Assembly of melleolide antibiotics involves a polyketide synthase with cross-coupling activity

Article abstract of DOI:10.1016/j.chembiol.2013.07.009

Summary Little is known about polyketide biosynthesis in mushrooms (basidiomycota). In this study, we investigated the iterative type I polyketide synthase (PKS) ArmB of the tree pathogen Armillaria mellea, a producer of cytotoxic melleolides (i.e., polyketides esterified with various sesquiterpene alcohols). Heterologously produced ArmB showed orsellinic acid (OA) synthase activity in vitro. Further, we demonstrate cross-coupling activity of ArmB, which forms OA esters with various alcohols. Using a tricyclic Armillaria sesquiterpene alcohol, we reconstituted the biosynthesis of melledonol. Intermolecular transesterification reactions may represent a general mechanism of fungal PKSs to create structural diversity of small molecules. Phylogenetic network construction of thioesterase domains of both basidiomycetes and ascomycetes suggests that the fungal nonreducing PKS family has likely evolved from an ancient OA synthase and has gained versatility by adopting Claisen-like cyclase or transferase activity.

Full text of DOI:10.1016/j.chembiol.2013.07.009

Please cite this article in press as: Lackner et al., Assembly of Melleolide Antibiotics Involves a Polyketide Synthase with Cross-Coupling Activity,
Chemistry & Biology (2013), http://dx.doi.org/10.1016/j.chembiol.2013.07.009
Chemistry & Biology
Brief Communication
Assembly of Melleolide Antibiotics Involves
a Polyketide Synthase with Cross-Coupling Activity
Gerald Lackner,1 Markus Bohnert,1 Jonas Wick,1 and Dirk Hoffmeister1,
*
Department of Pharmaceutical Biology, Hans Knoll Institute, Friedrich Schiller Universitat, Beutenbergstrasse 11a, Jena 07745, Germany
1
¨
¨
*Correspondence: dirk.hoffmeister@hki-jena.de
http://dx.doi.org/10.1016/j.chembiol.2013.07.009
SUMMARY
acid (OA) moiety esterified to various protoilludane sesquiter-
pene alcohols (Arnone et al., 1988; Bohnert et al., 2011; Donnelly
Little is known about polyketide biosynthesis in et al., 1982). Feeding studies unambiguously proved the poly-
ketide origin of the OA moiety (Misiek et al., 2009). Although
OA is a widespread building block for secondary metabolites in
mushrooms (basidiomycota). In this study, we inves-
tigated the iterative type I polyketide synthase (PKS)
ArmB of the tree pathogen Armillaria mellea, a
producer of cytotoxic melleolides (i.e., polyketides
esterified with various sesquiterpene alcohols).
Heterologously produced ArmB showed orsellinic
acid (OA) synthase activity in vitro. Further, we
demonstrate cross-coupling activity of ArmB, which
fungi, especially in lichen-forming ascomycetes (Schmitt et al.,
2008), only OrsA from Aspergillus nidulans (Sanchez et al.,
2010; Schroeckh et al., 2009) and enzyme CC1G_05377 from
Coprinopsis cinerea have been described as fungal OA syn-
thases (Ishiuchi et al., 2012). Recently, we identified and cloned
a genetic locus from Armillaria mellea (armB) coding for an
iterative type I PKS, which is expressed during melleolide
production (Lackner et al., 2012). The deduced protein ArmB
has an overall identity (similarity) of 26.5% (41.7%) with the
forms OA esters with various alcohols. Using a tricy-
clic Armillaria sesquiterpene alcohol, we reconsti-
tuted the biosynthesis of melledonol. Intermolecular orsellinic acid synthase OrsA of Aspergillus nidulans. ArmB is
composed of seven domains typically present in nonreducing
(NR) iterative type I PKSs (Figure 1B). In the absence of genetic
transesterification reactions may represent a general
mechanism of fungal PKSs to create structural
tools for Armillaria, functional evidence for ArmB activity was
still missing. Therefore, we aimed at the reconstitution of its
activity in vitro.
diversity of small molecules. Phylogenetic network
construction of thioesterase domains of both
basidiomycetes and ascomycetes suggests that
the fungal nonreducing PKS family has likely evolved
from an ancient OA synthase and has gained versa-
tility by adopting Claisen-like cyclase or transferase
activity.
