DOI: 10.1002/cbic.201200598

Nonribosomal Assembly of Natural LipocyclocarbamateLipoprotein-Associated Phospholipase InhibitorsChad W. Johnston, Rostyslav Zvanych, Nadiya Khyzha, and Nathan A. Magarvey*[a]

Lipocyclocarbamate natural products provided the inspirationfor the first-in-class synthetic phospholipase inhibitor darapla-dib, currently in phase III clinical trials for the treatment ofatherosclerosis. The natural lipocyclocarbamates SB-253514,SB-311009, and SB-315021 possess bicyclic ring systems inte-gral to their action (Figure 1). Genome sequencing and biosyn-

thetic analysis based on TE domain phylogeny afforded thecandidate nonribosomal peptide synthetase (NRPS) biosynthet-ic gene cluster. Confirmation of the nonribosomal origin ofthese molecules was confirmed by insertional inactivation ofthe corresponding NRPS, and the proposed biosynthesis ofthese nanomolar inhibitors of Lp-PLA2 is presented.

Lipoprotein-associated phospholipase A2 (Lp-PLA2) causesinflammation in atherosclerotic plaques and is a novel targetfor the treatment of atherosclerosis.[1] Early target-basedscreening campaigns of natural product extracts (34 000) atSmithKline Beecham against Lp-PLA2 identified a collection ofnatural cyclocarbamate compounds from a single organism,Pseudomonas fluorescens DSM 11579.[2, 3] Two classes of lipocy-clocarbamate compound were identified, one possessing a 5,5-the other a 5,7-fused bicyclic ring system, and both exhibitedselective inhibition of Lp-PLA2. These compounds were the in-spiration for the synthetic agent darapladib,[4, 5] which is now inphase III clinical trials as a first-in-class treatment for atheroscle-rosis.[6] Synthetic modifications to microbial natural productshave been critical in advancing their clinical use for treatingheart disease, with polyketide-derived statins providing an ex-cellent example.[7] The selectivity of natural product scaffoldshas been instilled through evolution, and the discovery ofnovel enzymes and chemical transformations associated withtheir assembly offers promise for chemoenzymatic approachesand diversity-oriented synthesis programs. Although the genesand enzymes responsible for natural cyclocarbamate Lp-PLA2

inhibitors are not known, identification of the likely nonriboso-mal peptide biosynthetic gene cluster will be important toinform the advancement of next-generation Lp-PLA2 inhibitors.

Pseudomonads are prolific producers of natural products,and many classes of compound, arising from highly homolo-gous biosynthetic gene clusters, are common within thisfamily.[8–10] For instance, specific types of lipopeptides (e.g. , sy-ringomycin) and siderophores (e.g. , pyoverdine) are created bynonribosomal synthetases (NRPSs) that have high levels ofsequence homology between enzymatic domains (e.g. , C, A,TE).[8–10] Common NRPS and polyketide synthase (PKS) geneclusters have also been used to classify Pseudomonas strainswith specific niche adaptations, such as plant commensals (rhi-zoxin) or plant pathogens (coronatine).[11–13] Diagnostic genecassettes and fragments are also used as markers to delineatebiosynthetic modes and programs, such as trans-acting acyl-transferase PKSs,[14] bifunctional epimerase/condensation NRPSdomains,[15] and fused sulfotransferase/thioesterase domainsthat create terminal olefins within metabolite end products.[16]

Thioesterase (TE) domains seemingly have an association withsmall-molecule products,[17–19] whereby TE sequences providedifferentiating value in connecting genes to small-moleculeproducts. Such information is useful in genomic and metage-nomic analysis to connect randomly sequenced NRPS and PKSgenes to small-molecule production capability. In pseudomo-nads, this value has been proven by the identification of thegenes for rhizoxin and pederin.[11, 12, 20] Here, we used a ge-nome-wide analysis of pseudomonad TE domains and inser-

Figure 1. Natural lipocyclocarbamates and their synthetic derivative darapla-dib inhibit Lp-PLA2 and limit leukocyte recruitment and atherosclerotic in-flammation.

