further decorate the ensuing diterpene, suggesting that these fungi might produce more elaborate diterpenoids. To probe the functional diversity of such biosynthetic capacity, an investigation of the phylogenetically diverse cyclases and associated CYPs from the fungal genus Aspergillus was undertaken, revealing their ability to produce isopimaradiene-derived diterpenoids. Intriguingly, labdane-related diterpenoid biosynthetic genes are largely found in plant-associated fungi, hinting that these natural products may play a role in such interactions. Accordingly, it is hypothesized here that isopimarane production may assist the plant-saprophytic lifestyle of Aspergillus fungi.

Full text of DOI:10.1021/acs.jnatprod.6b00764

Article
pubs.acs.org/jnp
Probing Labdane-Related Diterpenoid Biosynthesis in the Fungal
Genus Aspergillus
Meimei Xu, Matthew L. Hillwig,† Mollie S. Tiernan,‡ and Reuben J. Peters*
Roy J. Carver Department of Biochemistry, Biophysics & Molecular Biology, Iowa State University, Ames, Iowa 50011, United States
S
* Supporting Information
ABSTRACT: While terpenoid production is generally associated with plants, a
variety of fungi contain operons predicted to lead to such biosynthesis. Notably,
fungi contain a number of cyclases characteristic of labdane-related diterpenoid
metabolism, which have not been much explored. These also are often found near
cytochrome P450 (CYP) mono-oxygenases that presumably further decorate the
ensuing diterpene, suggesting that these fungi might produce more elaborate
diterpenoids. To probe the functional diversity of such biosynthetic capacity, an investigation of the phylogenetically diverse
cyclases and associated CYPs from the fungal genus Aspergillus was undertaken, revealing their ability to produce isopimaradiene-
derived diterpenoids. Intriguingly, labdane-related diterpenoid biosynthetic genes are largely found in plant-associated fungi,
hinting that these natural products may play a role in such interactions. Accordingly, it is hypothesized here that isopimarane
production may assist the plant-saprophytic lifestyle of Aspergillus fungi.
is terminated by deprotonation, either of the carbocation
roduction of terpenoid natural products is generally
Passociated with plants rather than microbes such as directly, yielding an olefin, or of a water molecule that is first
added to the carbocation, yielding a hydroxylated product.6
fungi. This is evident for the large superfamily of labdane-
In fungi, labdane-related diterpenoid biosynthesis is typically
carried out by bifunctional cyclases that represent fusion of a
CPS and a class I diterpene synthase. For example,
Phaeosphaeria nodorum contains a bifunctional cyclase that
produces ent-kaurene via ent-CPP (2),7 and this can be
separated into distinct polypeptides with CPS and kaurene
synthase (KS) activity,8 with functionally analogous CPS-KSs
reported from other fungi as well.9−11 In addition, it has been
shown that a related bifunctional cyclase from Phoma betae is a
syn-aphidicolan-16β-ol synthase (CPS-AoS), produced via the
distinct stereoisomer syn-CPP (3),12 while Diaporthe amygdali
contains a bifunctional phyllocladan-16α-ol synthase (CPS-
PoS), produced via (normal) CPP (4).13
Phylogenetic analysis of the identified fungal bifunctional
diterpene cyclases has shown that the previously characterized
CPS-KSs are closely related, and even the functionally distinct
CPS-AoS and CPS-PoS are relatively closely related to these as
well (i.e., these fall into two neighboring phylogenetic clades).2
Thus, much of the phylogenetic diversity of these fungal
labdane-related diterpenoid biosynthetic enzymes has not yet
been explored. The Aspergillus genus contains a number of
these cyclases, scattered over a significant portion of the
remaining, uncharacterized phylogenetic range (Figure 1A).
