Metagenomic natural product discovery in lichenprovides evidence for a family of biosyntheticpathways in diverse symbiosesAnnette Kampaa,1, Andrey N. Gagunashvilib,1, Tobias A. M. Guldera, Brandon I. Morinakaa, Cristina Daolioc,Markus Godejohannc, Vivian P. W. Miaod, Jörn Piela,e,2, and Ólafur S. Andréssonb,2

aKekule Institute of Organic Chemistry and Biochemistry, University of Bonn, 53121 Bonn, Germany; bFaculty of Life and Environmental Sciences, University ofIceland, 101 Reykjavik, Iceland; cBruker BioSpin GmbH, 76287 Rheinstetten, Germany; dDepartment of Microbiology and Immunology, University of BritishColumbia, V6T 1Z3 Vancouver, Canada; and eInstitute of Microbiology, Eidgenössische Technische Hochschule Zurich, 8093 Zurich, Switzerland

Edited by Nancy A. Moran, Yale University, West Haven, CT, and approved June 18, 2013 (received for review March 27, 2013)

Bacteria are a major source of natural products that provide richopportunities for both chemical and biological investigation. Al-though the vast majority of known bacterial metabolites derivefrom free-living organisms, increasing evidence supports the wide-spread existence of chemically prolific bacteria living in symbioses. Astrategy based on bioinformatic prediction, symbiont cultivation,isotopic enrichment, and advanced analytics was used to character-ize a unique polyketide, nosperin, from a lichen-associated Nostocsp. cyanobacterium. The biosynthetic gene cluster and the structureof nosperin, determined from 30 μg of compound, are related tothose of the pederin group previously known only from nonphoto-synthetic bacteria associated with beetles and marine sponges. Thepresence of this natural product family in such highly dissimilarassociations suggests that some bacterial metabolites may be spe-cific to symbioses with eukaryotes and encourages explorationof other symbioses for drug discovery and better understand-ing of ecological interactions mediated by complex bacterialmetabolites.

biosynthesis | Peltigera membranacea | trans-acyltransferase polyketidesynthase | 13C nuclear magnetic resonance

Symbiosis, defined by de Bary (1) as the “living together of twoorganisms,” includes a broad range of partnerships, from

loose associations to obligate interdependencies and host–para-site interactions. Many involve microbes, with perhaps the mostsuccessful—between bacteria and early nucleated cells in thePrecambrian—leading to mitochondria and chloroplasts in mod-ern eukaryotes (2). Symbiotic interactions are being examinedwith increasing molecular detail, focusing not only on attributesthat may be beneficial for each organism individually but also onwhat might be important for the association. It is increasinglybeing recognized that biosynthetic pathways leading to synthesisof specialized metabolites may play key roles in the biology ofsymbiosis (3).Lichens are ancient and physiologically highly integrated

symbioses between heterotrophic filamentous fungi (mycobionts)and cyanobacteria or coccoidal green algae (photobionts) thatmay date as far back as 600 Mya (4). The morphology of thecharacteristic and stable macroscopic body of a lichen, thethallus, typically bears little resemblance to the individualorganisms that form it and, in many cases, can be highly orga-nized: fungal cells on the periphery for physical support andprotection and photobiont cells inside, providing photosynthateor fixed nitrogen or both (5) (Fig. 1 A–C). Although the pho-tobionts can often be isolated in pure culture (Fig. 1D), mostmycobionts (almost exclusively from the Ascomycota) are re-fractory to propagation in vitro by standard methods, and intactlichens cannot be maintained artificially for long. Nevertheless,such limitations are gradually being overcome using advancedanalytical platforms, e.g., metagenomics in the characterization

of mycobiont lectin genes (6), and PCR-based phylogenetics ininvestigation of intrathalline bacterial diversity (7).In a number of bacterial–eukaryote symbioses, bacterial

partners have been implicated in the production of complexmolecules derived from polyketide synthase (PKS) and nonri-bosomal peptide synthetase (NRPS) pathways (3, 8, 9). Exam-ples include pederin, made by bacteria that live in rove beetles ofthe genus Paederus, and structurally related metabolites, theonnamides and psymberin, produced by bacteria that live inmarine sponges (Fig. 2). In general, metabolites known or sus-pected to be of symbiont origin show remarkably low structuraloverlap with natural products discovered in screening programsfrom free-living bacteria (10). This phenomenon raises the in-triguing question of whether symbiont chemistry might encom-pass structural scaffolds covering distinctive regions of chem-ical space.In this study, we applied a combination of metagenomic and

natural product discovery methods to identify nosperin, the first

Significance

Remarkable chemical families are being recognized by studyingdiverse symbioses. We identified, through metagenomics, thefirst cyanobacterial trans-AT polyketide biosynthetic pathwayin the Nostoc symbiont of the lichen Peltigera membranaceaand showed its expression in natural thalli. An isotope-basedtechnique designed for characterizing minute amounts of ma-terial confirmed predictions that its product, nosperin, is a dis-tinct member of the pederin family of compounds that waspreviously thought exclusive to animal–bacteria associations.The unexpected discovery of nosperin in lichen expands thestructural range and known distribution of this family of nat-ural products and suggests a role associated with symbiosis.

