Homologous NRPS-like gene clusters mediate redundant small-molecule biosynthesis in Aspergillus flavus

Article abstract of DOI:10.1002/anie.201207456

Biosynthetic crosstalk: Most gene clusters in fungi are orphans with no known associated metabolites. NMR-based comparative metabolomics was used to identify the products of two highly homologous orphan clusters in Aspergillus flavus. The two clusters enc

Full text of DOI:10.1002/anie.201207456

.
Angewandte
Communications
DOI: 10.1002/anie.201207456
Natural Products
Homologous NRPS-like Gene Clusters Mediate Redundant Small-
Molecule Biosynthesis in Aspergillus flavus**
Ry R. Forseth, Saori Amaike, Daniel Schwenk, Katharyn J. Affeldt, Dirk Hoffmeister,
Frank C. Schroeder,* and Nancy P. Keller*
Fungi are among the most prolific sources of pharmacolog-
ically relevant natural products.[1] This large diversity of
fungal small molecules serves important functions in fungal
ecology, for example as virulence factors and as chemical
defense agents. However, only a fraction of the biosynthetic
capabilities suggested by genomic analyses has been observed
under laboratory conditions, because expression of many,
perhaps even most biosynthetic pathways depends strongly
on environmental conditions.[2] Here we demonstrate the use
of comparative metabolomics[3] for the analysis of knock-out,
overexpression, and knock-down strains to identify metabo-
lites derived from two nonribosomal peptide synthetase
(NRPS) gene clusters in the aflatoxin-producing ascomycete
Aspergillus flavus, a crop contaminant[4] and opportunistic
pathogen that causes aspergillosis in immunocompromised
humans.[5]
Although the A. flavus genome encodes at least 25
polyketide synthase (PKS), 18 NRPS, and two hybrid
NRPS-PKS gene clusters,[6] assignments have been made for
only four metabolites.[7] Many A. flavus secondary-metabolite
pathways are under the control of the nuclear protein LaeA,
a global regulator of morphogenesis and virulence factor in
A. flavus and other pathogenic fungi.[8] Two LaeA-regulated
clusters, which were named lna and lnb, exhibit a striking level
of genetic similarity (Figure 1a, Table S1). The lna and lnb
clusters contain two noncanonical NRPS genes with high
sequence homology (58% identical at the amino acid level),
lnaA and lnbA, respectively, which are accompanied by
matching sets of genes likely coding for tailoring enzymes. lna
and lnb are orphan clusters that have no known associated
metabolites and belong to a family of noncanonical NRPS
genes that consist of an adenylation (A) domain, a peptidyl
carrier protein (PCP) domain, and a thioester reductase (R)
domain, but lack a canonical condensation (C) domain
(Figure 1a). The functions of this unusual family of NRPSs,
which share homology to reductases participating in the
biosynthesis of l-lysine in fungi,[9] have not been explored.
We here demonstrate that the lna and lnb clusters encode sets
of enzymes that produce overlapping sets of previously
undescribed metabolites, and show that one primary function
of the noncanonical NRPSs LnaA and LnbA likely consists in
the reduction of l-tyrosine. Furthermore, the lna and lnb
biosynthetic pathways appear to be part of a signaling
network that controls the formation of sclerotia, a resilient
overwintering structure.
To identify lna-associated metabolites by means of com-
parative metabolomics, we created deletion and overexpres-
sion[10] mutants of the NRPS gene lnaA (DlnaA and
OE::lnaA, respectively) as well as double-mutant strains
DlnaA, KD::lnbA and OE::lnaA, KD::lnaB, where KD
indicates a knock-down of gene expression using RNAi
(strains are listed in Table S3; for methods, see the Supporting
Information, Section 1). Unexpectedly, we noted that in the
DlnaA, KD::lnbA double-mutant strain, in which both the lna
and lnb pathways are disrupted, formation of sclerotia was
strongly suppressed relative to the wild-type(WT; see the
Supporting Information, Figure S1).