Moreover, the genes encoding these also are often found near
those for cytochrome P450 (CYP) mono-oxygenases, which
presumably further decorate the hydrocarbon backbone
produced by the diterpene synthases, suggesting that these
regions represent biosynthetic gene clusters that might produce
related diterpenoids (∼7000 members), which is defined by the
use of class II diterpene cyclases in their biosynthesis,1 from a
previous genome-mining report. In particular, from the ∼1000
sequenced fungal genomes available at NCBI, less than 100
such cyclases were found.2 Nevertheless, a number of bioactive
labdane-related diterpenoid natural products have been
identified from fungi,3 perhaps most prominently pleuro-
mutilin, various derivatives of which are in clinical use and
trials.4
Class II diterpene cyclases are characterized by a DxDD
motif and catalyze protonation-initiated bicyclization of the
general diterpene precursor (E,E,E)-geranylgeranyl diphosphate
(GGPP, 1), forming the eponymous labdadienyl+ diphosphate
intermediate.1 Direct deprotonation of this yields copalyl
diphosphate (CPP), which is generated as a distinct stereo-
isomer (Chart 1).1 Such cyclases are then termed CPP
synthases (CPSs).
CPP is almost invariably subsequently cyclized and/or
rearranged by class I diterpene synthases.1 These enzymes are
characterized by a D(D,E)xx(D,E) motif involved in binding
the Mg2+ cofactor5 and catalyze ionization of the allylic
diphosphate ester to trigger a carbocation cascade reaction that
Chart 1. Stereoisomers of Copalyl Diphosphate (CPP)
Received: August 19, 2016
© XXXX American Chemical Society and
American Society of Pharmacognosy
A
DOI: 10.1021/acs.jnatprod.6b00764
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
The cyclases from A. fumigatus and A. niger were directly
cloned from the relevant fungi, while synthetic genes were
obtained for those from A. oryzae and N. fischeri (these were
codon-optimized for expression in the targeted heterologous
host E. coli). These cyclases were then recombinantly expressed
in E. coli using a previously reported modular metabolic
engineering system, which provides the substrate 1 via
coexpression of a GGPP synthase.15 Analysis by GC-MS of
organic solvent extracts from the resulting recombinant cultures
and comparison to authentic standards (Figure 2) indicated
that the cyclases from A. fumigatus and A. oryzae produce
isopimara-7,15-diene (5), while those from A. niger and
N. fischeri produce sandaracopimaradiene (6; isopimara-
8(14),15-diene). Given that production of these tricycles
proceeds via initial cyclization of GGPP to CPP, these
bifunctional (iso)pimaradiene synthases were termed CPS-PS
(i.e., AfCPS-PS, AoCPS-PS, AnCPS-PS, and NfCPS-PS,
respectively).
To investigate the stereochemistry of the CPP intermediate
and, hence, that of the derived isopimaradienes, the class I
active site of each CPS-PS was inactivated by mutation of the
first aspartate (Asp) of the relevant D(D,E)xxE motif to alanine
(Ala) (see Table S1 for specific mutations). As expected, each
of these mutants no longer exhibits class I activity (i.e., acts as a
CPS only). Accordingly, when these were incorporated into the
modular metabolic engineering system, only copalol, derived
from CPP by endogenous phosphatases, is evident. These
PSinact mutants were further coexpressed with class I diterpene
synthases specific for either 2 or 4 and found to specifically
produce 4 (Figure S1). Thus, these fungal enzymes all produce
pimaradienes with normal configuration, as defined by
comparison to the analogous A/B rings in cholesterol,1 as
well as C13β-methyl configuration (i.e., isopimaradienes),
differing only in placement of the endocyclic double bond
that results from alternative sites for deprotonation of a
common isopimar-15-en-8-yl+ intermediate (Scheme 1).
