The molecular basis of conjugated polyyne biosynthesis in phytopathogenic bacteria

Article abstract of DOI:10.1002/anie.201403344

Polyynes (polyacetylenes), which are produced by a variety of organisms, play important roles in ecology. Whereas alkyne biosynthesis in plants, fungi, and insects has been studied, the biogenetic origin of highly unstable bacterial polyynes has remained a riddle. Transposon mutagenesis and genome sequencing unveiled the caryoynencin (cay) biosynthesis gene cluster in the plant pathogen B. caryophylli, and homologous gene clusters were found in various other bacteria by comparative genomics. Gene inactivation and phylogenetic analyses revealed that novel desaturase/acetylenase genes mediate bacterial polyyne assembly. A cytochrome P450 monooxygenase is involved in the formation of the allylic alcohol moiety, as evidenced by analysis of a fragile intermediate, which was stabilized by an in situ click reaction. This work not only grants first insight into bacterial polyyne biosynthesis but also demonstrates that the click reaction can be employed to trap fragile polyynes from crude mixtures. Triple trouble: The genes for a bacterial polyyne biosynthesis pathway were discovered in the plant pathogen Burkholderia caryophylli. Caryoynencin biosynthesis involves bacteria-specific desaturases/acetylenases and a cytochrome P450 monooxygenase. Highly fragile polyynes were successfully trapped and isolated from a crude metabolite mixture by an in situ click reaction. Homologous polyyne gene clusters were found in various bacterial genomes.

Full text of DOI:10.1002/anie.201403344

Angewandte
Chemie
DOI: 10.1002/anie.201403344
Biosynthesis
The Molecular Basis of Conjugated Polyyne Biosynthesis in
Phytopathogenic Bacteria**
Claudia Ross, Kirstin Scherlach, Florian Kloss, and Christian Hertweck*
Abstract: Polyynes (polyacetylenes), which are produced by
a variety of organisms, play important roles in ecology.
Whereas alkyne biosynthesis in plants, fungi, and insects has
been studied, the biogenetic origin of highly unstable bacterial
polyynes has remained a riddle. Transposon mutagenesis and
genome sequencing unveiled the caryoynencin (cay) biosyn-
thesis gene cluster in the plant pathogen B. caryophylli, and
homologous gene clusters were found in various other bacteria
by comparative genomics. Gene inactivation and phylogenetic
analyses revealed that novel desaturase/acetylenase genes
mediate bacterial polyyne assembly. A cytochrome P450
monooxygenase is involved in the formation of the allylic
alcohol moiety, as evidenced by analysis of a fragile inter-
mediate, which was stabilized by an in situ click reaction. This
work not only grants first insight into bacterial polyyne
biosynthesis but also demonstrates that the click reaction can
be employed to trap fragile polyynes from crude mixtures.
Whereas many polyacetylenes have been characterized,
especially from plants, basidiomycetes, and insects,[2,3c,5]
polyynes are a rarity among bacterial metabolites. The very
few partially characterized bacterial polyynes, such as Sch
31828 (1) from Microbispora sp.,[4c] the cepacins (2) from the
human pathogen Burkholderia cepacia,[4a] and the antibacte-
rial compound caryoynencin (3) from the carnation pathogen
Burkholderia caryophylli,[4b] possess a terminal alkyne func-
tion (Figure 1A). Because of their high instability, synthetic
P
olyynes are structurally intriguing molecules with alternat-
ing triple and single carbon–carbon bonds and they have
attracted considerable interest in organic chemistry because
of their unique rod-like architectures and high reactivity.[1]
Interestingly, various polyynes have also been found in
nature. Often imprecisely referred to as polyacetylenes,
these compounds constitute a family of natural products
from a variety of organisms, including plants, algae, insects,
lichen, fungi, and bacteria.[2] Naturally occurring polyynes
often have potent biological activities and are of ecological
relevance to the producing organism.[2,3] The unique molec-
ular scaffold of multiple conjugated triple bonds is usually
essential for bioactivity, yet these molecules are highly
unstable and their characterization poses a major challenge.[4]
In fact, numerous synthetic and natural polyynes undergo
exothermic crosslinking reactions and many polyyne natural
products are even considered to be explosives that need to be
handled with caution.