Author contributions: A.N.G. and Ó.S.A. initiated project; A.K., A.N.G., V.P.W.M., J.P., andÓ.S.A. designed research; A.K., A.N.G., B.I.M., V.P.W.M., J.P., and Ó.S.A. performed re-search; A.N.G. carried out bioinformatic analyses of WGS, isolated Nostoc strains andconducted gene expression studies; A.K. performed feeding studies and compound iso-lations; A.K., T.A.M.G., B.I.M., C.D., and M.G. performed metabolic analyses and elucidatedstructure; J.P. analyzed the trans-AT PKS genes and performed metabolic prediction; C.D.and M.G. contributed new reagents/analytic tools; A.N.G. and V.P.W.M. examined distri-bution of gene cluster; A.K., A.N.G., T.A.M.G., B.I.M., C.D., M.G., V.P.W.M., J.P., and Ó.S.A.analyzed data; A.K., A.N.G., T.A.M.G., V.P.W.M., J.P., and Ó.S.A. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The sequences reported in this paper have been deposited in the Gen-Bank database [accession nos. GQ979609 (nsp gene cluster), JQ975876 (second trans-ATgene cluster), GU591312 (nostopeptolide-like gene cluster), JX181775 (P. membranaceaWGS Nostoc rRNA genes), KC489223 (heterocyst glycolipid gene cluster), KC291407(rbcLXS operon), and JX975209 (Nostoc sp. N6 rRNA genes)].1A.K. and A.N.G. contributed equally to this work.2To whom correspondence should be addressed. E-mail: osa@hi.is or jpiel@ethz.ch.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1305867110/-/DCSupplemental.

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member of the pederin family from a lichenized cyanobacteriumand a further example toward the emerging concept of symbio-sis-associated natural product pathways (10).

ResultsDiscovery of Trans-AT PKS Genes in the Lichen Metagenome. Peltigeramembranacea is a widely distributed terrestrial lichen carryingNostoc sp. as its photobiont (Fig. 1 A–D). Total lichen DNAextracted from field samples collected in Iceland processed forwhole genome sequencing (WGS) (11) revealed approximatelyequal contributions from the mycobiont, the photobiont, and thecommunity of intrathalline microbes. Bioinformatic mining ofthe initial metagenome assembly yielded 18 candidate clusterscontaining genes that encode PKS enzymes (SI Appendix, TableS1). Among the putative bacterial gene clusters, two weremembers of the trans-acyltransferase (AT) PKS family (Fig. 3; SIAppendix, Fig. S1) in which AT domains are not encoded by thePKS genes but rather by a separate gene elsewhere: i.e., the ATsthat load the polyketide building blocks are not integral parts ofthe modules but act as free-standing units (10). This group ofenzymes is particularly interesting because many of them areresponsible for products made specifically by symbiotic bacteria(10). These gene clusters in the lichen are most likely derivedfrom the photobiont, as only Nostoc exhibited a high level clonalpresence, indicated by DNA sequence coverage in theWGS, and acommensurate level of coverage was found for diagnostic markers

of the Nostoc genome, such as hgl (involved in heterocyst gly-colipid biosynthesis; SI Appendix, Table S1). The longer of thetwo gene clusters in P. membranacea, designated “nsp” (Fig. 3)had significant homology to the gene clusters for the biosynthesisof pederin family compounds and therefore was selected forfurther investigation.The nsp gene cluster consists of a 59-kb region with 3 large

genes (nspA, nspC, and nspD) that encode multidomain PKS orPKS/NRPS proteins and a suite of 10 smaller genes that encodeaccessory enzymes (Fig. 3; Table 1). The multidomain proteinstogether comprise a “starter” module 0, followed by nine PKS orPKS/NRPS elongating modules (modules 1–9). The 5′ end of thegene cluster, i.e., nspA (modules 0–3), nspB, and the beginning ofnspC (module 4 and the KS region of module 5), as well as ac-cessory genes at the 3′ end of the cluster, have closely relatedcounterparts in biosynthetic gene clusters of pederin-type com-pounds (Fig. 3). The middle region, however, has primary affinitiesto NRPS–PKS biosynthetic pathways from other members ofProteobacteria or Cyanobacteria, viz., the end of nspC (modules5–7) is similar to the PKS genes of the rhizoxin (rhi) biosyntheticgene clusters from Burkholderia sp. (12) and Pseudomonas sp.(13). Further downstream, the PKS genes have resemblances togene clusters reported from various Nostocales or Oscillator-iales. An ∼3-kb region at the junction of nspC and nspD is es-pecially intriguing in bearing ∼80% identity at the DNA level toa portion of the nos-like gene cluster (a cis-AT PKS pathway) in

A B C D

Fig. 1. The foliose lichen Peltigera membranacea and Nostoc symbiont. (A) Lichen in situ. (Scale bar, 5 cm.) (B) Rhizines (Rhi) on lower surface and apothecia(Apo) protruding from thallus edge. (C) Thallus cross section illustrating stratified internal structure including photosynthetic cyanobiont layer (shown witharrows) between cortical and medullary mycobiont layers (above and below, respectively). (Scale bar, 100 μm.) (D) Nostoc sp. N6 in culture. (Scale bar, 100 μm.)(photograph for Fig. 1C, courtesy of Martin Grube).

Fig. 2. Pederin family compounds and symbioses. (Upper Left) Image of Paederus fuscipes courtesy of Christoph Benisch (www.kerbtier.de). (Upper Right)Image of Theonella swinhoei courtesy of Yoichi Nakao. (Lower Right) Image of Psammocinia aff. bulbosa adapted with permission from ref. 15. Copyright2007 American Chemical Society. (Lower Left) Image of Mycale hentscheli courtesy of Mike Page.

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P. membranacea (SI Appendix, Fig. S2); a homolog of this clusterin Nostoc sp. GSV224 is responsible for biosynthesis of nostopep-tolide (14), a cyclic peptide-polyketide (SI Appendix, Table S2).Altogether, the nsp locus appears to be an evolutionary mosaic oftrans- and cis-AT PKS fragments from diverse sources.Expression of the nsp pathway was detected by RNA-seq

analysis in P. membranacea thalli freshly collected from the samelocation as the source material for the WGS. Consistent withexpectations for a photobiont-specific gene cluster, nsp tran-scripts were observed in the main thallus tissue that containsboth mycobiont and photobiont cells, but not in apothecia orrhizines, which are lichen structures that are derived only fromthe mycobiont (Fig. 1B; SI Appendix, Table S3). Although trans-AT PKS systems have been found in a wide range of bacteria (16,17), none have been reported for cyanobacteria, which are oth-erwise rich sources of cis-AT PKSs (17, 18). These observationssuggested the possibility of metabolic products that might benovel, not only from structural but also from ecological andevolutionary perspectives.