To enable investigation of the substrate specificity of the
class I active site of these cyclases, the class II active site was
inactivated by mutation of Asp from the relevant DxDD motif
to Ala. As previously reported for a fungal CPS-KS,8 mutation
of the middle Asp was sufficient to abrogate the CPS activity of
NfCPS-PS. However, to block CPS activity, it proved necessary
to mutate all three Asp in the other fungal CPS-PS (i.e., only
these triple mutants completely lost CPS activity, as the single
and double mutants could still react with GGPP to some
extent; see Table S1 for final mutants). When these CPSinact
mutants were coexpressed in the modular metabolic engineer-
ing system with a CPS producing 4, the expected (iso)-
pimaradiene was produced, demonstrating that these retain
class I activity. Thus, these are suitable for investigating the
substrate specificity of the PS active site.
Figure 1. Fungal labdane-related diterpenoid biosynthetic capacity.
(A) Representative phylogenetic tree with a selected subset of fungal
bifunctional diterpene cyclases, based on a previously reported more
complete tree, named by species (letters refer to distinct enzymes
found in the same species).2 Biochemically characterized cyclases are
annotated as defined in the text (those identified here are highlighted
in bold). (B) Chromosome map for the diterpene cyclases and CYPs
investigated here.
more elaborated diterpenoids (e.g., Figure 1B). Here, to both
probe fungal labdane-related diterpenoid biosynthesis and
further investigate the utility of a previously developed modular
metabolic engineering system that relies on functional
recombinant expression in Escherichia coli14 for such explora-
tion, biochemical characterization of selected cyclases and
neighboring CYPs from the genus Aspergillus was carried out.
RESULTS AND DISCUSSION
■
To begin these studies, the bifunctional diterpene cyclases from
Aspergillus fumigatus strain AF293, A. niger strain CBS 513.88,
and A. oryzae strain RIB40, as well as the closely related fungi
Neosartorya fischeri strain NRRL 181, were chosen for analysis.
While the cyclases from A. fumigatus and A. oryzae are closely
related, these were selected to explore the hypothesis that their
phylogenetic relationship reflects conservation of function.
More importantly, together these cyclases cover a significant
portion of the remaining uncharacterized phylogenetic range of
this fungal enzymatic family (Figure 1A), indicating that
biochemical characterization of these, along with the nearby
CYPs, may provide insight into the functional diversity of
labdane-related biosynthesis in not only the genus Aspergillus
but fungi more broadly.
Accordingly, these CPSinact-PS mutants were coexpressed
with CPSs producing either 2 or 3. Those from A. fumigatus
and A. oryzae did not react with 2, while those from A. niger and
N. fischeri did and produced the diterpene alcohol ent-manool
7, along with small amounts of the diterpene olefin ent-
sandaracopimaradiene 8 (Figure S2). On the other hand, those
from A. fumigatus and A. oryzae react with 3, and both
produced an unknown olefin, while those from A. niger and
N. fischeri both produced an unknown alcohol (Figure S3A).
To identify these compounds, metabolic flux to terpenoids in
the engineered E. coli was increased as previously described,16
and the culture volumes were scaled up, enabling isolation of
B
DOI: 10.1021/acs.jnatprod.6b00764
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
Figure 2. Products of fungal bifunctional diterpene cyclases. GC-MS chromatograms of organic solvent extracts of recombinant E. coli expressing the
indicated enzyme and engineered to produce 1, along with those of authentic standards.
sufficient amounts for characterization of each compound by
NMR. This analysis indicates that the olefin is syn-isopimara-
7,15-diene 9 (Table S2 and Figures S4−6), while the alcohol is
syn-manool 10 (Table S3 and Figures S7−9).
Scheme 1. Related Reactions Catalyzed by the Bifunctional
Fungal Diterpene Cyclases Characterized Here
Hence, while the reaction catalyzed by the PS active site with
the normal substrate 4 is mechanistically very similar among all
four cyclases characterized here (Scheme 1), some differences
between them are revealed by their distinct reactivity with these
alternative substrates. In particular, the PS active sites from the
A. fumigatus and A. oryzae cyclases are more specific, and their
ability to react with 3 and produce syn-isopimaradiene suggests
that this alternative substrate is oriented in a very similar
manner to 4, enabling cyclization (Figure S3B). By contrast, the
greater promiscuity of the PS active sites in the cyclases from
A. niger and N. fischeri leads to the incorporation of water, in an
indirect hydrolysis reaction wherein water is added to the
Figure 3. Products from reaction of indicated fungal CYP503 family members with 6. GC-MS chromatograms of organic solvent extracts of
recombinant E. coli expressing the indicated CYP, along with the requisite CPR, and also engineered to produce 6, along with those of authentic
standards.