Figure 1. A) Structures of selected bacterial polyynes; Sch 31828 (1)
from Microbispora sp., cepacin (2) from Burkholderia cepacia, and
caryoynencin (3) from Burkholderia caryophylli. B) Sequential desatura-
tion of fatty acids by plant-derived desaturases (acetylenases for triple
bonds).
approaches to derivatives of 3 were developed to gain more
insight into their structure and function, and the results
indicate that the intact polyyne, in particular the terminal
triple bond, seems to be crucial for bioactivity.[6] Notably,
caryoynencin (3) is the only known bacterial polyyne to
possess four conjugated triple bonds; it thus represents an
intriguing molecule for studying the biogenetic origin of this
rare pharmacophore. In natural products, alkynes are typi-
cally formed by stepwise O2-dependent dehydrogenation of
aliphatic chains in a process catalyzed by di-iron enzymes
named desaturases (or acetylenases for triple bond forma-
tion; dual functions have been observed).[7] Compared to the
fairly high number of known polyynes, there have been only
few reports to date on plant-derived desaturases that mediate
the progressive desaturation of fatty acids into polyacety-
[*] C. Ross, Dr. K. Scherlach, F. Kloss, Prof. Dr. C. Hertweck
Department of Biomolecular Chemistry, Leibniz Institute for
Natural Product Research and Infection Biology, HKI
Beutenbergstrasse 11a, 07745 Jena (Germany)
E-mail: Christian.hertweck@hki-jena.de
Prof. Dr. C. Hertweck
Chair of Natural Product Chemistry, Friedrich Schiller University
Jena (Germany)
[**] We thank A. Perner and H. Heinecke for MS and NMR measure-
ments, and B. Urbansky and C. Karkowski for general lab assistance.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201403344.
Angew. Chem. Int. Ed. 2014, 53, 1 – 6
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
1
These are not the final page numbers!

.
Angewandte
Communications
lenes[2,8] (Figure 1B). A set of desaturases that evolved
independently of their plant congeners was shown to be
involved in alkyne formation in insects.[5b] In the fungus
Cantharellus formosus, a bifunctional desaturase as well as an
acetylenase, which is phylogenetically distinct from known
plant and fungal desaturases, were reported to catalyze alkyne
formation.[9] By contrast, the biogenetic origin of polyacety-
lenes in prokaryotes has remained completely enigmatic.
Herein, we report the discovery of the first bacterial gene
cluster coding for the biosynthesis of a polyyne, namely
caryoynencin in Burkholderia spp., and this discovery led to
the identification of numerous related clusters in other
bacteria. We also show that specialized desaturases and
a cytochrome P450 monooxygenase are involved in the
formation of the diene-tetrayne compound. Moreover, we
describe the application of a chemical trapping approach as
a strategy for isolating and characterizing these highly
unstable molecules directly from the fermentation broth
extract.
To identify the genes involved in the biosynthesis of the
unusual polyyne metabolite, we performed a random trans-
poson mutagenesis of the caryoynencin producer Burkholde-
ria caryophylli (DSM50341). We hypothesized that disruption
of caryoynencin biosynthesis genes would affect the pheno-
type. First, a null mutant could be detected by using an
antibacterial plate assay with B. subtilis as a sensitive test
strain (Figure 2A, trace I). Second, we reasoned that a visual
mutant screening would be viable since B. caryophylli
produces high amounts of polyynes even on agar plates and
these compounds tend to polymerize to form dark brown
polymerization products,[4b,6] thus resulting in brownish col-
onies. In the mutant library, we noticed one clone that
displayed white colonies instead of the usual brownish
phenotype (Figure 2B). Analysis of secondary metabolite
formation and a negative B. subtilis plate assay revealed that
caryoynencin production was fully abolished in the mutant
strain (Figure 2A, trace II), thus implying that a gene puta-
tively involved in polyyne biosynthesis had been disrupted by
the transposon. To locate its insertion site, we performed
transposon rescue. Sequence mapping of the transposon
insertion site revealed integration within a gene that shows
homology to D9 desaturases (Figure 3, clade II), which
catalyze the oxidation of stearic acid to oleic acid, for
example, and are involved in the formation of polyunsatura-
ted fatty acids.[2,10] A Blast homology search pointed to
orthologous genes in the genomes of the plant pathogens
Burkholderia gladioli BSR3[11] and Burkholderia gladioli pv.