Prediction of the Compound Structure.Detailed examination of theketosynthase (KS) domains in PKS gene clusters using phylo-genetic methods and comparisons of module architecture in

pathways with similar products can often facilitate prediction ofnatural product structures generated by trans-AT PKSs (16, 19).When the Nsp KS sequences (KS1–KS9, referring to the modulenumber in which the domain occurs) were aligned and comparedwith 494 homologs using KSs from cis-AT systems as an out-group, the resulting clades were generally consistent with respectto KS functions (SI Appendix, Fig. S3–S5). For example, all KSswith known function in the same group as KS1 accept acetylstarters incorporated by domains of the GCN5-related N-ace-tyltransferase family (GNAT) (20). In this way, partial structuresfor the substrates of KS1–3, 5, 7, and 9 were predicted (Table 2).As expected from the earlier analyses, KS1–5 (nspA, nspC) weremost similar to KSs of the pederin (21, 22) and/or onnamide (23)PKS, and a full domain analysis revealed virtually complete ar-chitectural identity with corresponding portions of the ped andonn PKS–NRPS clusters over the first six modules, ending withKS5. The region also included an NRPS (module 4a) that cat-alyzes the insertion of a glycine residue (Fig. 4). This observationindicated that a large part of the polyketide product would re-semble pederin and onnamides. The remainder of the corestructure was more difficult to predict, because two of the fourKSs (KS6 and KS8) fell into clades consisting of KS0s, which arenonelongating KS variants that usually show little consistency

Table 1. List of the genes present in the nsp gene cluster and their predicted functions

ORFProteinsize Proposed function Closest homolog (protein,origin)

Percentidentity

Accessionnumber*

nspA 5,320 PKS PedI, Paederus fuscipes symbiont 42 AAR19304nspB 371 Flavin-dependent oxygenase PedJ, P. fuscipes symbiont 66 AAR19305nspC 8,252 PKS-NRPS OnnI, Theonella swinhoei symbiont 49 AAV97877nspD 2,206 PKS JamP, Lyngbya majuscula 60 AAS98787nspE 474 MatE efflux transporter SxtM1, Lyngbya wollei 54 ACG63829nspF 285 O-Methyltransferase OnnH, T. swinhoei symbiont 57 AAV97876nspG 86 ACP Cpap_1683, Clostridium papyrosolvens DSM 2782 58 EGD47495nspH 411 β-Ketoacyl synthase Cpap_1682, C. papyrosolvens DSM 2782 60 EGD47494nspI 420 HMG-CoA synthase PksG, Bacillus subtilis subsp. subtilis SC-8 72 EHA29460nspJ 262 Enoyl-CoA hydratase Cpap_1678, C. papyrosolvens DSM 2782 52 EGD47490nspK 442 Acyltransferase PedD, P. fuscipes symbiont 49 AAS47563nspL 464 Cytochrome P450 PPSIR1_33239, Plesiocystis pacifica SIR-1 35 EDM78481nspM 647 Asparagine synthase Acid_5610, Candidatus Solibacter usitatus Ellin6076 48 ABJ86557

*Accession numbers are for the GenBank database.

Fig. 3. Nosperin biosynthetic gene cluster nsp and flanking regions. Microsynteny and homology with pederin and onnamide biosynthetic gene clusters areindicated in gray. Similarity of nsp to other PKS biosynthetic gene clusters is indicated by double-headed arrows. Numbers denote individual modules. Geneswith similar proposed functions (Table 1) are indicated with identical colors. β, genes involved in β-branch formation; T, transposon. See SI Appendix, Figs. S15and S16 for details of regions flanking the nsp locus.

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between phylogeny and substrate structure (16). KS06 was po-sitioned in a small subclade containing homologs from the rhi-zoxin and bacillaene PKSs that are involved in shifting doublebonds from the α,β- to the β,γ-position (24, 25). These KSs arefound in modules harboring, in addition to the KS0 and the acylcarrier protein (ACP), a dehydratase (DH) domain postulated tocatalyze double bond isomerization and characterized by aNSAF/YL instead of the usual DxxxQ/H motif involved in de-hydration (26). The same elements are present in the nspmodule

encoding KS06; moreover, KS7, encoded by the module imme-diately downstream, is highly similar to rhizoxin KS12, whichaccepts a substrate with a shifted double bond (25). Togetherwith the upstream NRPS module, these features strongly sug-gested the presence of an enamide moiety, which is not presentin pederin or onnamides. KS9, associated with KSs elongat-ing chains of amino acid residues, consistent with its positionC-terminal to a second NRPS module (8a), was the only otherKS with predictable function. An analysis of residues lining the

Table 2. Analysis of KS domains present in the Nsp PKSs

Domain Closest characterized relative (substrate specificity) Predicted specificity of KS clade Moiety present in nosperin

KS1 pederin KS1 (acetyl starter) Acetyl AcetylKS2 onnamide KS2 (α-L-methyl + β-D-OH) α-L-methyl + β-D-OH α-L-methyl + β-D-OH (anti configured)KS3 onnamide KS3 (β-exomethylene) mostly β-exomethylene β-exomethyleneKS4 onnamide KS4 (KS0) KS0 KS0

KS5 pederin KS5 (amino acid) amino acid glycineKS6 rhizoxin KS11 (KS0, double bond) KS0, double bond KS0, double bondKS7 rhizoxin KS12 (shifted double bond) shifted double bond shifted double bondKS8 bryostatin KS8 (KS0) KS0 KS0