RESULTS AND DISCUSSION
ArmB Produces Orsellinic Acid In Vitro
The armB gene is interrupted by nine introns. We therefore
assembled the 6630 bp coding sequence from synthesized
fragments and cloned it into expression vector pET28a for
production of N-terminally hexahistidine-tagged ArmB. SDS-
INTRODUCTION
Fungal polyketides include a remarkable repertoire of biologi- PAGE and MALDI-mass spectrometry (MS) (Figure S1 available
cally active compounds, ranging from drugs to detrimental online) confirmed expression of the full-length protein. For
toxins (Cox, 2007; Hertweck, 2009). Contrasting Aspergillus reconstitution of activity, the enzyme was incubated with acetyl
and other ascomycetes, only a few polyketides have been coenzyme A (acetyl-CoA) and malonyl-CoA. Afterward, the
isolated from the basidiomycetes, in particular from the agari- products were extracted and analyzed by liquid chromatography
comycotina, that is, the mushroom-type fungi. However, as (LC)-MS. Notably, in samples containing ArmB, an additional
high-throughput sequencing of basidiomycetes makes genomic compound (m/z = 167.034) appeared that was not detectable
data readily accessible, we become increasingly aware that in control experiments (Figure 2A). Comparison with a synthetic
basidiomycetes are more prolific in their capacity to synthesize standard and high-resolution MS proved that the reaction
polyketides than expected (Lackner et al., 2012). For more pro- product was identical with OA. So far, only ascomycete PKSs
found insight into mushroom polyketide synthase (PKS) genes, have been expressed in a heterologous host and shown to be
we investigated the tree pathogen Armillaria mellea, colloquially active in vitro. After pioneering work by Fujii et al. (2000), who
referred to as ‘‘honey mushroom’’. A. mellea also attacks grape- demonstrated tetrahydroxy naphthalene production by PKS1
vine, fruit, and nut crops by eliciting Armillaria root disease from Colletotrichum lagenarium in an Aspergillus oryzae cell
(Baumgartner et al., 2011). These infamous pathogens produce free extract, bikaverin synthase (PKS4) from Gibberella fujikuroi
a range of antifungal and phytotoxic polyketide derivatives, was the first fungal PKS to be functionally produced in E. coli
termed melleolides (Figure 1A). Their production increases in (Ma et al., 2007). More recently, Ishiuchi et al. (2012) expressed
the presence of host plant cells or competing fungi (Peipp and a PKS gene from Coprinopsis cinerea in Saccharomyces
Sonnenbichler, 1992). Melleolides are composed of an orsellinic cerevisiae. However, this protein was not isolated for in vitro
Chemistry & Biology 20, 1–6, September 19, 2013 ª2013 Elsevier Ltd All rights reserved
1

Please cite this article in press as: Lackner et al., Assembly of Melleolide Antibiotics Involves a Polyketide Synthase with Cross-Coupling Activity,
Chemistry & Biology (2013), http://dx.doi.org/10.1016/j.chembiol.2013.07.009
Chemistry & Biology
Assembly of Polyketide-Terpene Esters
Figure 1. Structure of Melleolides and ArmB
(A) Chemical structures of OA and selected mel-
leolides.
OH
A
OH
H
CH3
O
HO
OH
(B) Domain structure of ArmB and proposed
biosynthetic steps of orsellinic acid.
OH
O
SAT, starter unit: ACP transacylase; MAT, malonyl-
CoA: ACP transacylase; PT, product template
domain; KS, ketosynthase; TE, thioesterase.
R
C
O
O
O
OH
O
C
C
OH
H
HO
HO
HO
R1
OR2
OH
OH
(orsellinic acid)
HO
offload the finished ketide from the PKS
assembly line. To test whether ArmB cata-
lyzes transesterification in the absence
of an auxiliary enzyme, we set up the
PKS reactions in the presence of alcohols.
Strikingly, ArmB did exhibit strong cross-
coupling activity. Although free OA re-
mained the major product, esters were
formed in significant yields and were
identified by (1) the UV/visible spectrum,
(2) the shift in retention time (Figure S3),
and (3) high-resolution MS (Table S1).