cocovenenans.[12] To investigate whether B. gladioli is also able
to produce polyacetylenes, we analyzed its secondary metab-
olome by LC–MS. Indeed, after optimization of the media
and culture conditions, we could show for the first time that B.
gladioli produces a polyyne derivative as indicated by the
characteristic UV spectrum. LC–HRESI-MS, MS/MS, and co-
elution experiments with an authentic standard unequivocally
identified the polyacetylene as caryoynencin (see the Sup-
porting Information). With two caryoynencin producers in
hand, we aimed at comparative genomics analyses. Therefore,
we submitted genomic DNA from Burkholderia caryophylli
for 454 shotgun sequencing, and by matching the genome
Figure 2. A) HPLC analyses (PDA 200–800 nm) of microbial cultures
and bioactivity of the culture extracts against Bacillus subtilis: I) B.
caryophylli wild type, II) B. caryophylli TM1, III) B. caryophylli DcayB/C,
IV) B. caryophylli DcayG. B) Transposon mutagenesis and targeted
gene knockouts. The plate shows the results of in vivo mutagenesis of
B. caryophylli, including a white phenotype mutant (TM1). Shown
below are the phenotypes of targeted knockouts of cayB/C (a, white
phenotype) and cayG (b, yellow phenotype) compared to the wild type
(c). C) The organization of the caryoynencin biosynthesis gene cluster
in B. caryophylli (Bc) and comparative genomics among Burkholderia
spp. (amino acid sequence similarities computed by BlastP). Bg=Bur-
kholderia gladioli pv. cocovenenans, Bg’= Burkholderia gladioli BSR3. The
black pin indicates the site of transposon integration.
Figure 3. A phylogenetic tree (neighbor joining algorithm) of desatur-
ases/acetylenases from plants (green), fungi (brown), insects
(orange), and bacteria (blue). Caryoynencin gene products are high-
lighted in bold, triple-bond introducing enzymes from studied polyyne
pathways are marked with an asterisk (*), and desaturases from
cryptic bacterial polyyne gene clusters are indicated with a hash (#).
The outgroup is marked with a dashed line (for a detailed tree, see the
Supporting Information).
2
www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 1 – 6
These are not the final page numbers!

Angewandte
Chemie
data, we found that the genes that putatively code for the
desaturases are located within a conserved gene cluster.
Besides three desaturase genes (cayB, cayC, and cayE), we
identified genes encoding a fatty acyl-AMP ligase (cayA),
a hydrolase (cayF), and a cytochrome P450 monooxygenase
(cayG). Additional genes for transport and efflux (cayHIJ)
were found downstream of the biosynthesis genes (Figure 2C,
and Table S1 and Figure S3 in the Supporting Information).
A phylogenetic analysis including the deduced cay gene
products and characterized and tentative desaturases (acety-
lenases) from plant, insect, and microbial pathways revealed
that the bacterial enzymes are clearly distinct from their
eukaryotic counterparts, thus suggesting an independent
evolution (Figure 3). Notably, CayB and CayC form a sepa-
rate clade with the closest homologue JamB, which has been
suggested to mediate alkyne formation in the jamaicamide
pathway of Lyngbya majuscula.[13] CayB and CayC are thus
the best candidates for enzymes involved in triple-bond
formation. To confirm their involvement in caryoynencin
biosynthesis, we created a targeted knockout of the predicted
desaturase (acetylenase) genes cayB and cayC through
insertion of a kanamycin resistance cassette. Metabolic
profiling of the resultant mutant strain revealed that polyyne
formation was fully abolished (Figure 2A, trace III). Further-
more, the mutant completely lost its antibacterial potency,
another indication for loss of the polyyne residue. These
findings indicate that the disrupted genes are crucial for
alkyne formation. Given that no enrichment of unusual
unsaturated fatty acid precursors could be detected, it appears
that the endogenous fatty acid pool is tapped for the
formation of the polyynes. Furthermore, since a ligase
(CayA), an acyl carrier protein (ACP, CayD) and a hydrolase
(CayF) are encoded in the cay gene cluster, the fatty acids are
likely ACP-bound during desaturation, thus preventing the
detection of intermediates in the broth.