KS9 oxazolomycin KS9 (serine) amino acid proline

O

HO

HN

O

MeO

O

S

O

HO

HN

O

MeO

HO

O

S

O

HO

HN

O

MeO

HO

O

N

S

O

O

HO

HN

O

MeO

HO

O

N

S

O

HO

O

OHHN

O

MeO

O

N

OH

OH

OH OO

OHHN

O

MeO

O

N

OH

NH2

OH O

HO

S

OS

O

O

HO

S

O

MeO

S

HOO

O

HO

HN

O

MeO

O

S

O

HO

HN

O

MeO

O

S

O

OHHN

O

MeO

O

N

OH OH

HO

NosperinNH2

O

NspL

NspM

Release

I II III

KS KS C CR

CR KR A GNAT

?

translation

KS KR

MT KS0

KR

KS

DH MT KS0 DH KS

KR MT C A KS0

KR

KS

AT ER TE

EDCBApsn FG H I J K L M

pederin orthology

1 2 3 4 5 6 7 8 9 4a 8a 0 module

Fig. 4. The nsp gene cluster, deduced architecture of the PKS proteins NspA, NspC, and NspD, and proposed biosynthesis of nosperin. GNAT, GCN5-relatedN-acetyltransferase family (20); KS, β-ketoacyl synthase; KR, ketoreductase; MT, C-methyltransferase; CR, crotonase superfamily (also known as enoyl-CoAhydratase) (30); KS0, nonelongating KS; C, nonribosomal peptide synthetase (NRPS) condensation domain; A, NRPS adenylation domain; DH, dehydratase; AT,acyltransferase; ER, enoyl reductase; TE, thioesterase; ?, unknown. Small black circles symbolize acyl and peptidyl carrier proteins. The positions of ampliconsused for the nsp screening are shown with black boxes and roman numerals.

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substrate pocket of the adenylation domain, known as thenonribosomal code (27, 28), returned a perfect match to pro-line-activating domains (SI Appendix, Table S5). In the absenceof diagnostic downstream KS domains, the portions of the pol-yketide generated by modules 5, 7, and 9 were predicted usingclassical PKS colinearity rules (29), although they often applypoorly to trans-AT PKSs. These rules indicated the presence oftwo additional methyl groups and a hydroxyl function. The ter-minal elongation step was predicted to be catalyzed by module 9in NspD, a cis-AT PKS module with an integrated AT domain.This step remained obscure, because the module architecture(KS-AT-KR-ER-ACP-TE) contrasts with the canonical order(KS-AT-DH-ER-KR-ACP-TE) and lacks a DH domain to pro-vide the substrate for the subsequent enoyl reduction. This fea-ture suggested either that the ER domain is nonfunctional,despite the presence of key amino acid residues, or that the DHactivity is provided in trans.Further structural predictions were possible by comparison of

the accessory and post-PKS nsp genes to known pathways. Thegenes nspGHIJK resembled those typically involved in the gen-eration of polyketide β-branches (Table 1), indicating the pres-ence of a pederin-type exomethylene bond (30). Because theclosest relatives of nspB and nspF in the ped and onn clustersencode an oxygenase and a methyltransferase (31), responsible,respectively, for one oxygenation (at C7) and one methylation (atthe C6 acetal oxygen) within the corresponding moiety of ped-erin, similar units in the nsp product were expected. The putativeasparagine synthetase (NspM) and cytochrome P450 (NspL)enzyme homologs, however, remained without counterpart inpederin-type pathways.

Isolation and Characterization of the Polyketide Nosperin. Using thepreliminary structural information as a guide, total extracts ofwhole lichens were examined for the presence of the predictedmetabolites. Due to copious amounts of diverse glycolipids andother metabolites, however, LC-MS and extensive NMR-guidedsubfractionation failed to detect a pederin-type polyketide. AsNostoc symbionts have often been cultured from lichens (5), analternative approach focusing only on the cyanobacterium wastaken. Macerated thalli of P. membrancacea were plated on BG-110,a minimal medium lacking nitrogen, and cyanobacteria identifi-able as Nostoc sp. by microscopic examination were establishedin pure culture (Fig. 1D). The presence of the nsp cluster in threerandom isolates was confirmed by PCR for amplicons repre-senting the PKS genes nspA and nspC, and the accessory gene,nspF (Fig. 4; SI Appendix, Table S7). One strain, designated N6,also characterized by sequencing of 16S and 23S ribosomalRNAs, was grown in BG-11 liquid medium for 4 wk to evaluategene expression in culture. Transcription of the nsp gene clusterin Nostoc sp. N6 was confirmed by mapping RNA-seq data andfound to be fivefold higher than in the thallus, relative to ex-pression of rbcLXS, a Nostoc reference marker (SI Appendix,Table S3). When extracts were prepared from scaled up cultures,numerous metabolites were observed in small amounts. How-ever, due to the unusual architecture of the terminal Nspdomains and the unknown nature of post-PKS modifications,prediction of the mass of the compound was challenging andconvincing candidates were not identified by MS analysis.In light of the challenges imposed by multicomponent trace

mixtures and the absence of a known mass, a strategy of stableisotopic enrichment followed by HPLC-SPE-NMR to addressproblems of sensitivity and complexity while allowing detectionof predicted structural moieties was used. Nostoc sp. was cul-tured in 25 L of BG-11 supplemented with 13C-labeled NaHCO3,and after 5 wk, cyanobacterial biomass from 10 L of culture wasfreeze-dried and extracted. HPLC-electrospray ionization (ESI)-MS analysis confirmed that most components in the extracts hadmultiple 13C atoms incorporated into individual molecules. To