The WT, DlnaA, and OE::lnaA metabolomes were
compared using differential analysis by 2D NMR spectrosco-
py (DANS) as described previously.[3b–d] Comparison of the
OE::lnaA spectra with either WT or DlnaA revealed a large
number of spin systems present only in the OE::lnaA spectra
(see Figure S2). Available structure databases indicated that
most of the OE::lnaA-specific signals did not correspond to
[*] R. R. Forseth,[+] Prof. Dr. F. C. Schroeder
Boyce Thompson Institute and Department of Chemistry and
Chemical Biology, Cornell University
D. Schwenk, Prof. Dr. D. Hoffmeister
Department of Pharmaceutical Biology, Hans Knçll Institute
Friedrich Schiller Universitꢀt
1 Tower Road, Ithaca, NY 14850 (USA)
E-mail: fs31@cornell.edu
Homepage: http://www.bti.cornell.edu/schroeder/
Beutenbergstrasse 11a, 07745 Jena (Germany)
[+] These authors contributed equally to this work.
[**] We thank J. A. Baccile, Y. Rokhlenko, and J. A. Bernstein for
assistance with synthesis and M. Kukula for help with mass
spectrometry. Support from the U.S. National Institutes of Health
(GM008500 to R.R.F. and GM084077 to N.P.K.), the International
Leibniz Research School Jena (to D.S.), DuPont Corp. (to F.C.S.),
the U.S. Department of Agriculture (WIS01200 to N.P.K.), and the
Food Research Institute, National Science Foundation subagree-
ment (IOS-0965649 to N.P.K) is gratefully acknowledged.
K. J. Affeldt, Prof. Dr. N. P. Keller
Department of Medical Microbiology and Immunology
University of Wisconsin-Madison
3476 Microbial Sciences Building, Madison, WI 53706 (USA)
E-mail: npkeller@wisc.edu
Dr. S. Amaike[+]
Department of Plant Pathology
University of Wisconsin-Madison (USA)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201207456.
Prof. Dr. N. P. Keller
Department of Bacteriology
University of Wisconsin-Madison (USA)
1590
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2013, 52, 1590 –1594

Angewandte
Chemie
hydroxy proton with the axial proton at C-6 defined the
relative configuration of 7 as (2R*,5S*). We further identified
a fully deaminated “monomer”, 3-(p-hydroxyphenyl)-1,2-
propanediol (8), a previously described fungal metabolite,[12]
and four pyrazine derivatives. These include the known
actinopolymorphol C (3),[13] an O-sulfonated derivative (4),
as well as two other highly polar derivatives. The NMR
spectra of one of these suggested N-oxidation which was
confirmed by HRMS; the molecular formula of C18H16N2O4
found corresponded to an unusual N,N-dioxide (5; for
a related synthetic compound, see Ref. [14]). NMR and
HRMS spectra of the second derivative of 3 indicated
sulfonylation at C-3 in this metabolite (!6). In addition to
1–8, which were consistently produced by OE::lnaA but not
DlnaA, we detected occasional production of several other
metabolites, whose structures (10–13) appeared unrelated to
those of 1–8, suggesting that they are not derived from the lna
biosynthetic pathway. Among these, compound 10 is a novel
metabolite featuring an intriguing ring system that is likely
derived from the known A. flavus terpenoid aflavinine (see
the Supporting Information, Section 9).[15]
Next we investigated whether production of 1–8 can be
elicited under specific conditions in wild-type A. flavus, and
whether these metabolites are in fact strictly lna-dependent
(see Table 1). Highly sensitive single-ion-monitoring MS
Table 1: A. flavus strains (also see Table S3) and occurrence of lna/lnb-
associated metabolites 1–8.[a]
Cmpd OE::lnaA
(rich
OE::lnaA OE::lnaA, Wild
KD::lnaB type
DlnaA DlnaA,
KDlnbA
Figure 1. a) The lna and lnb gene clusters in A. flavus.[2c] Both lna and
lnb include noncanonical NRPS genes encoding adenylation (A),
carrier protein (PCP), and a short-chain dehydrogenase/reductase
domain (R-domain). There is no lnaD orthologue in the lnb gene
cluster. See also Table S1 in the Supporting Information. b) Metabo-
lites identified in this study and the known piperazinomycin (9).
Compounds marked with an asterisk have been previously described.
medium)
1
2
3
4
5
6
7
8
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
+++
–
+++
+++
+++
–
++
++
++*
–
+
+
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
+++
+++
+++
+++
–
–
any known metabolites from Aspergilli or other fungi.