With respect to alcohol selectivity, primary
alcohols were strongly preferred over sec-
ondary alcohols (Figure 2B). Tert-butanol
was not turned over. Regarding chain
lengths, we observed increasing yields
(melledonol) R1 , R2 = H
(melleolide D) R1 = Cl, R2 = CH3
(armellide A) R = H
(armellide B) R = Cl
B
SAT
KS
MAT
PT
ACP
ACP
TE
O
O
S
acetyl-CoA
O
O
3 x malonyl-CoA
OH
C
HO
O
OH
activity assays. Here, we report the reconstitution of a mush- from methanol to pentanol. A further increase to hexanol was
room polyketide synthase in vitro. Apparently, ArmB represents not detected. Cyclic alcohols were relatively well accepted,
a tetraketide synthase catalyzing three rounds of chain elonga- with cyclobutanol being preferred over cyclopentanol and
tion followed by a C2–C7 ring closure and off-loading of the cyclohexanol (Figure 2C). The esterification of cyclobutanol
product to form free OA. Notably, ArmB was active without is particularly intriguing, as it may, to a degree, mimic the cyclo-
prior incubation with a phosphopantheinyl transferase con- butanol ring of the protoilludane, the preferred attachment site
verting the (apo)-acyl carrier protein (apo-ACP) to holo-ACP. of OA in melleolides. We therefore hypothesized that ArmB
Apparently, holo-ArmB was formed by the action of the has the capacity to assemble melleolides in the presence of a
holo-ACP synthase of E. coli. In this respect, ArmB resembles suitable sesquiterpene alcohol. Hydroxy protoilludanes are not
bikaverin synthase (PKS4) from Gibberella fujikuroi (Ma et al., commercially available, nor does synthesis represent a viable
2007). Furthermore, the addition of the putative starter unit route. Consequently, we procured melleolide D from A. mellea
acetyl-CoA was not required for OA production by ArmB. liquid cultures, followed by hydrolysis of the ester bond to obtain
Following the substrate requirements of ascomycete PKSs submilligram amounts of free 1,4,5,10,13-pentahydroxy-D2,3
-
(Fujii et al., 2000; Ma et al., 2007), we assumed that loading protoilludene (PHP). When the PKS reaction was run in the
of malonyl-CoA and subsequent decarboxylation primes the presence of PHP, a product appeared that was identical to
ArmB-mediated reaction.
melledonol regarding retention time, UV absorption, and m/z
(Figure 3A). Because recording nuclear magnetic resonance
spectra was not feasible, given the small amounts of product
Ester Formation by ArmB
In Armillaria, OA is esterified to various protoilludane sesquiter- formed in vitro, we cannot ultimately determine the exact
pene alcohols constituting the diverse class of melleolide antibi- attachment site of OA. However, because C-5 represents the
otics. Although we detected unesterified OA in mycelial extracts predominant position for OA attachment in vivo, it is plausible
of Armillaria (see Figure S2), it seems to be a minor product. that melledonol is the main product (Figure 3B). It should be
Whereas the protoilludane ring system of the melleolide terpene noted that esters can be established via carbon C-1 in vivo, as
portion is produced by a terpene synthase (Engels et al., 2011), shown for armellide B (Arnone et al., 1988). However, among
the enzymatic activity that couples the sesquiterpene to OA the 56 described melleolides (Bohnert et al., 2011; Misiek and
has not been identified to date. It appears plausible that an Hoffmeister, 2012), only three involve an ester other than C-5.
acyl transferase catalyzes the transesterification, as demon- This finding hints to a strongly preferred but not exclusive
strated for the lovastatin (Kennedy et al., 1999) and fumagillin regioselectivity of ArmB toward the alcohol substrate. This
(Lin et al., 2013) biosyntheses. In these cases, however, the is probably reflected by trace amounts of an earlier eluting
PKSs do not include a thioesterase (TE) domain. Thus, in the (13.9 min; Figure 3A) product of identical mass and
case of ArmB, we assumed that the TE domain of the polyketide UV spectrum, which may represent a concurrently produced
synthase accepts the sesquiterpene alcohol as a substrate to C-1 ester.
2
Chemistry & Biology 20, 1–6, September 19, 2013 ª2013 Elsevier Ltd All rights reserved

Please cite this article in press as: Lackner et al., Assembly of Melleolide Antibiotics Involves a Polyketide Synthase with Cross-Coupling Activity,
Chemistry & Biology (2013), http://dx.doi.org/10.1016/j.chembiol.2013.07.009
Chemistry & Biology
Assembly of Polyketide-Terpene Esters
A
B
C
[M-H]-
0.6
250
200
150
100
50
m/z 221.08
O
O
C
0.5
0.4
0.3
0.2
0.1
0
HO
I
OH
[M-H]-
[M-H]-
m/z 249.11
m/z 235.10
relative
yield
O
O
C
O
O
HO
C
HO
II
OH
OH
III
0
0
5
10
15
20
25
30
20
21
22
23
24
25
retention time (min)
retention time (min)
Figure 2. In Vitro Activity of ArmB
(A) Organic extracts of in vitro reactions analyzed by HPLC (l = 254 nm). I, synthetic OA standard; II, PKS reaction; III, negative control (vector).