C
DOI: 10.1021/acs.jnatprod.6b00764
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
tertiary position of the allylic carbocation generated by initial
ionization of the diphosphate ester (Figure S3C). This
presumably results from water molecules that are appropriately
positioned for such addition in the active site in the presence of
the alternative substrates 2 and 3, but not the native substrate 4.
Beyond the production of isopimaradienes, the presence of
genes encoding CYPs near those encoding the CPS-PS suggests
that these fungi may produce more elaborated isopimarane
diterpenoids. In A. fumigatus and A. oryzae these CYPs are
members of the CYP58 family, while in A. niger and N. fischeri
these are members of the CYP503 family (Figure 1B).
However, the putative CYP58 family member in A. fumigatus
was predicted to contain a premature stop codon, which was
verified here by cloning and sequencing, and this CYP was not
further investigated.
The ability of the full-length CYPs to react with the
isopimaradiene (5 or 6) produced by the neighboring CPS-PSs
was investigated with the modular metabolic engineering
system. On the basis of previous experience with plant
CYPs,14 synthetic genes codon-optimized for expression in
E. coli were used in place of the native CYP genes for this
purpose. In addition, to provide the requisite electrons, a
similarly optimized synthetic gene also was obtained for the
CYP reductase from A. oryzae. Each CYP was then coexpressed,
along with the reductase, as well as the relevant CPS-PS. While
no activity was observed with the CYP58D2 from A. oryzae
(data not shown), with both CYP503 family members a
hydroxylated derivative of 6, the product of their neighboring
CPS-PSs, was observed by GC-MS (Figure 3). That produced
by the CYP503C1 from A. niger was determined to be
sandaracopimaradien-18-ol (11) by comparison to a previously
reported CYP product (i.e., CYP720B417). By contrast, the
CYP503B4 from N. fischeri was found to instead produce
sandaracopimaradien-9α-ol (12), again identified by compar-
ison to a previously reported CYP product (i.e., CYP76M818).
Thus, despite the common production of 6 by their CPS-PS,
the different activity of the nearby CYP503 family members
indicates that A. niger and N. fischeri produce distinct derived
isopimarane diterpenoids (Scheme 2).
noting that in at least four cases now it has been shown that
members of the CYP503 family can carry out initial
oxygenation reactions, suggesting a key early role for this
CYP family in fungal labdane-related diterpenoid biosynthesis.
It has been previously suggested that a bifunctional diterpene
cyclase from Aspergillus nidulans produces ent-pimara-8(14),15-
diene.23 However, the analytical methods used in that report
cannot distinguish between enantiomers. Particularly given that
this cyclase is closely related to NfCPS-PS (84% amino acid
sequence identity, greater than the 74% identity between the
functionally analogous AfCPS-PS and AoCPS-PS characterized
here), it seems likely that this A. nidulans cyclase also may
produce (normal) sandaracopimaradiene (i.e., 6 via 4).
Moreover, the presence of a neighboring CYP503 family
member (CYP503B1) similarly closely related to the
CYP503B4 from N. fischeri further suggests that this fungus
also produces a more elaborated derived isopimarane as well.