[a] C. W. Johnston, R. Zvanych, N. Khyzha, Prof. N. A. MagarveyDepartment of Chemistry and Chemical Biology andDepartment of Biochemistry and Biomedical SciencesM. G. DeGroote Institute for Infectious Disease ResearchMcMaster University1280 Main Street West, Hamilton, Ontario, L8N 3Z5 (Canada)E-mail : magarv@mcamaster.ca

Supporting information for this article is available on the WWW underhttp ://dx.doi.org/10.1002/cbic.201200598.

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tional-gene-inactivation studies to reveal the cluster of genesassociated with the natural lipocyclocarbamate Lp-PLA2 inhibi-tors. This lipocyclocarbamate NRPS cluster provides key infor-mation on how nature constructs these low-nanomolar inhibi-tors of Lp-PLA2.

The lipocyclocarbamate producer P. fluorescens DSM 11579was subjected to Illumina whole-genome sequencing and as-sembled by using the ABySS genome-assembly program andGeneious bioinformatic software.[21] Based on the chemical ar-chitecture of the lipocyclocarbamate, a bimodular NRPS assem-bly can be inferred, along with genes for rhamnosyl transferand carbamate formation. Homology search analysis of theassembled genomic contigs revealed at least six independentNRPS clusters. To classify these gene clusters and their prod-ucts in terms of known pseudomonad chemistry, their TE do-mains were compared through primary sequence alignmentsby using the amino acid sequences associated with the TE do-mains involved in elaborating known pseudomonad secondarymetabolites, including pyoverdine, pyochelin, coronatine, andrhizoxin (Figure 2 A). Additionally, NRPS and PKS TE domainsfrom the genomes of other Pseudomonas spp. that did notproduce lipocyclocarbamates were included to assist in dis-cerning the candidate lipocyclocarbamate TE from P. fluores-cens DSM 11579. A phylogenetic tree was constructed fromthe multiple TE domain sequence alignments; its brancheswere seen to sort according to the class of secondary metabo-lite, regardless of the organism of origin. From this tree, three

P. fluorescens DSM 11579 NRPS gene clusters were seen togroup with TE domains associated with large lipopeptides,[8, 9]

including two that contained tandem thioesterase domainscommonly observed in such pathways. One P. fluorescens DSM11579 TE domain grouped with pyoverdine TEs, particularlywith a candidate pyoverdine cluster from Pseudomonas brassi-cacearum strain NFM421.[22] Another TE domain sorted witha P. brassicacearum strain NFM421 cluster that appears toencode an uncharacterized siderophore, as evidenced by flank-ing genes involved in iron metabolism machinery and sidero-phore uptake. Apart from these common TE-encoding biosyn-thetic clusters, one P. fluorescens DSM 11579 TE domain sortedas a distinct branch, and was found to associate with a uniquebimodular NRPS gene cluster. This 9.8 kb gene cluster alsoincluded genes for a flavin-dependent monooxygenase (71 %identical and 81 % similar) and a rhamnosyltransferase withhigh sequence homology to RhlB (45 % identical and 61 % sim-ilar to RhlB, Pseudomonas aeruginosa PAO1), which is involvedin rhamnolipid creation in Burkholderia and Pseudomonas spe-cies. Interestingly, this gene cluster was flanked on either sideby genes homologous to those at the end of the syringomycingene cluster, on one side by the NRPS encoded by SyrE (77 %identical and 87 % similar) and on the other by the transcrip-tion regulator SyrF (69 % identical and 81 % similar) and anassociated NodT outer-membrane protein (82 % identical and89 % similar) (Figure 2 B), thus suggesting the candidate lipocy-clocarbamate bimodular NRPS cluster had inserted within

a pre-existing syringomycin-like biosynthetic genecluster.[23] This insertion is further supported by thepresence of noncoding sequences (NCS) at the boun-daries of the candidate lipocyclocarbamate clusterwith no homology to pseudomonad DNA and havinga percentage GC content that is inconsistent with therest of the genome and the predicted biosyntheticgenes.