(B) Relative yields of cross-coupling activity of ArmB with selected alcohols (20 mM). Error bars indicate the standard deviation (n = 3).
(C) HPLC profiles and proposed structures of OA esters formed with cyclic alcohol substrates. This panel has been composed out of three chromatograms, which
are provided as Supplemental Information (see Figure S1 for data on ArmB identification and Figure S3 and Table S1).
In conclusion, our results indicate that ArmB catalyzes the Table S2). This network demonstrates that ascomycete
transesterification during melleolide biosynthesis. The fact that sequences can be grouped into lactonizing TEs (blue), hydrolyz-
free OA but not melledonol is the major product in vitro points ing TEs (violet), and CLC domains (red). Subclades of CLC
to auxiliary factors that modulate substrate access to the active domains have partially been described in previous studies (Vag-
site of the enzyme in vivo, where melleolides are the main stad et al., 2012). The basidiomycete TE domains are monophy-
products.
letic and further diverged into two major clades (depicted in dark
and light green). PKS1 from the white-rot fungus Stereum
hirsutum should be highlighted, as it clusters closely with ArmB.
This is remarkable in that the genera Stereum and Armillaria are
Alternative Offloading Mechanisms Mediate Polyketide
Diversity in Fungi
In exceptional cases, ketosynthase (KS) domains are capable of only very distantly related from a phylogenetic standpoint.
forming C-O bonds (Bretschneider et al., 2012; Kwon et al., Because Stereum species produce OA butyl ester and methyl
2002). However, we assume it is the TE domain that provides ester (Li et al., 2006), we expect that the PKS1 possesses
the enzymatic activity for ester bond formation. Notably, unlike cross-coupling activity as well. Thus, phylogenetic network con-
bacterial PKSs (Staunton and Weissman, 2001), TE domains of struction of TE domains might be a suitable tool for the prediction
fungal NR-PKSs often catalyze Claisen-like cyclization (CLC) re- of domain function from genomic data. Furthermore, our phylo-
actions rather than hydrolysis. Thus, they accomplish C-C bond genetic studies suggest a scenario where the ancestral NR-PKS
formation during cyclization of the second ring (Fujii et al., 2001). of the fungal kingdom was an OA synthase (i.e., a monocyclic
Consequently, CLC activity should be a hallmark of polycyclic PKS, including a TE domain with hydrolase activity). At a later
PKSs. Well-studied fungal PKSs producing monocyclic polyke- point in evolution, TE domains have taken two routes to diversify
tides are resorcylic acid lactone synthases. Indeed, their TE polyketide products. Whereas the evolution of CLC activity
domain is involved in macrolactonization, as shown for the TE enabled polycyclic polyketides, the evolution of transferase ac-
domain of zearalenone (Wang et al., 2009; Zhou et al., 2008) tivity allowed for the coupling of monocyclic polyketides (OA)
and radicicol synthase (Zhou et al., 2010). Although these two with hydroxylated molecules. Lactonization is a specialized
TEs can catalyze cross-coupling with alcohols in vitro (Wang case of the latter variant and requires a hydroxylated starter
et al., 2009), we have now identified an example where an inter- unit. Both ways are mutually exclusive but vastly helped increase
molecular transesterification occurs in a natural system to yield the structural diversity of fungal polyketides.
PKS-derived hybrid metabolites. Given that further examples
of OA esters are known from fungi (Figure 3C; e.g., the lichen SIGNIFICANCE
¨
metabolite lecanoric acid [Stocker-Worgotter, 2008] or the
¨
glyoxylase inhibitor MS3 from Stereum spp. [Kurasawa et al., We have expressed the gene armB of the basidiomycete
1975; Li et al., 2006]), ArmB cross-coupling may represent a Armillaria mellea in E. coli and demonstrated that it is a fully
more generic mechanism, which contributes to the diversity of functional OA synthase in vitro. Furthermore, we revealed a
fungal polyketide derivatives.