Intriguingly, almost all of the fungi predicted to produce
labdane-related diterpenoids (i.e., contain the relevant
diterpene cyclases) are associated with plants as either
pathogens or saprophytes,2 and such biosynthesis (i.e.,
production of gibberellin phytohormones) by Fusarium
fujikuroi has been shown to contribute to the virulence of
this rice plant pathogen.24 Thus, it seems at least plausible that
biosynthesis of such labdane-related diterpenoids is relevant to
the interaction of these fungi with their plant hosts, and the
conserved biosynthesis of isopimaranes across the genus
Aspergillus suggests a role for these diterpenoid natural products
in the plant-saprophytic lifestyle associated with these fungi.
Indeed, given the ease with which these cyclases can be diverted
to alternative reactions by even single amino acid changes,21 it
is striking that these phylogenetically disparate enzymes
produce such similar products. Regardless, given the
phylogenetic range of the diterpene cyclases characterized
here (Figure 1A), these studies provide insight into the
labdane-related diterpenoid biosynthetic capacity of not only
the fungal genus Aspergillus but that of (plant-associated) fungi
more generally as well.
EXPERIMENTAL SECTION
■
Scheme 2. Alternative Hydroxylation of
Sandaracopimaradiene (6) Catalyzed by CYP503B4 and
CYP503C1
General Experimental Procedures. NMR spectra were acquired
on a Bruker AVIII-700 spectrometer equipped with a 5 mm HCN
cryogenic probe, using TopSpin v1.4 software. Analysis was carried out
at 25 °C. Chemical shifts were calculated by reference to those known
for CDCl3 signals offset from tetramethylsilane (13C 77.23 ppm, H
1
7.24 ppm). All spectra were acquired using standard programs from
1
the TopSpin v1.4 software, with collection of 1D H NMR and 2D
double-quantum filtered correlation spectroscopy (DQF-COSY),
heteronuclear single-quantum coherence (HSQC), heteronuclear
multiple-bond correlation (HMBC), HMQC−COSY, and ROESY
(700 MHz), as well as 1D 13C NMR (174 MHz) and distortionless
enhancement of polarization transfer (DEPT) spectra. GC-MS
analyses were carried out using a 3900 GC with Saturn 2100T ion
trap MS (Varian), equipped with an HP-5MS column (Agilent, 0.25
μm, 0.25 i.d., 30 m) with a He flow rate of 1.2 mL/min and the
following oven temperature program: 50 °C for 3 min, 15 °C/min to
300 °C, hold 3 min. Samples (1 μL) were injected via splitless
injection at 250 °C. Flash chromatography was carried out with a 4 g
silica gel column (80−200 mesh) using a Reveleris automated system
(Grace, Deerfield, IL, USA) with a 15 mL/min flow rate, 5 mL
injections, and UV detection at 200 nm, with the following stepwise
gradient: 0%, 5%, 15%, 25% ethyl acetate (in hexane) for 1 min each
and a final wash with 100% ethyl acetate for 3 min. HPLC was carried
out with an Agilent Poroshell 120 EC-C18 column (4.6 × 150 mm, 4
μm) on an Agilent 1200 series system equipped with fraction collector
Notably, the other previously characterized CYP503 family
members both target the same C4α-methyl substituent of a
labdane-related diterpene as CYP503C1, albeit in either ent-
kaurene or syn-aphidicolan-16β-ol (i.e., CYP503A1 and
CYP503N1, respectively).19,20 By contrast, CYP503B4 exhibits
distinct regiospecificity in targeting C-9 instead. Regardless,
while previous work had shown that fungal diterpene cyclases
can be functionally expressed in E. coli,9,12,13,21,22 these results
further demonstrate that at least some fungal CYPs can be
incorporated as well. Although a role for CYP58D2 in
elaboration of 5 cannot be ruled out (e.g., this may not be
amenable to functional expression in E. coli), it seems worth
D
DOI: 10.1021/acs.jnatprod.6b00764
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
and diode array detector and run at a flow rate of 1 mL/min. The
column was pre-equilibrated, and the sample was injected, washed with
50% acetonitrile/dH2O (0−2 min), and eluted with 50−100%
acetonitrile (2−7 min), followed by a 100% acetonitrile wash (7−23
min), with peak-based fraction collection. All reagents were purchased
from Thermo-Fisher Scientific unless noted otherwise.
relied on dephosphorylation of the CPP product by endogenous
phosphatases from E. coli, which yield the derived primary alcohol
(normal) copalol 4′. The various observed enzymatic products were
first tentatively identified by comparison of the associated mass spectra
(MS) to the available MS libraries and then direct comparison of
retention time and MS to that of authentic standards where available.