Detailed NMR spectroscopic and high-resolution (HR) MS
analysis of purified fractions containing the OE::lnaA-specific
components suggested a pair of diastereomeric piperazines,
1 and 2, as major lnaA-dependent compounds (Figure 1b; see
Tables S4–S10 for spectroscopic data). Comparison with
synthetic samples prepared from cyclo(l-Tyr-l-Tyr) and
cyclo(d-Tyr-l-Tyr) derivatives confirmed these structural
assignments (Supporting Information, Section 8). The struc-
tures of 1 and 2 are reminiscent of piperazinomycin (9),[11]
a bacterial metabolite first identified from Streptomyces
olivoreticuli, which may be derived from oxidative macro-
cyclization of 1.
[a] “+++”=abundant, detected by DANS and MS, “++”=detected by
MS only (“*”=occasionally detected), and “+”=detected by MS,
roughly 10-old less abundant than in WT. Strains were cultured on GMM
unless indicated otherwise.
(SIMMS) analysis of DlnaA, WT, and OE::lnaA cultures
grown using conditions previously shown to support lnaA
expression[2c] revealed the presence of piperazines 1 and 2 in
WT, although at roughly 100-fold lower concentrations than
in OE::lnaA (see the Supporting Information, Section 11).
Pyrazine 3 was found only in some, but not all WT extracts,
whereas 4–8 were not detected. OE::lnaA produced com-
pounds 1–3, 7, and 8, whereas 4–6 were absent under these
culturing conditions. Notably, SIMMS also detected very
small quantities of 1 and 2 in DlnaA extracts, at levels roughly
10 times lower than in WT. This suggested that 1 and 2 are
perhaps also produced by enzymes encoded by the homolo-
gous lnb cluster. To test this hypothesis, extracts derived from
two different DlnaA, KD::lnbA double-mutant strains were
The piperazines 1 and 2 are accompanied by a derivative
that includes a quaternary carbon (d = 93.1 ppm), two iso-
lated methylene groups, and an OCH2-CH(N)-CH2-phenyl
fragment. HRMS indicated
a
molecular formula of
C18H21NO4, which in conjunction with 2D NMR spectroscopic
data (Table S9) led to the identification of the hemiacetal-
containing morpholine 7. ROESY correlations between the
Angew. Chem. Int. Ed. 2013, 52, 1590 –1594
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
1591

.
Angewandte
Communications
analyzed by HPLC/ESI+-SIMMS, which showed that com-
pounds 1–8 are completely absent in the double mutants,
whereas WTand DlnaA, grown as positive controls in parallel,
produced 1 and 2 as before (Figure S4). These results indicate
that the amounts of 1 and 2 found in DlnaA are derived from
lnbA expression and thus the lna and lnb clusters (Figure 1a)
encode partially redundant biosynthetic pathways.
mation of the most abundant lna- or lnb-associated metab-
olites (1, 2, 7, 8) requires additional reduction from aldehydes
or imines to the corresponding amines and alcohols. These
reductive steps seemed likely to involve putative NmrA-like
proteins encoded by lnaB and lnbB (Figure 1a), as NmrA-like
proteins are believed to serve enzymatic functions as epimer-
ases or reductases,[18] in addition to possible roles in gene
regulation.[19]
Since the structures of 1–8 appear to be tyrosine (Tyr)
derived, the two putative NRPS proteins LnaA and LnbA
were tested for their ability to specifically activate Tyr by
assaying amino acid dependent ATP-[32P]-pyrophosphate
exchange activity.[16] Both LnaA and LnbA specifically
activated l-Tyr, although LnaA was somewhat d/l-unspecific
and also activated d-Tyr (Figure 2a). However, neither LnaA
nor LnbA include a condensation domain, and correspond-
ingly, none of the lna- or lnb-associated metabolites identified
in the WT nor in the OE::lnaA background feature a peptide
bond. In addition, neither protein activated the l-Tyr-l-Tyr
dipeptide (Figure S5). However, both LnaA and LnbA
include C-terminal R- (putative short-chain reductase and/
or epimerase) domains, with amino acid sequence similarity
to other microbial reductase domains.[17] Based on the
chemical structures of the identified lna- and lnb-dependent
metabolites, it appeared likely that the R-domains in LnaA
and LnbA are involved in the reduction of l-Tyr derivatives.