remarkable cross-coupling activity of ArmB as it catalyzes
the transesterification of OA with various alcohols. Using
a rare natural sesquiterpene alcohol as a substrate, we
provide evidence that this activity is indeed connected to
Evolution of Aromatic Polyketide Diversity in the Fungal
Kingdom
To test if functional categories of PKSs can be predicted by melleolide biosynthesis. Thus, ArmB represents an example
phylogenetic clustering, we constructed a phylogenetic network where inherent intermolecular transferase activity is rele-
using TE domains from characterized ascomycete PKSs as well vant in a natural biosynthetic route to yield complex poly-
as from recently sequenced basidiomycete PKSs (Figure 4; ketide derivatives. Phylogenetic analysis of thioesterase
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Please cite this article in press as: Lackner et al., Assembly of Melleolide Antibiotics Involves a Polyketide Synthase with Cross-Coupling Activity,
Chemistry & Biology (2013), http://dx.doi.org/10.1016/j.chembiol.2013.07.009
Chemistry & Biology
Assembly of Polyketide-Terpene Esters
domains suggests that OA synthase is the ancient form of
[M-H]-
A
fungal aromatic PKSs, which have later evolved complexity
by either acquiring Claisen-like cyclization activity (poly-
cycles) or transferase activity (esters/lactones). Further-
more, intermolecular transesterification might be a common
mechanism to add diversity to fungal aromatic polyketides.
Consequently, ArmB-mediated cross-coupling might also
expand the toolbox of synthetic biologists for the creation
of novel bioactive compounds by pathway engineering.
Finally, our results open perspectives toward a better
understanding of Armillaria root disease. Some melleolides
show phytotoxic effects on plant cells in vitro. Once stable
gene inactivation in Armillaria becomes feasible, armB will
be a promising candidate gene to study the contribution of
small molecule toxins on the virulence of this serious plant
pathogen.
m/z 433.19
166.9
MS/MS
OA
148.0
122.8
100 150 200
m/z
melledonol
PKS reaction
control
EXPERIMENTAL PROCEDURES
11
12
13
14
15
Gene Expression and Purification of ArmB
retention time (min)
Fragments of the armB coding sequence (GenBank accession JQ801748.1)
were synthesized at Genscript. The coding sequence was cloned into vector
pET28a as a SpeI/NotI fragment to create plasmid pGL77 carrying the (His)6-
armB gene. E. coli BL21 (DE3) transformed with pGL77 was grown in 2 l auto-
induction medium at 37ꢀC to an optical density of OD600 = 0.5. Subsequently,
cultures were grown at 16ꢀC for further 18 hr. His-tagged protein was purified
by Ni2+-affinity chromatography using an imidazole gradient followed by gel
B
OH
OH
3
1
2
14
15
13
¨
filtration on an Akta Explorer (Amersham Pharmacia Biotech) fast protein
9
4
10
HO
7
liquid chromatography (FPLC) using a Superdex 200 10/300 GL column
(GE Healthcare) at a flow rate of 0.5 ml/min in reaction buffer (100 mM
NaH2PO4, pH 7.4). In order to obtain highly active preparations of enzyme,
Ni2+-nitrilotriacetic acid (NTA)-purified fractions were rebuffered on a PD-10
column (GE Healthcare), concentrated, and freshly used for in vitro assays.
5
6
H
OH
PHP
HO
OH
OH
H
ACP
S
HO
Polyketide Synthase In Vitro Reaction and Product Analysis
O
OH
Approximately 20 mg ArmB was incubated with acetyl-CoA (1 mM) and
malonyl-CoA (3 mM) in 100 ml reaction buffer (100 mM NaH2PO4, pH = 7.4).
As a negative control, we used a protein fraction obtained from E. coli BL21
transformed with the empty vector pET28a instead of the armB expression
plasmid pGL77. For the formation of esters, 20 mM alcohol was added
from 200 mM stock solutions. Relative yields of alcohols were calculated
from areas under curve (AUCs; l = 254 nm) of OA derivatives as follows:
AUCEster/(AUCEster + AUCOA). For melleolide assembly in vitro, the sesquiter-
pene alcohol was directly dissolved in reaction buffer to obtain an approximate
5 mM solution. After 20 hr, the reaction mixture was extracted twice with one
volume of ethyl acetate/acetic acid (99:1), evaporated to dryness, and solved
in 100 ml MeOH for high-performance liquid chromatography (HPLC) and MS
measurements. Analytical HPLC was performed on an Agilent 1200 instrument
equipped with a diode array detector, set to detect the wavelength range from
210 to 400 nm, and a Zorbax Eclipse XDB-C18 column (150 3 4.6 mm). HPLC
conditions were as follows: gradient elution with 0.1% trifluoroacetate in H2O/
MeCN 99:1 to 100% MeCN in 30 min, MeCN 100% for 12 min, and flow rate
1 ml/min. High-resolution electrospray ionization-MS was measured on a
Thermo Accela instrument (LC) and a Thermo Exactive orbitrap spectrometer
(high-resolution MS) in negative mode.