Given the consistency of this unique gene clusterwith respect to the biosynthesis of the Lp-PLA2 inhib-itors, including a phylogenetically distinct TE domain,adenylation domains for activating proline and serine(possibly leading to a dehydroalanine; Tables S1 andS2 in the Supporting Information), along with a rham-nosyltransferase, we created an insertionally inactivat-ed lpiB NRPS gene with a chloramphenicol resistancecassette (cat) to confirm its role in lipocyclocarba-mate biosynthesis. The plasmid used for creating theinactive NRPS gene consisted of a 2 kb fragment oflpiB within pBlueScript KSII with a chloramphenicolresistance cassette inserted within an internal HindIIIrestriction site (Figure 3 A). This gene inactivationvector was introduced to wild-type P. fluorescens DSM11579 by electroporation, and a double crossovermutant was identified through chloramphenicol re-sistance and confirmed by PCR for genomic integra-tion. Comparing the metabolites produced by P. fluo-rescens DSM 11579 wild-type and the DlpiB strainshowed that the mutant had lost the ability to pro-duce lipocyclocarbamate compounds (Figure 3 B).

Figure 2. Thioesterase domain alignment identifies a unique bimodular NRPS gene clus-ter for lipocyclocarbamate biosynthesis. A) Thioesterase-based phylogeny of distinctpseudomonad NRPS- and PKS-derived secondary metabolites. B) P. fluorescens DSM11579 bimodular NRPS gene cluster candidate for lipocyclocarbamates. Colors of the ter-minal branches of the tree indicate the respective organisms in which the TE domainswere obtained. In red are the areas of significant homology flanking the candidate lipo-cyclocarbamate gene cluster with the known genes identified by syringomycin biosyn-thesis.

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Considering that the boundaries of the cluster aredelineated by flanking genes for another knownPseudomonas natural product, a proposed biosynthe-sis based on the available genes can be generated(Scheme 1). We propose that the synthesis of thelipocyclocarbamates is initiated by the condensationof CoA-bound 3-hydroxyoctanoic acid with serineloaded onto the first thiolation (T) domain, in a reac-tion catalyzed by the first condensation (C) domainof LpiB. The amino acylated product tethered to thefirst T domain would then be condensed with theproline of the second. We propose that, during thiscondensation, a second reaction leads to dehydrationof the serine, and this leads to a dehydroalanine-con-taining acylated dipeptide tethered to the secondT domain. Although precedent is lacking for the re-sulting ring closure, two reaction mechanisms maybe envisioned. The first—although less likely—possi-bility is a Diels–Alder cycloaddition reaction, wherebythe dehydroalanine exo-methylene side chain reactswith the keto-thioester. Although we cannot directlyevoke known TE-based catalytic logic in this process,evidence for the reaction has been postulated in thelinkage of diketopiperazine containing an exo-methyl-ene side chain serving as the nucleophilic compo-nent in an asynchronous cycloaddition with a ketonewithin the construction of the natural product varie-colortide.[24]

However, a more likely TE-based ring-closure mechanismwould be the migration of the lone pair of the enamine nitro-gen, which would, in effect, result in the b-carbon of the dehy-droalanine becoming nucleophilic ; this may be used by the TEto catalyze C�C bond formation with the thioester carbon. Al-though dehydroalanine side chains are typically electrophilic, itis possible that the above-mentioned enamine lone pair migra-tion arises, and that the TE is able to use the dehydroalanineb-carbon as a nucleophile for ring closure, leading to the aza-quinone species (Scheme 1). This would be analogous to TE-mediated nucleophilic attack from the b-carbon enolate carb-anion on the enzyme-tethered activated carbonyl group,which liberates terraquinones from their NRPS assembly linethrough C�C bond formation.[25] The tautomeric form of thequinone, 2-hydroxypyridone may then be acted upon by LpiC,the predicted flavin-dependent monooxygenase, which cata-lyzes a Baeyer–Villiger oxidative ring-expansion reaction lead-ing to the formation of a des-glyco SB-315021 5,7-fused ringsystem. Although Baeyer–Villiger ring expansions are some-what rare, monooxygenases are invoked in ring-expansion re-actions in the cases of mithramycin and urdamycin.[26, 27] The 3-hydroxy of the N-acyl chain could then be acted upon by theLpiA rhamnosyltransferase leading to the glycosylated prod-ucts (Scheme 1). Although the cluster does not encode en-zymes that would be responsible for the activation of rham-