caryoynencin (3) disappeared with formation of a new com-
pound (5b) that matches the expected mass of the proposed
triazole (Figure 4). By performing preparative HPLC, we
were able to purify 5b, which was sufficiently stable to be fully
characterized. 1D and 2D NMR spectra were in full agree-
ment with the proposed structure. Moreover, we succeeded in
elucidating the absolute configuration of the hydroxymethine
In order to provide additional functional evidence for the
identity of the genes belonging to the cay biosynthesis gene
cluster, we inactivated cayG, the putative cytochrome P450
monooxygenase gene, through insertional disruption. HPLC-
DAD–HRESI-MS analysis of the DcayG mutant extract
showed the accumulation of two metabolites, with m/z =
265.1238 [MÀH]À (calcd. for C18H17O2: 265.1234; 4) and m/
z = 285.1499 [MÀH]À (calcd. for C18H21O3: 285.1496; 7) as
shown in Figure 2A, trace IV. Both compounds display
a shifted UV spectrum, which indicates a change in the
degree of unsaturation of the fatty acid backbone.
To fully elucidate their structures, we attempted the
challenging isolation of the fragile polyyne intermediates.
Typically, the inherent instability of polyynes with a terminal
alkyne moiety prevents their isolation and purification. To
overcome this hurdle, we intended to chemically trap the
polyynes. Derivatization of the terminal alkyne by CuI-
catalyzed azide–alkyne cycloaddition (CuAAC, “click reac-
tion”)[14] appeared to be most promising. As a proof of
principle, we selectively trapped caryoynencin from the crude
fermentation extract of the wild type Burkholderia caryo-
phylli. Using a protocol that selectively targets terminal
alkynes,[15] we treated the crude mixture with benzyl azide and
the CuI catalyst. LC–MS analysis of the mixture revealed that
Figure 4. Chemical trapping of the tetraynes by an in situ click reaction
with benzyl azide. Shown are a schematic representation of the general
approach and HPLC profiles of the culture extracts (PDA 200–
800 nm): I) B. caryophylli wild type, II) B. caryophylli wild type subse-
quent to click reaction, III) B. caryophylli DcayG, and IV) B. caryophylli
DcayG subsequent to click reaction. Shown below are the structures of
7 and triazoles 5a,b and 6, and the inferred structures of 3a,b and 4.
DMF=N,N-dimethylformamide.
group (6S) by using the Mosher method (see the Supporting
Information).
Since the polyyne click method proved to be viable for
stabilizing the polyyne residue, we used the same approach
for the DcayG mutant. The intermediate (4) was derivatized
in situ, which led to the formation of a new compound (6) at
the expense of 4. The triazole was isolated, purified, and fully
characterized by NMR spectroscopy. Compared to 5b, the
lack of two olefinic methine signals pointed to the reduction
of a double bond. Furthermore, the 1H NMR data supported
the idea that compound 6 is not hydroxylated. In agreement
with the HRESI-MS and 2D NMR data for 6, metabolite 4
features an alkyl chain instead of the allylic alcohol motif,
while the tetraalkyne-ene residue remained congruent
Angew. Chem. Int. Ed. 2014, 53, 1 – 6
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
3
These are not the final page numbers!

.