obtain insights into structural features of these compounds, thecrude extract was subjected to repetitive HPLC-solid phase ex-traction (SPE) purification with subsequent NMR analysis oftarget molecules eluted with fully deuterated solvent (SI Ap-pendix, Figs. S6–S9). This method allowed collection of high-quality 1H- and 13C-spectra, as well as COSY-, HSQC-, andHMBC-2D-NMR data from microgram amounts of the 13C-labeled material (SI Appendix, Figs. S8–S13). NMR signalscharacteristic of the predicted exomethylene, methoxy, and C-methyl functions were detected for a component eluting at 30.3min (SI Appendix, Figs. S6–S9), of which only 30 μg wereobtained. Further support for the identity of the compound camefrom ESI(+)-MS analysis, which indicated a multiply labeled minorcomponent with an exact unlabeled mass of m/z = 564.2926, wellwithin the predicted range and best fitting a calculated atomiccomposition of C26H43N3O9Na (calculated: m/z = 564.2897).MS/MS analysis of the molecular ion peak additionally revealeda daughter ion consistent with the formal loss of MeOH (m/z =532.2664; calculated for C25H39N3O8Na: m/z = 532.2635), sug-gesting a methoxy group in the predicted structure. Several otherindicative fragments were also visible in the MS/MS data, e.g.,an additional cleavage of an acetamide functionality (m/z =473.2260; calculated for C23H34N2O7Na: m/z = 473.2264). TheNMR data fully supported the identity of the compound as amember of the pederin group and allowed elucidation of itsconstitution. In combination with bioinformatic analysis it waspossible to predict most of the stereogenic elements, except theconfiguration at C12 and C14 (Fig. 5; for a full description of theMS and NMR-based characterization see SI Appendix, Data S2).The structure of the compound is in almost perfect agreementwith the predicted features and represents a hybrid of pederinand an unusual proline-containing terminal moiety not pre-viously observed in this group of natural products. Two devia-tions from the incomplete product prediction are the terminalamide function, most likely generated by the asparagine syn-thase-like protein NspM, and the hydroxyl moiety at C20, in-dicating post-PKS oxidation by the P450 homolog NspL.Altogether, the data show that this compound, which has beendesignated nosperin, represents a unique member of thepederin family of natural products.

Distribution of the nsp Locus in Cyanobacteria. Although the culti-vated strain Nostoc sp. N6 carried the nsp cluster, it alsoexhibited a distinctive 23S rRNA polymorphism and originatedfrom a specimen of P. membranacea independent from that usedin metagenome sequencing. These observations suggested thatthe nsp pathway might be common in P. membranacea photo-bionts or in Nostoc and possibly even other cyanobacteria. APCR-based survey for nspA, nspC, and nspF indicated that thensp cluster was present in P. membranacea from several locationsin Iceland, but samples of this lichen in British Columbia, Can-ada, also included specimens where the targeted sequences werenot detected (Table 3). Three nsp amplicons obtained froma specimen near Vancouver on the mainland were nearly iden-tical (99.9%) to those from a Vancouver Island sample, butshowed ∼3% divergence from the Icelandic reference (SI Ap-pendix, Fig. S14).A bioinformatic search for nsp-like sequences in GenBank (SI

Appendix, Table S2) and a PCR survey for amplicons of nspA,nspC, and nspF in 26 cyanobacterial strains representing 15genera from all cyanobacterial orders (Materials and Methods)did not return any positive results except in the genus Nostoc,suggesting that the nsp pathway per se may have a phylogeneti-cally restricted distribution. Within Nostoc there appeared to bean association with lichens: results from PCR testing of fourstrains (Nostoc sp. PCC9709, AR10B, AR9A, and WL-1) iso-lated from Peltigera spp. (32, 33) were positive for nsp, whereasfour other strains not associated with lichens (Nostoc muscorum

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PCC7906, Nostoc punctiforme PCC73102, Nostoc spp. PCC6705,and PCC7107) were negative.The limited distribution within Cyanobacteria and the appar-

ent absence of the nsp gene cluster in some samples from Can-ada suggested that rather than being a core part of the genome,the nsp genes may have been introduced horizontally. Inves-tigation of regions flanking the nsp cluster revealed IS4 elementson both sides and linkage to genes associated with plasmidreplication and partitioning (including parA, parB, and parMhomologs and a gene encoding a DNA helicase), suggesting thepossibility of an extrachromosomal source (Fig. 3; SI Appendix,Figs. S15 and S16).

DiscussionIn this report, we describe identification of the nsp genes in theP. membranacea lichen metagenome, the first trans-AT PKS genecluster from a cyanobacterium, and the application of a strategyconsisting of bioinformatic prediction, symbiont cultivation, iso-tope enrichment, and 13C-NMR that enabled characterization ofa unique symbiosis-associated natural product, nosperin, fromthe photobiont.Lichens have long been known for distinctive mycobiont-pro-

duced compounds, such as depside and depsidone polyketides(34), but unique structures and pathways are now also emergingfrom studies of the photobionts. Two conventional cis-AT PKS–NRPS biosynthetic pathways have recently been described fromcyanobacteria associated with lichens: the mcy gene cluster (35,36) involved in synthesis of microcystins, notorious hepatotoxinstypical of many cyanobacteria, and the crp gene cluster, re-sponsible for production of cryptophycins (37), anticancer agentsof more limited distribution. The elements of the nosperin bio-synthetic pathway, in contrast, are similar to the less common

trans-AT PKS–NRPS systems responsible for a number of animal–bacteria symbiosis-associated compounds including pederin, the-opederins, onnamides, mycalamides, psymberin (irciniastatin A),and others (10) (Fig. 2). These compounds, with almost identicalcore regions but different biosynthetic starter regions and/ortermini, are often highly toxic to eukaryotes and some have beenconsidered promising candidates for anticancer drug de-velopment (38–40). Notably, this group of compounds has neverbeen recovered from screening free-living bacteria, despiteconspicuous pharmacological activities. Studies of mycalamide A(Fig. 2), which binds in the E-site of the ribosome normally oc-cupied by the tRNA-terminal CCA (41), and synthetic analogstogether with molecular modeling, have identified the N-acyllinked tetrahydropyran structure as central to binding and ac-tivity (42). The presence of the N-acyl linked tetrahydropyran innosperin suggests it might have similar bioactivity; however, theamounts available were too small for testing.The discovery of nosperin not only increases the number of

chemical scaffolds and biosynthetic enzymes encompassed by thepederin group but also expands the remarkable range of symbi-oses associated with this natural product family (Fig. 2). Fur-thermore, although the taxonomic identities of their producersare unknown, with the exception of pederin, which is producedby a close relative of Pseudomonas aeruginosa (21, 43, 44), bothlichen metagenomic data and expression and product charac-terization in Nostoc sp. N6 clearly show that nosperin derivesfrom the cyanobacterial photobiont of P. membranacea. Althoughthis cyanobacterium is essential to every phase of thallus growthand development, it is also a facultative symbiont, being cultur-able by itself on basic mineral salts media. This first individuallyidentified and culturable producer of a pederin family naturalproduct provides new opportunities to study the biochemistry and