Analysis of Unknown Enzymatic Products. To obtain sufficient
Cloning. To clone their diterpene cyclases (see Table S4 for
accession numbers), cultures of Aspergillus fumigatus AF293 and
A. niger CBS 513.88 were obtained from the Fungal Genetics Stock
Center (Kansas State University). These fungi were inoculated onto
potato dextrose agar plates (39 g/L) and grown at 28 °C for 4 days.
These cultures were then harvested by scraping and total RNA isolated
using the Concert Plant RNA Reagent (Invitrogen). The genes for the
cyclases and CYP58p were amplified by targeted RT-PCR (see Table
S5 for primers) and cloned by directional topo-isomerization into
pENTR/SD/d-TOPO vectors (Invitrogen). Complete gene sequenc-
ing demonstrated that these matched the predicted genes. Thus, the
cyclases from A. oryzae and N. fischerii were simply obtained by gene
synthesis. These were amplified by PCR (see Table S5 for primers)
and again subcloned into pENTR/SD/d-TOPO vectors. Genes for the
targeted CYPs and the CYP reductase from A. oryzae (AoCPR) also
were obtained by synthesis (see Table S4 for accession numbers). All
synthetic genes, including codon optimization for expression in E. coli,
were purchased from Genscript (the corresponding sequences can be
found in the Supporting Information). CPS-PS mutants were
generated by whole-plasmid PCR amplification using overlapping
mutagenic primers (see Table S5 for primer sequences). All mutants
were verified by complete gene sequencing. For CPS-PS expression,
wild-type and mutant genes were subcloned by directional
recombination into a previously described pGG/DEST vector,15
which contains a synthase for production of the general diterpene
precursor GGPP (1). To enable functional CYP expression, AoCPR
was first cloned into pET-Duet (Novagen), which is compatible with
the pGG/DEST constructs, specifically into the first multiple cloning
site, using the NcoI and NotI restriction sites, which were introduced
on the 5′ and 3′ ends of AoCPR, respectively, by PCR amplification
(see Table S5 for primers). The targeted CYPs were then subsequently
cloned into the resulting pET-Duet/AoCPR vector, specifically into
the second multiple cloning site, using the NdeI and XhoI restriction
sites; again these were introduced on the 5′ and 3′ ends of the CYPs
by PCR amplification (see Table S5 for primers). Previous work has
suggested that replacement of the N-terminal transmembrane helix
sequence found in eukaryotic CYPs with a lysine-rich leader sequence
enables functional expression in E. coli,14 which also was attempted
here (see Table S5 for relevant primers), but found not to improve
activity relative to the full-length constructs. All constructs were
verified by complete sequencing of the introduced genes. Additional
coexpression of the ent-kaurene synthase from Arabidopsis thaliana
(AtKS), which is specific for ent-CPP 2, or the D404A mutant of the
abietadiene synthase from Abies grandis (AgAS:D404A; lacking the
CPS activity otherwise associated with this bifunctional diterpene
cyclase25), which is specific for (normal) CPP 4, utilized previously
described pET-Duet-based constructs.16
quantities of the unknown products of AfCPSinact-PS and NfCPSinact
-
PS with syn-CPP 3 for NMR analysis, 3 L of the relevant bacterial
culture was grown as described above. This was extracted twice with
equal volumes of hexanes, the phases separated in a separatory funnel,
and the pooled hexanes dried by rotary evaporation. The resulting
extract was redissolved in 10 mL of fresh hexanes and fractionated via
flash chromatography. The resulting fractions were analyzed by GC-
MS. Those containing the enzymatic products were dried under N2
and dissolved in 5 mL of methanol. The compounds were then further
purified via HPLC. Again, the fractions containing the enzymatic
products were identified by GC-MS analysis. Those containing pure
compounds were pooled, dried under N2, and dissolved in 0.5 mL of
CDCl3 (Aldrich) for NMR analysis. Observed HMBC correlations
were used to propose the majority of the structures, while COSY
correlations between protonated carbons were used to complete the
structures, which were further verified by HSQC correlations.