Figure 3. Genetic inactivation of the lpiB NRPS and lpiC monooxygenasegenes confirms their involvement in lipocyclocarbamate biosynthesis. A) Aninsertional inactivation system was constructed to inactivate lpiB (and lpiC)through homologous recombination. B) Extracted cultures of wild-type,DlpiB, and DlpiC P. fluorescens DSM 11579 reveal that lpiB is responsible forlipocyclocarbamate biosynthesis.

Scheme 1. Proposed biosynthesis of the lipocyclocarbamate Lp-PLA2 inhibitors.

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nose, we were able to identify enzymes within the genomethat are analogous to those associated with activation of rham-nose as a nucleotide sugar (50 % identical and 62 % similar toRmlD, P. aeruginosa PAO1).[28] Interconversion of the 5,7 ringsystem with the 5,5 system observed in SB-253514 could beafforded by acid-enabled reversible allylic 1,3 transposition re-actions leading to a ring rearrangement.

To verify the proposed role of LpiC in lipocyclocarbamatebiosynthesis, an insertional inactivation strategy similar to theone used to inactive lpiB was employed. Double crossover andgenomic integration of the chloramphenicol resistance cas-sette into lpiC was confirmed by PCR (Figure S1), and DlpiCmutants were subsequently fermented, extracted, and ana-lyzed by LCMS. This inactivation completely abolished the ofthe production of the SB series of lipocyclocarbamates (Fig-ure 3 B). Subsequent principal component analysis of DlpiB andDlpiC extracts failed to detect any differences (Figure S2). IfLpiC’s role is exclusive to post-assembly-line action, one mayanticipate that an intermediate would accumulate, such as theproposed aza-quinones. Our inability to detect any differencesleads us to infer that the action of LpiC might include a stepprior to liberation from the assembly line. One suggestion con-sistent with our biosynthetic hypothesis is that it might cata-lyze a flavin-dependent dehydration of the serine. The minimalarchitecture of the lipocyclocarbamate gene cluster, includinglpiABC leads us to infer that LpiC acts more than once, and wetherefore suggest that it is likewise a Baeyer–Villiger monooxy-genase operating on the supposed aza-quinone species.

In conclusion, we present a unique biosynthetic cluster thatseemingly elicits new reactions associated with the NRPSmachinery and subsequent product tailoring. Included in theapparent logic associated with the lipocyclocarbamate naturalproducts is a TE that may use a b-carbon from an enamine asthe nucleophile to attack the thioester and afford a C�C bondring closure. Although C�C bond formation facilitated by a TEdomain has been observed in a select number of cases inwhich an enolate serves as the nucleophile,[25] in this case wecould observe different but analogous use of the TE by usingan enamine-led activation of a b-carbon of the exo-methyleneside chain of a dehydroalanine. Further implied in this highlyunusual natural pharmacophore construction is a plausibleBaeyer–Villiger ring expansion. The initial discoveries madehere in unveiling the biosynthetic cluster for lipocyclocarba-mate natural products open up natural routes to constructingnew analogues of these highly privileged scaffolds for nano-molar Lp-PLA2 inhibition. Moreover, the phylogenetic parsingof Pseudomonas TE domains along chemical scaffold linesmight also be useful with respect to identifying new chemis-tries by genome mining and perhaps specifically to revealother Lp-PLA2 inhibitors.