O

OHHN

N

OH OMeO

OHO

OH201

24

10132 NH2

O

Fig. 5. Stereochemical characterization of nosperin by NMR and bioinformatic analysis. The absolute configurations at the chiral centers were predicted byanalysis of the stereospecificity of KR domains (blue), the NRPS domain structure (orange), the overall domain organization in comparison with other pederin-type biosynthetic gene clusters (red), and NMR coupling constants and/or chemical shifts (green).

Table 3. Detection of nsp amplicons in P. membranacea from Iceland and two locations inBritish Columbia, Canada

Region Locality Number of samples nspA nspC nspF

Reykjavík Grafarholt 1 + + +Keldur 3 + + +Mosfellsbaer 1 + + +Öskjuhlid 5 + + +Raudavatn 1 + + +Ulfarsfell 1 + + +

Vancouver (Mainland) Belcarra 1 + + +Black Mountain 1 − − −Brothers Creek 1 + + +Eagle Ridge 2 + + +Eagle Ridge 3 − − −

Vancouver Island Horth Hill 2 + + +Roche Cove 2 − − −

For positions of the amplicons see Fig. 4. +, a PCR product of the expected size was observed; −, no PCRproduct was observed.

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physiology of the biosynthetic pathway in vivo, as well as to im-prove metabolite yield through optimization of production pro-tocols and strain improvement.Study of genes, gene clusters, and biosynthetic pathways in

diverse symbiotic associations may help clarify their functions oridentify metabolic products that are essential. Some polyketidesproduced by trans-AT PKSs, such as pederin (21, 22, 43),bryostatin (45, 46), and rhizoxin (12, 47), are known to partici-pate in host defense and pathogenicity in symbiotic associations.It has also been suggested that PKS–NRPS compounds such asthe microcystins, sometimes produced by cyanobacterial sym-bionts, may contribute to the chemical defense of lichens againstgrazers (35, 36). Expression of the nsp genes in P. membranaceaand their presence in all Icelandic specimens tested suggest thatit is a beneficial trait, although its role is unclear. In this regard, itmay be significant that no microcystin pathway homolog wasidentified in P. membranacea, leaving open the possibility thatnosperin might have a similar function in the lichen. Examina-tion of geographically distant populations of P. membranaceawas informative, as absence of nsp amplicons from some samplesfrom Canada indicates that these genes are not essential for thelichen symbiosis although nosperin may confer advantage undersome conditions. This provision may also apply to pederin, onna-mides, and psymberin, where the metazoan hosts can be foundwith or without the metabolites (23, 43, 48). In the case ofP. membranacea, additional field studies may help elucidatewhether there is a primary cause, e.g., a founder effect, a par-ticular environmental condition, or an interplay of other factorsthat underlie the distributional differences observed.The presence of similar trans-AT PKS–NRPS gene clusters in

different groups of bacteria has suggested that these clusters arehorizontally transferred (44). The flanking of the nsp cluster bytransposable elements is consistent with this hypothesis, and themosaic of homologies across the gene cluster suggests involve-ment of several intergenomic and intragenomic recombinationevents. The homology of NspE and part of NspD to proteinsfrom Oscillatoriales (Fig. 3) suggests that an ancestral ped-likeoperon, specifying the conserved core part the molecule, mayhave been introduced into and modified by oscillatorean cya-nobacteria: the position of nspE and nspD between sequenceswith high homology to the ped gene cluster and the orientation ofnspE opposite to the ped-like genes suggest an intragenomicrearrangement mediated by genetic similarity of PKS–NRPSmodules. Transfer to Nostocales and subsequent recombinationresulted in the present domain organization that includes the ∼3-kbcis-AT containing fragment from a Nostoc nos-like cluster. Thepresence of a cis-AT domain is unusual in a PKS relying ontrans-ATs, with few occurrences among the ∼40 large trans-ATPKS complexes with known products (10, 49). A relatively recentinsertion of this ∼3-kb fragment is indicated by the high aminoacid and nucleotide similarities to the nos cluster and suggeststhat near relatives of the nsp pathway may exist. It will be in-teresting whether a gene cluster similar to nsp, but without thecis-AT encoding region, or with other types of inserts and sub-stitutions, will be found in other bacteria. Study of such examplesof naturally engineered multidomain genes and gene clustersinvolving distantly related participants may not only generateuseful hypotheses for further understanding their evolution, butthe phylogenetic reconstruction may also be informative inidentifying models of successful architectures for application incombinatorial biosynthesis industrially.The metabolic options offered by symbiotic associations pro-

vide exciting potential for drug development and highlight theneed for new discovery strategies applicable to these complexsystems. Although individual steps of the present procedure havebeen used previously in natural product research (15, 19, 47,50–52), the combination of methods has not been reportedand should be applicable to many further organisms. The

13C-NMR–based technique and other recent methods such as im-aging MS (53) can detect low concentration signatures ofnosperin and facilitate investigation of its role in symbiosis.These approaches could also identify molecular variants: e.g.,recently studied specimens of P. membranacea that appearnegative for only one or two of the three primer sets used for nspscreening may present variants of nosperin. This possibility isakin to the situation of the microcystins and cryptophycins forwhich a large number of structural variants have been found (36,37). A thorough study of the >1,500 species of cyanobacteria-bearing lichens and the multitude of other organisms includingbryophytes, ferns, cycads, and angiosperms (54) that harborcyanobacterial symbionts may yield many new biosyntheticpathways and metabolites to provide both alternative chemistryfor potential pharmacological applications and a wealth of in-formation on the chemical biology of symbiosis.