Angewandte
Communications
(Figure 4). By contrast, compound 7 was not susceptible to
derivatization by benzyl azide, which indicates the absence of
a terminal alkyne moiety. By using preparative HPLC, we
achieved the isolation of trace amounts of this compound,
which finally allowed its structural elucidation. 13C NMR
analysis clearly revealed that the terminal triple bond was
missing. Instead, the presence of a methyl carbon atom and an
additional methine carbon atom was noticed; a chemical shift
of d = 56.7 ppm for C-17 pointed to proximity to an oxygen
atom. H,H-COSY coupling of the methyl protons H-18 and
H-17 and HMBC coupling of H-18 and H-17 with C-16 and C-
posed as defensive chemicals, since most polyynes possess
strong antimicrobial properties.[2,5b] The discovery of the
molecular basis of polyyne biosynthesis in bacteria will now
help in the identification of further bacterial polyacetylenes
by genome mining and may thus lead to a better under-
standing of their ecological function.
In conclusion, we have identified the first biosynthesis
gene cluster coding for a polyyne biosynthetic pathway in
bacteria. A crucial role for novel desaturase genes in bacterial
polyyne formation was revealed, and these enzymes seem to
have evolved independent of the equivalent genes known
from plant metabolism. Bacterial polyyne pathways appear to
be rare but comparative genome analyses unveiled numerous
orthologues in diverse bacteria, and through metabolic
profiling, we verified that the important phytopathogen B.
gladioli produces caryoynencin too. Knowledge of the
molecular basis of bacterial polyyne biosynthesis and the
discovery of several polyyne biosynthesis gene clusters will
greatly aid functional studies and set the basis for the
genomics-based discovery of new bacterial polyynes. To
tackle the challenging handling of fragile polyynes, we
successfully employed an in situ click reaction. Although
Huisgen azide-alkyne cycloadditions have been used for the
chemical derivatization of biomolecules and their chemical
derivatives,[21] this is the first report of the successful trapping
of highly reactive polyynes from the crude metabolite mixture
of a microbial culture. This generally applicable approach
enabled the isolation and full characterization of the polyyne
natural products, thus providing spectral data that is other-
wise difficult or impossible to retrieve. As a proof of concept,
we could rigorously determine the structures of a tetrayne
pathway intermediate from a targeted knockout mutant
(DcayG) and elucidate the absolute configuration of caryoy-
nencin. Furthermore, analysis of the metabolites of the
mutant revealed that a cytochrome P450 monooxygenase
plays a key role in the formation of the allylic alcohol residue
of caryoynencin and that the presence of a terminal alkyne
residue is not crucial for bioactivity. These results thus not
only shed light on bacterial polyyne biosynthesis but also
demonstrate the power of combining synthetic methods and
genetics to analyze the biosynthesis of highly reactive path-
way intermediates.
15, respectively, finally established the structure of
7
(Figure 4). The discovery of an alcohol residue in lieu of
a triple bond can be rationalized by the mechanistic link
between hydroxylation and desaturation.[16] Alcohol 7 thus
likely represents a shunt product of terminal alkyne forma-
tion.
It is remarkable that the deletion of the cytochrome P450
enzyme (CayG) results in the accumulation of compounds
lacking not only the hydroxy group but also one double bond.
To test whether CayG could belong to the family of CYP450
desaturases, we performed a phylogenetic analysis including
representatives from secondary metabolite pathways (see the
Supporting Information). CayG is most closely related to
polyketide tailoring enzymes, in particular to the multifunc-
tional CYP450 monooxygenase AurH[17] from the aureothin
pathway, and it is only remotely related to studied CYP450
desaturases from plants and yeasts. However, in geldanamy-
cin biosynthesis, deletion of a CYP450 monooxygenase gene
(gdmP) leads to the accumulation of a dihydro variant of
geldanamycin, which suggests that GdmP acts as a desatur-
ase.[18] By analogy, CayG could introduce the double bond by
desaturation or through
a
hydroxylation–dehydration
sequence (potentially leading to the observed E/Z-isomers),
followed by hydroxylation at the allylic position (Scheme S1).