TE
OH
O
O
C
HO
OH
OH
melledonol
C
OH
OH
O
OH
HO
OH
O
O
O
O
O
O
OH
OH
OH
OH
MS3
OH
OA butyl ester
OH
lecanoric acid
Preparation of PHP
Melleolide D was isolated from A. mellea as described (Bohnert et al., 2011).
For hydrolysis of the ester, melleolide D (1 mg) was stirred in 250 ml KOH
(1 M) for 2 days. The reaction mixture was extracted five times with 250 ml
ethyl acetate. The organic phase was dried in vacuo, dissolved in MeOH,
and fractionated by silica gel chromatography (MeOH/chloroform gradient).
Fractions were again evaporated to dryness, dissolved in MeOH, and analyzed
by LC-MS to identify the fractions containing PHP (m/z 283 [M-H]À). Carbon
atoms are numbered according to Donnelly et al. (1982).
Figure 3. Melleolide Formation by ArmB
(A) In vitro cross coupling of orsellinyl-ACP with PHP yielded a product
exhibiting identical UV absorption and mass fragmentation as melledonol.
(B) Proposed scheme of melleolide assembly in vitro.
(C) Selected OA esters found in fungi.
See Figure S2 for in vivo data.
4
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Please cite this article in press as: Lackner et al., Assembly of Melleolide Antibiotics Involves a Polyketide Synthase with Cross-Coupling Activity,
Chemistry & Biology (2013), http://dx.doi.org/10.1016/j.chembiol.2013.07.009
Chemistry & Biology
Assembly of Polyketide-Terpene Esters
OH
O
O
R
TE (cross-coupling)
HO
orsellinic acid esters
OH
O
O
O
HO
0.1
resorcylic acid
lactones
TE (lactonizing)
polycycles
OH
O
TE (hydrolyzing)
HO
OH
orsellinic acid
OH OH
O
O
CLC
HO
OH
naphthopyrone
CLC
OH OH
ATHN
O
CLC
OH
CLC/
O
O
O
deacetylase
HO
OH
HO
OH OH
OH
OH
HO
OH
Norsolorinic acid
THN
Figure 4. Phylogenetic Network of Thioesterase Domains of Fungal NR PKSs
Monocyclic PKSs include TE domains with hydrolase (violet) or transferase (i.e., lactonization) (blue) or cross-coupling (bold species names) activity. Polycyclic
PKSs from ascomycetes feature a CLC domain and can be further subdivided (Vagstad et al., 2012). Basidiomycete enzymes (green) are monophyletic (except
Ustilago PKS2) and have diverged into two major subclades (light and dark green). The asterisk denotes the function is not verified. The scale bar indicates the
uncorrected pairwise distance. For accession numbers, see Table S2.
Phylogenetic Network Construction
for providing access to FPLC instruments. D.H. acknowledges support for
¨
this study by the Hans Knoll Institute. M.B. is a predoctoral fellow of the
Sequences of product template and thioesterase domains were aligned with
the MUSCLE algorithm implemented in MEGA5 (Tamura et al., 2011). The
obtained alignment was used for phylogenetic network construction using
Splitstree4 (Huson, 1998), applying the built-in neighbor-net method.
Jena School for Microbial Communication. This paper is dedicated to Profes-
sor Wolfgang Steglich on the occasion of his 80th birthday.
Received: April 27, 2013
Revised: July 15, 2013
Accepted: July 16, 2013
Published: August 29, 2013
SUPPLEMENTAL INFORMATION
Supplemental Information includes three figures and two tables and can
be found with this article online at http://dx.doi.org/10.1016/j.chembiol.
2013.07.009.
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ACKNOWLEDGMENTS
We thank Andrea Perner and Dr. Andreas Habel (Hans Kno¨ ll Institute, Jena) for
¨
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Please cite this article in press as: Lackner et al., Assembly of Melleolide Antibiotics Involves a Polyketide Synthase with Cross-Coupling Activity,
Chemistry & Biology (2013), http://dx.doi.org/10.1016/j.chembiol.2013.07.009
Chemistry & Biology
Assembly of Polyketide-Terpene Esters
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