Materials and MethodsIdentification of PKS Gene Clusters in the P. membranacea Metagenome andExpression Analysis of Whole Thalli. Metagenomic DNA was processed forsequencing at commercial facilities via Roche 454 and Illumina Solexa 2 ×35-bp methodology generating 1.76 GB of 454 data and 1.4 GB of Illuminadata, yielding ∼50× coverage of the Nostoc genome. A draft assembly of theP. membranacea metagenome was constructed with MIRA v3.2.1 (www.chevreux.org/projects_mira.html). To search for PKS gene clusters, concate-nated consensus sequences of the KS (N terminus, pf00109; C terminus,pf02801; http://pfam.sanger.ac.uk/) and ACP domains (pf00698) were used ina TBLASTN search (55) to retrieve all relevant contigs from the metagenomicdatabase. Accuracy of the assembly was verified by visual inspection of thecontigs in GAP4 (Staden package) (56) based on a mapped 3.5-kb paired-endlibrary. Portions of the nsp sequence were verified by PCR amplification andsequenced directly using BigDye chemistry (Applied Biosystems; MacroGen).RNA-seq data sets from field samples of lichen thalli, apothecia, and rhizineswere previously generated (6) and used for mapping in this study withBowtie (57).

Structure Prediction. Amino acid sequences of 503 KS domains from trans-ATand cis-AT PKSs were retrieved from GenBank and aligned using the MUSCLEalgorithm with a gap open score of −1, as implemented in Geneious 5.5.3(Biomatters Ltd.). After manual improvement of the alignment, phyloge-netic reconstruction was performed by means of the Geneious softwareusing the neighbor joining algorithm with a Jukes-Cantor distance method.KS domains of cis-AT PKSs were used as an outgroup. Bootstrap analysis wasdone with 1,000 pseudoreplicate sequences.

Chemical Analysis of Whole Lichen. Air-dried lichen (30 g) was ground to a finepowder in liquid nitrogen using a mortar and pestle and stirred for 24 h atroom temperature in MeOH. The mixture was filtered, and the solid materialwas extracted a second time. The solvent of the combinedMeOH extracts wasremoved under reduced pressure. The crude extract was partitioned between10:1 MeOH/H2O (300 mL) and n-hexane (3 × 100 mL). The solvent was re-moved from the aqueous MeOH layer under reduced pressure, and theresidue was further fractionated by silica gel column chromatography. Thefollowing solvents (0.5 L each) were used to elute compounds: petroleumether, petroleum ether/EtOAc (1:1), EtOAc, EtOAc/MeOH (9:1; 8:2; 7:3, 1:1),and MeOH. The fractions were evaporated under reduced pressure andanalyzed by LC-MS using a Phenomenex Luna C18 column with a mobilephase gradient of 1:9 CH3CN/H2O + 0.1% TFA to 100% acetonitrile over30 min and a flow rate of 1 mL/min.

Isolation of Nostoc sp. N6. Lichen thalli collected from the same location asmaterial used for WGS and expression studies were macerated betweensterile microscope slides (58), and cells were plated on BG-110 (withoutNaNO3) agar medium (59) and incubated at 20 °C with a 12/12-h day/nightcycle. Nostoc colonies were purified by repeated streaking on the samemedium and maintained at room temperature. Analysis of the 16S and 23SrRNA sequences in the RNA-seq library (below) confirmed both the purity ofthe culture and its identification as a Nostoc sp.

RNA Extraction and RT-PCR of Nostoc sp. N6. Total RNAwas isolated from 1 L ofBG-11 medium incubated at 20 °C under constant illumination for 4 wk.Cyanobacteria were retained on Miracloth (Calbiochem) after culture fil-tration, rinsed with water, blotted with paper towels, flash frozen in liquid

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nitrogen, and crushed to a fine powder. TRIzol reagent (Life Technologies)was added to the powder, and it was ground again. The mixture wastransferred to a 15-mL polypropylene tube and processed according to theTRIzol protocol. Before RT-PCR, the RNA was treated with DNase I (RNase-free) (Fermentas) to remove residual genomic DNA. First-strand cDNA wassynthesized from 1 μg total RNA using SuperScript II Reverse Transcriptase(Invitrogen). RNA-seq data were obtained using Illumina Solexa GenomeAnalyzer IIx at the deCODE Genetics facility (Reykjavik, Iceland). RNA-seqmapping was done with Bowtie (57) and visualized in Geneious 5.5.3.

Culture of Nostoc sp. N6 for Natural Product Analysis. Twenty-five liters of cellswere grown in an illuminated (5,200 lm) bubble-column bioreactor in BG-11liquid medium, optionally enriched with 3 mM 13C-labeled NaHCO3, for 5 wkat pH 7.8 and 25 °C. The cyanobacteria from 10- and 5-L portions of theculture were collected by filtration, frozen in liquid nitrogen, freeze-dried,and stored at −20 °C.

Chemical Extractions and Analysis of Nostoc sp. N6, Unlabeled Culture. Freeze-dried cyanobacteria (above) were homogenized in 50 mL CH2Cl2/MeOH (2:1)and stirred for 15 min at room temperature. Biomass was filtered andtreated again with the same amount of CH2Cl2/MeOH for 30 min at 30 °C.This procedure was repeated twice. The combined extracts were dried underreduced pressure. The crude extract was dissolved in MeOH and subjected toLC-MS analysis using an Agilent 1200 series HPLC and Bruker DaltonicsmicrOTOF-Q-spectrometer. HPLC was carried out with a Phenomenex LunaC18 column (5 μm, 250 × 2.00 mm), a mobile phase gradient of CH3CN/H2O(20:80) to (80:20) over 45 min, and a flow rate of 1 mL/min.