Experimental Section

Bacterial strains and culture conditions: P. fluorescens strain DSM11579 was ordered from the German Resource Centre for BiologicalMaterial (DSMZ). P. fluorescens strain DSM 11579 was cultured onLB agar plates at 37 8C. Strain identity was confirmed by 16S se-

quence alignment with 16S sequences that were amplified fromsingle colonies by using the 16S primers: 27f (5’-AGAGTT TGATCMTGGCTC AG-3’) and 1525r (5’-AAGGAG GTGATC CAGCC-3’).[29] Toproduce lipocyclocarbamates, P. fluorescens DSM 11579 was cul-tured in F12 production medium (20 % glucose, 1 % soy flour, 0.3 %corn steep liquor, 0.9 % (NH4)2SO4, 2 % CaCO3, 0.05 % MgSO4·7 H2O,0.325 % NaHPO4, pH 7.0, 28 8C).[2]

Genome sequencing: A single colony of P. fluorescens DSM 11579was grown overnight in lysogeny broth (LB; 3 mL) at 37 8C and250 rpm. Genomic DNA was harvested by using a GenElute Bacteri-al Genomic DNA Kit (Sigma). Genomic DNA was sent for librarypreparation and Illumina sequencing with an Illumina MiSeq DNAsequencer at the Farncombe Metagenomics Facility at McMasterUniversity. Contigs were assembled by using the ABySS genomeassembly program and with Geneious bioinformatic software.[21]

Assembly of the thioesterase domain phylogenetic tree: Thioes-terase domains from P. aeruginosa strain PA7, P. fluorescens strainSBW25, P. fluorescens strain Pf-5, P. brassicacearum strain NFM421,Pseudomonas putida strain KT2440, Pseudomonas syringae pv.tomato strain DC3000, and P. syringae pv. tomato strain K40 wereidentified by using the BLAST function from the Integrated Micro-bial Genomes database (http://img.jgi.doe.gov/). The thioesterasedomain of the PvdL NRPS was used as a query to identify thioes-terase domains directly. The PKS/NRPS protein PksJ was also usedas a query to identify all remaining PKS/NRPS proteins that mighthave thioesterase domains with low homology to the PvdL thioes-terase domain. To extract thioesterase sequences from PKS/NRPSproteins, domains were identified automatically by using the PKS/NRPS program, and Geneious software was used to constructa phylogenetic tree.[31] The tree alignment was performed by usingan identity matrix with a gap open penalty of 9 and a gap exten-sion penalty of 3. The tree construction used a Jukes–Cantor ge-netic distance model, and a Neighbor-Joining method was used toconstruct the tree without an out group.

Prediction of adenylation domain specificities: Adenylationdomain specificities for lpiB were determined using NRPS Predictoror PKS/NRPS programs,[31, 32] and the ten residue codes of eachentry and its top scoring hit were recorded (Table S1).[32] A collec-tion of adenylation domain codes for domains that activate serineor dehydroalanine is also provided (Table S2).

Construction of the DlpiB and DlpiC P. fluorescens DSM 11579strains: Knockout plasmids for P. fluorescens DSM 11579 were con-structed by inserting a 2 kb PCR product of lpiB (primers PFKOR3-ClaI : 5’-TTTTAT CGATGA TCCGAC TGTGCT CG-3’ and PFKOF2SacI:5’-TTTTGA GCTCGA CGTACT TTACCC GC-3’) or lpiC (primersLpiC2kbHindIIIF: 5’-TTTTAA GCTTCC AGGTCC ATCTCT ATG-3’ andLpiC2kbSacIR: 5’-TTTTGA GCTCTG CTTTTT CGGCGA TGG-3’) intopBlueScript KSII (+) by using ClaI/SacI, or HindIII/SacI restrictiondigest sites, respectively. Inserts were ligated into pBlueScript KSII(+) with T4 ligase, transformed into chemically competent DH5a

(Invitrogen), and plated on LB with ampicillin (100 mg mL�1), isopro-pyl-b-d-thiogalactopyranoside (IPTG; 100 mg mL�1), and X-GAL(100 mg mL�1). Positive clones were identified by blue/white screen-ing, and verified through digestion following an overnight growthand plasmid miniprep with a QIAprep Spin Miniprep Kit (Qiagen).A clone containing a 2 kb insert was digested with HindIII (for lpiB)or StuI (for lpiC) to cut in the middle of the 2 kb insert, treatedwith CIP, and gel-extracted to remove the remaining CIP. A chlor-amphenicol resistance cassette was amplified from pRE112 (primersHindIIIChlorF: 5’-TTTTAA GCTTCT AAATAC CTGTGA CGG-3’ and Hin-dIIIChlorR: 5’-TTTTAA GCTTCT ATCACT TATTCA GGC-3’; or StuI-