This model is in agreement with reported cases of competing
desaturation and hydroxylation by CYP450 enzymes.[19]
In terms of bioactivity, it is surprising that 5b, in which the
terminal alkyne was converted into a triazole moiety, is still
active in antibacterial assays (minimum inhibitory concen-
tration (MIC): B. subtilis 3.12 mgmLÀ1, 7.6 mm; MRSA
12.5 mgmLÀ1, 30.3 mm). This finding contrasts with earlier
studies on structure–activity relationships, carried out with
synthetic model compounds, which suggested that the termi-
nal alkyne is indispensable for the antibacterial activity.[6]
The discovery of the novel polyyne biosynthesis gene
cluster in three plant-pathogenic Burkholderia spp. raises the
question of how widespread related gene clusters are amongst
other bacteria. In a broader genome-mining analysis, we
found six orthologous gene clusters in other bacterial species
from the genera Burkholderia, Pseudomonas, Collimonas,
and Mycobacterium (Figure S3). This result is particularly
interesting since many Burkholderiales and Pseudomonales
live in close association with eukaryotes and their metabolic
capabilities are often essential for the eukaryotic partner.[20]
The potent antibacterial activity of 3 and the specific
occurrence of polyyne gene clusters among these genera
may suggest an ecological function for the producing organ-
isms. In plants and insects, polyacetylenes have been pro-
Received: March 14, 2014
Published online: && &&, &&&&
Keywords: biosynthesis · caryoynencin · click chemistry ·
.
polyynes · transposon mutagenesis
[1] a) M. Brøndsted Nielsen, F. Diederich, Chem. Rec. 2002, 2, 189 –
198; b) A. L. Shi Shun, R. R. Tykwinski, Angew. Chem. 2006,
118, 1050 – 1073; Angew. Chem. Int. Ed. 2006, 45, 1034 – 1057.
[2] R. E. Minto, B. J. Blacklock, Prog. Lipid Res. 2008, 47, 233 – 306.
[3] a) L. P. Christensen, K. Brandt, J. Pharm. Biomed. Anal. 2006,
41, 683 – 693; b) V. M. Dembitsky, Lipids 2006, 41, 883 – 924;
c) D. V. Kuklev, A. J. Domb, V. M. Dembitsky, Phytomedicine
2013, 20, 1145 – 1159.
[4] a) W. L. Parker, M. L. Rathnum, V. Seiner, W. H. Trejo, P. A.
Principe, R. B. Sykes, J. Antibiot. 1984, 37, 431 – 440; b) T.
4
www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 1 – 6
These are not the final page numbers!

Angewandte
Chemie
Kusumi, I. Ohtani, K. Nishiyama, H. Kakisawa, Tetrahedron
Lett. 1987, 28, 3981 – 3984; c) M. Patel, M. Conover, A. Horan,
D. Loebenberg, J. Marquez, R. Mierzwa, M. S. Puar, R. Yar-
borough, J. A. Waitz, J. Antibiot. 1988, 41, 794 – 797.
[5] a) I. W. Farrell, J. W. Keeping, M. G. Pellatt, V. Thaller, J. Chem.
Soc. Perkin Trans. 1 1973, 2642 – 2643; b) V. S. Haritos, I. Horne,
K. Damcevski, K. Glover, N. Gibb, S. Okada, M. Hamberg, Nat.
Commun. 2012, 3, 1150.
817 – 833; b) A. C. Jones, L. Gerwick, D. Gonzalez, P. C.
Dorrestein, W. H. Gerwick, BMC Microbiol. 2009, 9, 247.
[14] a) V. V. Rostovtsev, L. G. Green, V. V. Fokin, K. B. Sharpless,
Angew. Chem. 2002, 114, 2708 – 2711; Angew. Chem. Int. Ed.
2002, 41, 2596 – 2599; b) C. W. Tornøe, C. Christensen, M.
Meldal, J. Org. Chem. 2002, 67, 3057 – 3064.
[15] T. Luu, R. McDonald, R. R. Tykwinski, Org. Lett. 2006, 8, 6035 –
6038.
[6] M. Yamaguchi, H. J. Park, S. Ishizuka, K. Omata, M. Hirama, J.
Med. Chem. 1995, 38, 5015 – 5022.
[16] J. A. Broadwater, E. Whittle, J. Shanklin, J. Biol. Chem. 2002,
277, 15613 – 15620.
[7] J. Shanklin, J. E. Guy, G. Mishra, Y. Lindqvist, J. Biol. Chem.