In a biosynthetic model, LnaA- and/or LnbA-derived l-Tyr
aldehyde (15) may form a dimer such as 17, perhaps via
a tethered intermediate, for example, 16 (Figure 2b). For-
To examine the putative role of LnaB in the biosynthesis
of 1 and 2, the metabolite profile of an OE::lnaA, KD::lnaB
double-mutant strain was compared to that of OE::lnaA by
2D NMR and HPLC/ESI+-SIMMS studies. These analyses
showed that the morpholine 7 is much more abundant in
OE::lnaA, KD::lnaB than in the OE::lnaA background,
relative to the piperazines 1 and 2 (Figure 2c). This shift
towards a relatively greater production of morpholine 7,
whose biosynthesis requires one reduction step less than
production of 1 and 2, suggests that LnaB participates in the
reduction of LnaA-derived intermediates. Knock-down of
lnaB would result in increased accumulation of 17, which may
partially undergo hydrolysis leading to increased production
of 7 (Figure 2b, Figure S6). Notably, production of pipera-
zines 1 and 2 is not fully abolished in the OE::lnaA, KD::lnaB
strain, and formation of morpholine 7 from l-Tyr aldehyde
(15) or 17 still requires one reductive step. The residual
reductase activity in the OE::lnaA, KD::lnaB double mutant
could result from expression of the second nmrA-like gene
lnbB (or another related gene proximal to lnbB, Table S1) or
Figure 2. a) Substrate specificity of LnaA (black bars) and LnbA (gray bars). Recombinant LnaA and LnbA were tested for their ability to adenylate
various amino acids. Shown are relative activity values normalized to l-Tyr activity. Error bars: standard deviation. b) Simplified model for the
biosynthesis of lnaA-dependent metabolites by lna and lnb- cluster genes (see also Figure S6). Tethered l-Tyr (14) is reduced to 15 by the R-
domains of LnaA or LnbA, resulting in the formation of a dimeric imine (17) potentially via a tethered intermediate (16). Cyclization and reduction
by LnaB (and perhaps LnbB) leads to formation of 1 or 2, whereas oxidation yields 3. In the absence of LnaB, isomerization of 17 (or a related
intermediate) to the enamine followed by loss of ammonia leads to increased formation of 7. c) Single-ion chromatograms from HPLC/ESI+-
SIMMS analysis corresponding to 1, 2, and 7 for the OE::lnaA and OE::lnaA, KD::lnaB strains. The OE::lnaA, KD::lnaB strain shows a dramatic
increase in production of 7 relative to 1 and 2, compared to the OE::lnaA strain.
1592
www.angewandte.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2013, 52, 1590 –1594

Angewandte
Chemie
incomplete silencing of lnaB. Lastly, whereas cyclization of 17
to a corresponding diimine followed by LnaB- (or LnbB-)
mediated reduction leads to piperazines 1 and 2, oxidation,
non-enzymatically or catalyzed by putative oxidases LnaC,
LnaD, or LnbC, would explain formation of pyrazine 3.
HPLC/ESI+-SIMMS analysis further showed that the
ratio of the two diastereomers 1 and 2 is significantly greater
in WT than in DlnaA (Figure 3a and Figure S7), suggesting
that the lnb pathway produces relatively larger amounts of the
genomes. The piperazinomycin-producing bacteria (Strepto-
myces sp.) have not been sequenced; however, available
Streptomyces genomes contain a number of putative genes
encoding proteins with high (up to 38%) amino acid sequence
similarity to LnaA. The close similarity of some bacterial
NRPS-like proteins to LnaA/LnaB despite bacteria not
synthesizing lysine by the Lys2 pathway suggests a complex
evolution of these proteins.[21]
Our work provides the first evidence that Lys2-type
enzymes also function in secondary metabolism and
additionally suggests that NRPS-like proteins may serve
as amino acid reductases, indicating that a reaction
hitherto considered as an NRP-offloading mode can
stand by itself. This study also demonstrates the con-
sequences of lnaA/lnbA gene disruption/silencing and
the concomitant loss of piperazine/pyrazine/morpholine
secondary products on fungal development, linking
sclerotia formation in A. flavus and the lna/lnb meta-
bolic pathways. This observation adds a unique role to
previously known functions of NRPS-like genes in fungi.