ASSOCIATED CONTENT
■
S
* Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jnat-
prod.6b00764.
Details of the described structural characterization of
enzymatic products, along with other supplemental
figures and the sequences of the synthetic genes utilized
here (PDF)
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: rjpeters@iastate.edu.
ORCID
Reuben J. Peters: 0000-0003-4691-8477
Present Addresses
†Chemistry Department, St. Vincent College, Latrobe,
Pennsylvania 15650, United States.
‡Integrated DNA Technologies, Coralville, Iowa 52241, United
States.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Enzymatic Characterization. The targeted enzymes were
characterized using a previously described modular metabolic
engineering system in E. coli.15 Accordingly, the relevant constructs
were transformed into the C41 OverExpress strain of E. coli (Lucigen)
and heterologously expressed under the appropriate antibiotic
selection. Briefly, the recombinant strains were grown in liquid TB
media (12 g casein, 24 g yeast extract, and 8 mL 50% glycerol per L
H2O, with the pH adjusted to 7.0) at 37 °C with shaking at 200 rpm to
OD600 = 0.6, then transferred to 16 °C, with continued shaking at 200
rpm, for an hour and induced with 1 mM IPTG. At the time of
induction, cultures were supplemented with phosphate buffer (pH 7.0)
to 100 mM final concentration, as previously described.16 After 3 days
of continued shaking at 200 rpm and 16 °C, enzymatic products were
extracted by addition of an equal volume of hexanes and gentle
swirling. The organic solvent was separated out and then dried under
N2, with the residue resuspended in fresh hexanes and analyzed by
GC-MS. Note that direct analysis of the PSinact mutants described here
The authors thank Prof. B. Hamberger (Michigan State
University) for the kind gift of CYP720B4. This work was
supported by grants from the NIH to R.J.P. (GM076324 and
GM109773).
REFERENCES
■
(1) Peters, R. J. Nat. Prod. Rep. 2010, 27, 1521−1530.
(2) Fischer, M. J.; Rustenhloz, C.; Leh-Louis, V.; Perriere, G. BMC
Microbiol. 2015, 15, 221.
(3) Quin, M. B.; Flynn, C. M.; Schmidt-Dannert, C. Nat. Prod. Rep.
2014, 31, 1449−73.
(4) Shang, R.; Wang, J.; Guo, W.; Liang, J. Curr. Top. Med. Chem.
2013, 13, 3013−25.
(5) Aaron, J. A.; Christianson, D. W. Pure Appl. Chem. 2010, 82,
1585−1597.
E
DOI: 10.1021/acs.jnatprod.6b00764
J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products
Article
(6) Zi, J.; Mafu, S.; Peters, R. J. Annu. Rev. Plant Biol. 2014, 65, 259−
286.
(7) Xu, M.; Wilderman, P. R.; Peters, R. J. Proc. Natl. Acad. Sci. U. S.
A. 2007, 104, 7397−7401.
(8) Kawaide, H.; Sassa, T.; Kamiya, Y. J. Biol. Chem. 2000, 275,
2276−2280.
(9) Toyomasu, T.; Kawaide, H.; Ishizaki, A.; Shinoda, S.; Otsuka, M.;
Mitsuhashi, W.; Sassa, T. Biosci., Biotechnol., Biochem. 2000, 64, 660−
664.
(10) Tudzynski, B.; Kawaide, H.; Kamiya, Y. Curr. Genet. 1998, 34,
234−240.