2009, 284, 18559 – 18563.
[8] S. J. Gagnꢀ, D. W. Reed, G. R. Gray, P. S. Covello, Biochemistry
2009, 48, 12298 – 12304.
[9] B. J. Blacklock, B. E. Scheffler, M. R. Shepard, N. Jayasuriya,
R. E. Minto, J. Biol. Chem. 2010, 285, 28442 – 28449.
[10] B. Behrouzian, P. H. Buist, Prostaglandins Leukotrienes Essent.
Fatty Acids 2003, 68, 107 – 112.
[11] Y. S. Seo, J. Lim, B. S. Choi, H. Kim, E. Goo, B. Lee, J. S. Lim,
I. Y. Choi, J. S. Moon, J. Kim, I. Hwang, J. Bacteriol. 2011, 193,
3149.
[12] a) N. X. Zhao, M. S. Ma, Y. P. Zhang, D. C. Xu, Int. J. Syst.
Bacteriol. 1990, 40, 452 – 455; b) N. Moebius, C. Ross, K.
Scherlach, B. Rohm, M. Roth, C. Hertweck, Chem. Biol. 2012,
19, 1164 – 1674.
[13] a) D. J. Edwards, B. L. Marquez, L. M. Nogle, K. McPhail, D. E.
Goeger, M. A. Roberts, W. H. Gerwick, Chem. Biol. 2004, 11,
[17] a) M. E. A. Richter, N. Traitcheva, U. Knꢁpfer, C. Hertweck,
Angew. Chem. 2008, 120, 9004 – 9007; Angew. Chem. Int. Ed.
2008, 47, 8872 – 8875; b) G. Zocher, M. E. Richter, U. Mueller, C.
Hertweck, J. Am. Chem. Soc. 2011, 133, 2292 – 2302.
[18] T. Li, S. Ni, C. Jia, H. Wang, G. Sun, L. Wu, M. Gan, G. Shan, W.
He, L. Lin, H. Zhou, Y. Wang, J. Nat. Prod. 2012, 75, 1480 – 1484.
[19] S. G. Bell, R. Zhou, W. Yang, A. B. Tan, A. S. Gentleman, L. L.
Wong, W. Zhou, Chem. Eur. J. 2012, 18, 16677 – 16688.
[20] a) S. J. Pidot, S. Coyne, F. Kloss, C. Hertweck, Int. J. Med.
Microbiol. 2014, 304, 14 – 22; b) K. Scherlach, K. Graupner, C.
Hertweck, Annu. Rev. Microbiol. 2013, 67, 375 – 397.
[21] a) S. G. Agalave, S. R. Maujan, V. S. Pore, Chem. Asian J. 2011, 6,
2696 – 2718; b) E. Lallana, R. Riguera, E. Fernandez-Megia,
Angew. Chem. 2011, 123, 8956 – 8966; Angew. Chem. Int. Ed.
2011, 50, 8794 – 8804; c) D. M. Beal, L. H. Jones, Angew. Chem.
2012, 124, 6426 – 6432; Angew. Chem. Int. Ed. 2012, 51, 6320 –
6326.
Angew. Chem. Int. Ed. 2014, 53, 1 – 6
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.angewandte.org
5
These are not the final page numbers!

.
Angewandte
Communications
Communications
Biosynthesis
Triple trouble: The genes for a bacterial
polyyne biosynthesis pathway were dis-
covered in the plant pathogen Burkholde-
ria caryophylli. Caryoynencin biosynthesis
involves bacteria-specific desaturases/
acetylenases and a cytochrome P450
monooxygenase. Highly fragile polyynes
were successfully trapped and isolated
from a crude metabolite mixture by an
in situ click reaction. Homologous poly-
yne gene clusters were found in various
bacterial genomes.
C. Ross, K. Scherlach, F. Kloss,
C. Hertweck*
&&&& — &&&&
The Molecular Basis of Conjugated
Polyyne Biosynthesis in Phytopathogenic
Bacteria
6
www.angewandte.org
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Angew. Chem. Int. Ed. 2014, 53, 1 – 6
These are not the final page numbers!