The occurrence of a functionally duplicated biosynthetic
pathway can be interpreted as a safeguard to ensure
timely sclerotial production and hence, persistence
during unfavorable environmental conditions. Our
results are suggestive of a complex signaling network
regulating the biosynthesis of the lna- and lnb-pathway
metabolites, which may include cross-pathway interactions
mediated by sensing of 1 and 2 or shared biosynthetic
intermediates. Detailed analysis of the metabolomes and
associated phenotypes of lna/lnb single and double knock-out
strains will be required to clarify the extent of interactions
between the lna/lnb clusters and determine the roles of the
identified metabolites for sclerotia formation and other
aspects of A. flavus biology.
Figure 3. a) Comparison of the diastereomeric ratios of compounds 1 and 2
in WT and DlnaA metabolite extracts. Error bars: standard deviation. b) Effect
of supplementation with synthetic 1 or 2 on lna gene cluster expression.
Northern analysis showed that addition of 1, but not 2 increased lnaB
expression. rRNA served as loading control.
(2R*,5S*) isomer, 2, than the lna pathway. Given that LnbA
activates l-Tyr with high selectivity, it seems unlikely that this
greater relative abundance of 2 results from incorporation of
both d-Tyr and l-Tyr. Instead, these stereochemical differ-
ences probably originate from different degrees of epimeri-
zation at the aldehyde or imine stage. Next, we asked whether
the ratio of diastereomers is actively regulated at the gene
expression level. Northern analysis of WT cultures incubated
with synthetic 1 or 2 showed that treatment with 2 did not lead
to any significant changes in lna gene expression, whereas
addition of 1 to WT cultures greatly increased expression of
lnaB (Figure 3b). This result strongly suggests metabolite-
mediated crosstalk between the lna and lnb pathways.
Received: September 15, 2012
Revised: November 8, 2012
Published online: December 20, 2012
Keywords: comparative metabolomics · genome mining ·
In conclusion, comparative metabolomics revealed eight
.
natural products · piperazines · secondary metabolism
lna-associated metabolites, 1–8, of which 1–3 could be
detected in WTA. flavus. The two most consistently produced
lna metabolites, 1 and 2, are also produced under participa-
tion of lnb genes, providing a first example for partially
redundant biosynthesis involving an NRPS-like pathway.
LnaA and LnbA deviate from canonical NRPS domain
structures in that they lack condensation domains, and thus, as
the structures of identified metabolites suggest, do not act as
peptide synthetases. Instead, LnaA and LnbA follow the
domain layout of Lys2 and related fungal enzymes that serve
as a-aminoadipate aldehyde reductases in fungal l-lysine
biosynthesis (24% and 25% identity of LnaA and LnaB to
S. cerevisiae Lys2, respectively[9]). Examination of fungal
genomes showed that LnaA-like proteins are primarily
confined to the Ascomycete taxon Plectomycetes, with
between one and five different LnaA-like proteins in differ-
ent Aspergilli. Recently published genomes (e.g., of Serpula
lacrymans and Heterobasidion annosum[20]) show that LnaA/
LnbA-type enzymes are also encoded in basidiomycete
[1] P. Schneider, M. Misiek, D. Hoffmeister, Mol. Pharm. 2008, 5,
234.
[2] a) J. M. Winter, S. Behnken, C. Hertweck, Curr. Opin. Chem.
Biol. 2011, 15, 22; b) A. A. Brakhage, V. Schroeckh, Fungal
Genet. Biol. 2011, 48, 15; c) D. R. Georgianna, N. D. Fedorova,
J. L. Burroughs, A. L. Dolezal, J. W. Bok, S. Horowitz-Brown,
C. P. Woloshuk, J. Yu, N. P. Keller, G. A. Payne, Mol. Plant
Pathol. 2010, 11, 213.
[3] a) G. L. Challis, J. Med. Chem. 2008, 51, 2618; b) R. R. Forseth,
E. M. Fox, D. Chung, B. J. Howlett, N. P. Keller, F. C. Schroeder,
J. Am. Chem. Soc. 2011, 133, 9678; c) R. R. Forseth, F. C.
Schroeder, Curr. Opin. Chem. Biol. 2011, 15, 38; d) C. Pungaliya,
J. Srinivasan, B. W. Fox, R. U. Malik, A. H. Ludewig, P. W.
Sternberg, F. C. Schroeder, Proc. Natl. Acad. Sci. USA 2009, 106,
7708.