Chemical Extractions and Analysis of Nostoc sp. N6, Labeled Culture. Freeze-dried cyanobacteria were extracted with stirring for 24 h in 2 LMeOH at roomtemperature. After filtration, the methanolic fraction was dried by evapo-ration and redissolved in 0.5 L MeOH/H2O (10:1) followed by liquid-liquidextraction with 0.5 L cyclohexane. The cyclohexane fraction was discarded.The remaining MeOH/H2O fraction was dried and stored at −20 °C. Thismaterial was directly used for LC-SPE-NMR analyses.

HPLC-SPE-NMR. The solvent system consisted of eluent A (H2O + 0.1% deu-terated formic acid) and eluent B (acetonitrile) with a linear gradient startingwith 10% of B up to 90% B in 30 min. The flow rate was 0.8 mL/min at 25 °C,and the injection volume was 50 μL. The chromatography was monitored at210, 220, and 254 nm, and thesewavelengthswere used to define absorbancethresholds to trigger SPE trapping. The HPLC eluate was diluted with H2O(2.4 mL/min) before trapping on SPE cartridges (Spark Holland), and indi-vidual peaks were trapped four times to increase concentration on cartridge.The cartridges were dried with pressurized nitrogen gas for 30 min each, andthe analytes were eluted with 190 μL CD3CN (99.8 atom %; Deutero GmbH)into 3-mm match tubes from Bruker BioSpin GmbH.

NMR. All NMR experiments were acquired on an AVANCE III 600 MHz NMRspectrometer equipped with a 5-mm QNP cryo probe head (Bruker Biospin).Standard parameter sets created for the Bruker SELU (structure elucidation)program were uniformly used. Gradient correlation spectroscopy (COSY) and

heteronuclear multiple-bond correlation spectroscopy (HMBC) were carriedout using 4,000 complex data points in F2 and 512 points in the F1 di-mension. The multiplicity edited gradient heteronuclear single quantumcorrelation (HSQC) was acquired with 2,000 data points in F2 and 400 pointsin the F1 dimension. The COSY experiment was acquired with 32 scans, theHSQC with 64 scans, and the HMBC with 128 scans per increment, resulting inexperiment times of 8 h 46 min (COSY), 12 h 4 min (HSQC), and 1 d 11 h(HMBC). A C13 spectrum with composite pulse decoupling on the protonchannel was acquired by collecting 4,096 scans with 131,072 complex datapoints at a sweep width of 40,761 Hz and with a relaxation delay of 5 s. Theexperiment time was 7 h 11 min.

Distribution Survey. P. membranacea thalli were collected at several localitiesin Iceland (Reykjavik area) and in British Columbia (North Shore mountainsnear Vancouver; Vancouver Island), and DNA was extracted using the pre-viously described methods (60). DNA samples representing cyanobacterialstrains other than those newly isolated from P. membranacea for this studywere prepared and described previously (32, 61) and stored at −20 °C. Theyinclude Anabaena sphaerica UTEX1616, Chlorogloeopsis fritschii PCC6718,Cylindrospermum stagnale PCC7417, Fischerella muscicola PCC7414,Geitlerinema sp. PCC7105, Gloeobacter violaceus PCC7421, Leptolyngbya sp.PCC7104, Leptolyngbya sp. PCC7375, Lyngbya kuetzingii UTEX1547,Myxosarcina sp. PCC7325, Nodularia spumigena PCC73104, Nodulariaharveyana UTEX2093, Pleurocapsa sp. PCC7315, Pleurocapsa sp. PCC7324,Pleurocapsa sp. PCC7321, Scytonema hofmanni PCC7110, SynechocystisPCC6803, Nostoc punctiforme PCC73102A, Nostoc sp. PCC6705, Nostoc sp.PCC9709, Nostoc sp. AR10B, and Nostoc sp. AR9A. In addition, DNA samplesfrom Calothrix sp. PCC7601, Nostoc muscorum PCC7906, and Nostoc sp.PCC7107 (originally obtained from the Pasteur Culture Collection of Cya-nobacteria) and Nostoc sp. WL-1 (kindly provided by E. Loos, University ofRegensburg, Regensburg, Germany) were prepared from cultures usingsimilar methods (32). Amplification of rbcLX (62) or rnpB (63) regions (SIAppendix, Table S7) (and in some cases, also the 16S rRNA gene) was used asa positive control to ensure DNA quality before screening with primer setstargeting the nsp gene cluster (Fig. 4). DNA from the Nostoc sp. N6 strainwas used as an nsp positive control. Conditions for nsp screening were 94 °Cfor 2 min, then 94 °C for 10 s, 55 °C for 30 s, and 72 °C for 30 s (35×), and then72 °C for 7 min. For rbcLX primers, the extension time was 1 min. EppendorfMasterMix 2.5× (Eppendorf) was used according to the manufacturer’sprotocol in a final volume of 50 μL. All negative samples were repeated atleast once. PCR amplicons from two samples were sequenced directly(MacroGen). The bioinformatic search was conducted in November 2012.

ACKNOWLEDGMENTS. We thank T. Taylor and W. Loos for a gift of lichensand Nostoc isolate WL-1; K. Anamthawat-Jónsson for help with microscopy;deCODE Genetics (D. N. Magnúsdóttir, G. P. Örlygsdóttir, S. Snorradóttir, andÓ. T. Magnússon) for sequencing; G. König for providing a fermentor;H. Gross for sharing knowledge on culturing cyanobacteria; and K. Peters-Plaumbaum and M. Engeser for MS support. This work was financially sup-ported in part by the DFG (SFB 642 to J. P. and Emmy Noether fellowship toT.A.M.G.), the EU (BlueGenics to J.P.), the Alexander von Humboldt Founda-tion (B.I.M.), and the Icelandic Research fund (to Ó.S.A.).

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Kampa et al. PNAS | Published online July 29, 2013 | E3137

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