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ChlorF: 5’-TTTTAG GCCTTC ATTCGA CTCCTG GGA-3’ and StuI-ChlorR: 5’-TTTTAG GCCTAA TGAGCA GACATC CCC-3’), purified, di-gested with HindIII or StuI, and ligated with the digested vector.This ligation was transformed into chemically competent DH5a (In-vitrogen) and plated on LB with ampicillin (100 mg mL�1) and chlor-amphenicol (30 mg mL�1). Colonies were confirmed by PCR, andverified with digestion following an overnight growth and plasmidminiprep. Electro-competent P. fluorescens DSM 11579 were pre-pared by taking P. fluorescens culture (10 mL) with OD600 = 1, sepa-ration in a centrifuge at 2095 g for 5 min at 4 8C, washing with su-crose (2 � 300 mm), resuspension in sucrose (100 mL, 300 mm), andchilling on ice. The lpiB or lpiC gene inactivation plasmids (1 mL,350 ng) of were added, and cells were transferred to a 0.1 mm cuv-ette and electroporated with one pulse at 1.8 kV. Electroporatedcells were shaken briefly with LB and plated on LB agar with chlor-amphenicol (60 mg mL�1) at 37 8C. Genomic integration was con-firmed by colony PCR by using primers with homology 100 bp out-side the homology arms of lpiB (5’-CGAAAC GCTACC AGGTCG CA-3’) or lpiC (5’-GCGCTG GTGGAC TTCAAG AA-3’) and chlorampheni-col cassette-specific primers HindIIIChlorR or ChlorOutF (5’-GAATGCTTAATG AATTAC AA-3’).

Extraction and detection of lipocyclocarbamates: P. fluorescensDSM 11579 colonies from LB agar plates were inoculated into LBcultures (50 mL) in sterile 250 mL Erlenmeyer flasks and grownovernight at 250 rpm and 37 8C. These overnight cultures wereused to inoculate sterile 250 mL Erlenmeyer flasks containing F12production medium (50 mL). Cultures were grown at 28 8C withshaking at 200 rpm for 72 h, and then adjusted to pH 4.5 and incu-bated at 70 8C for 1 h. After being cooled to room temperature,cells were pelleted by centrifugation at 4713 g for 30 min. Cell pel-lets from each flask were resuspended in methanol (25 mL), andthe solution was stirred vigorously for 1 hour at room temperature.Methanolic extract was analyzed directly by LCMS. LCMS data wascollected on a Bruker micrOTOF II mass spectrometer with an Agi-lent 1200 series HPLC by using an Ascentis Express C18 column(150 mm � 2.1 mm, 2.7 mm, Sigma) with acetonitrile (0.1 % formicacid) and water (0.1 % formic acid) as the mobile phase at0.25 mL min�1. Separation was achieved by with 5 % acetonitrile for5 min, ramping to 100 % acetonitrile by 45 min, holding at 100 %for 10 min, then returning to 5 % acetonitrile by 60 min, and re-equilibrating at 5 % acetonitrile until 65 min. SB-253514 elutedafter 35 min.

Principal component analysis (PCA): PCA of DlpiC and DlpiB F12cultures (n = 5) was carried out using Bruker Daltonics Profile Anal-ysis with the following parameters: tR range: 0–65 min; massrange: m/z 300–1000; rectangular bucketing: 0.3 min (delta m/z0.5) ; normalized by using the sum of bucket values in the analysis.

Acknowledgements

This work was supported by generous gifts from McMaster Uni-versity and the Canadian Institute of Health Research. We aregrateful for the help of Dr. Xiang Li in this work.

Keywords: biosynthesis · darapladib · lipocyclocarbamates ·nonribosomal peptides · Pseudomonas spp. · synthetases

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Received: September 14, 2012Published online on February 10, 2013

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