(11) Bomke, C.; Rojas, M. C.; Gong, F.; Hedden, P.; Tudzynski, B.
̈
Appl. Environ. Microbiol. 2008, 74, 5325−5339.
(12) Oikawa, H.; Toyomasu, T.; Toshima, H.; Ohashi, S.; Kawaide,
H.; Kamiya, Y.; Ohtsuka, M.; Shinoda, S.; Mitsuhashi, W.; Sassa, T. J.
Am. Chem. Soc. 2001, 123, 5154−5155.
(13) Toyomasu, T.; Niida, R.; Kenmoku, H.; Kanno, Y.; Miura, S.;
Nakano, C.; Shiono, Y.; Mitsuhashi, W.; Toshima, H.; Oikawa, H.;
Hoshino, T.; Dairi, T.; Kato, N.; Sassa, T. Biosci., Biotechnol., Biochem.
2008, 72, 1038−47.
(14) Kitaoka, N.; Lu, X.; Yang, B.; Peters, R. J. Mol. Plant 2015, 8, 6−
16.
(15) Cyr, A.; Wilderman, P. R.; Determan, M.; Peters, R. J. J. Am.
Chem. Soc. 2007, 129, 6684−6685.
(16) Morrone, D.; Lowry, L.; Determan, M. K.; Hershey, D. M.; Xu,
M.; Peters, R. J. Appl. Microbiol. Biotechnol. 2010, 85, 1893−1906.
(17) Hamberger, B.; Ohnishi, T.; Seguin, A.; Bohlmann, J. Plant
Physiol. 2011, 157, 1677−95.
(18) Wang, Q.; Hillwig, M. L.; Okada, K.; Yamazaki, K.; Wu, Y.;
Swaminathan, S.; Yamane, H.; Peters, R. J. J. Biol. Chem. 2012, 287,
6159−6168.
(19) Tudzynski, B.; Hedden, P.; Carrera, E.; Gaskin, P. Appl. Environ.
Microbiol. 2001, 67, 3514−3522.
(20) Fujii, R.; Minami, A.; Tsukagoshi, T.; Sato, N.; Sahara, T.;
Ohgiya, S.; Gomi, K.; Oikawa, H. Biosci., Biotechnol., Biochem. 2011, 75,
1813−7.
(21) Mafu, S.; Potter, K. C.; Hillwig, M. L.; Schulte, S.; Criswell, J.;
Peters, R. J. Chem. Commun. (Cambridge, U. K.) 2015, 51, 13485−7.
(22) Kawaide, H.; Imai, R.; Sassa, T.; Kamiya, Y. J. Biol. Chem. 1997,
272, 21706−21712.
(23) Bromann, K.; Toivari, M.; Viljanen, K.; Vuoristo, A.; Ruohonen,
L.; Nakari-Setala, T. PLoS One 2012, 7, e35450.
(24) Wiemann, P.; Sieber, C. M.; von Bargen, K. W.; Studt, L.;
Niehaus, E. M.; Espino, J. J.; Huss, K.; Michielse, C. B.; Albermann, S.;
Wagner, D.; Bergner, S. V.; Connolly, L. R.; Fischer, A.; Reuter, G.;
Kleigrewe, K.; Bald, T.; Wingfield, B. D.; Ophir, R.; Freeman, S.;
Hippler, M.; Smith, K. M.; Brown, D. W.; Proctor, R. H.;
Munsterkotter, M.; Freitag, M.; Humpf, H. U.; Guldener, U.;
Tudzynski, B. PLoS Pathog. 2013, 9, e1003475.
(25) Peters, R. J.; Ravn, M. M.; Coates, R. M.; Croteau, R. B. J. Am.
Chem. Soc. 2001, 123, 8974−8978.
F
DOI: 10.1021/acs.jnatprod.6b00764
J. Nat. Prod. XXXX, XXX, XXX−XXX