[4] a) M. A. Klich, Mol. Plant Pathol. 2007, 8, 713; b) T. Michailides,
T. Thomidis, Plant Pathol. 2007, 56, 352.
Angew. Chem. Int. Ed. 2013, 52, 1590 –1594
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
1593

.
Angewandte
Communications
[5] a) M. T. Hedayati, A. C. Pasqualotto, P. A. Warn, P. Bowyer,
D. W. Denning, Microbiology 2007, 153, 1677; b) A. Chakra-
barti, S. S. Chatterjee, A. Das, M. R. Shivaprakash, Med. Mycol.
2011, 49, S35.
[6] T. E. Cleveland, J. Yu, N. Fedorova, D. Bhatnagar, G. A. Payne,
W. C. Nierman, J. W. Bennett, Trends Biotechnol. 2009, 27, 151.
[7] J. Marui, N. Yamane, S. Ohashi-Kunihiro, T. Ando, Y. Terabaya-
shi, M. Sano, S. Ohashi, E. Ohshima, K. Tachibana, Y. Higa, M.
Nishimura, H. Koike, M. Machida, J. Biosci. Bioeng. 2011, 112,
40.
[8] a) S. Amaike, N. P. Keller, Eukaryotic Cell 2009, 8, 1051; b) J. W.
Bok, S. A. Balajee, K. A. Marr, D. Andes, K. F. Nielsen, J. C.
Frisvad, N. P. Keller, Eukaryotic Cell 2005, 4, 1574; c) D. Wu, S.
Oide, N. Zhang, M. Y. Choi, B. G. Turgeon, PLoS Pathog. 2012,
8, e1002542.
[13] a) E. Powell, S. X. Huang, Y. Xu, S. R. Rajski, Y. Wang, N.
Peters, S. Guo, H. E. Xu, F. M. Hoffmann, B. Shen, W. Xu,
Biochem. Pharmacol. 2010, 80, 1221; b) S. X. Huang, E. Powell,
S. R. Rajski, L. X. Zhao, C. L. Jiang, Y. Duan, W. Xu, B. Shen,
Org. Lett. 2010, 12, 3525.
[14] J. H. Zhang, Q. R. Peng, S. X. Zhang, Y. B. Li, S. Q. Li, H. X.
Gao, Z. Q. Zhou, J. Mol. Struct. 2011, 987, 34.
[15] J. B. Gloer, M. R. TePaske, J. S. Sima, J. Org. Chem. 1988, 53,
5457.
[16] P. Schneider, M. Weber, K. Rosenberger, D. Hoffmeister, Chem.
Biol. 2007, 14, 635.
[17] a) A. Pospiech, B. Cluzel, J. Bietenhader, T. Schupp, Micro-
biology 1995, 141, 1793; b) N. Kessler, H. Schuhmann, S.
Morneweg, U. Linne, M. A. Marahiel, J. Biol. Chem. 2004, 279,
74137419.
[18] K. L. Kavanagh, H. Jornvall, B. Persson, U. Oppermann, Cell.
Mol. Life Sci. 2008, 65, 3895.
[19] A. Andrianopoulos, S. Kourambas, J. A. Sharp, M. A. Davis,
M. J. Hynes, J. Bacteriol. 1998, 180, 1973.
[9] D. E. Ehmann, A. M. Gehring, C. T. Walsh, Biochemistry 1999,
38, 6171.
[10] S. Bergmann, J. Schꢀmann, K. Scherlach, C. Lange, A. A.
Brakhage, C. Hertweck, Nat. Chem. Biol. 2007, 3, 213.
[11] D. L. Boger, J. C. Zhou, J. Am. Chem. Soc. 1993, 115, 11426.
[12] W. A. Ayer, P. P. Singer, Phytochemistry 1980, 19, 2717.
[20] a) D. C. Eastwood, et al., Science 2011, 333, 762; b) A. Olson,
et al., New Phytol. 2012, 194, 1001.
[21] K. E. Bushley, B. G. Turgeon, BMC Evol. Biol. 2010, 10, 26.
1594
www.angewandte.org
ꢀ 2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2013, 52, 1590 –1594