Lichen Biosynthetic Gene Clusters. Part I. Genome ... - ACS Publications

Article Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Lichen Biosynthetic Gene Clusters. Part I. Genome Sequencing Reveals a Rich Biosynthetic Potential Robert L. Bertrand, Mona Abdel-Hameed, and John L. Sorensen* Department of Chemistry, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada S Supporting Information *

ABSTRACT: Lichens are symbionts of fungi and algae that produce diverse secondary metabolites with useful properties. Little is known of lichen natural product biosynthesis because of the challenges of working with lichenizing fungi. We describe the first attempt to comprehensively profile the genetic secondary metabolome of a lichenizing fungus. An Illumina platform combined with the Antibiotics and Secondary Metabolites Analysis Shell (FungiSMASH, version 4.0) was used to sequence and annotate assembled contigs of the fungal partner of Cladonia uncialis. Up to 48 putative gene clusters are described comprising type I and type III polyketide synthases (PKS), nonribosomal peptide synthetases (NRPS), hybrid PKS-NRPS, and terpene synthases. The number of gene clusters revealed by this work dwarfs the number of known secondary metabolites from C. uncialis, suggesting that lichenizing fungi have an unexplored biosynthetic potential.

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No lichen secondary metabolite has been definitively linked to a biosynthetic gene cluster via either gene knockout or heterologous expression. Three lichen products, grayanic acid,7 usnic acid,8 and 6-hydroxymellein,9 have been linked through nondefinitive experiments. Transcriptional heterologous expression of three lichen polyketide synthase (PKS) genes has been achieved, though de novo metabolite biosynthesis was not observed in any case.10,11 We estimate the number of complete and GenBank-deposited type I PKS genes from lichenizing fungi to be around 50. Genetic information about the accessory genes located with these PKS is known for only seven of these PKS.7−9,12−15 Biosynthetic gene clusters comprising type III PKS, nonribosomal peptide synthetase (NRPS), hybrid PKSNRPS, and terpene synthases have not been described in lichenizing fungi. Two NRPS and two PKS-NRPS genes (it is not known whether accessory tailoring genes are present) have been reported,16 and polymerase chain reaction (PCR) experiments have also suggested that type III PKS populate mycobiont genomes.17 Profiling the secondary metabolite gene clusters of lichenizing fungi would therefore advance our understanding of how lichens produce natural products and facilitate future functional characterization experiments. We report whole-genome sequencing of the fungal partner of the lichen Cladonia uncialis and the first attempt to comprehensively profile all biosynthetic gene clusters within a

ichens are the product of a remarkable symbiosis between algae and fungi, a symbiosis that first emerged around 600 million years ago.1 The algal partner provides food through photosynthesis, while the fungal partner provides protection through its filamentous layers and an anchor to the ground from which to draw water and nutrients. This symbiosis allows lichens to thrive in virtually any terrestrial habitat, including Antarctica.2 Lichens are vulnerable to predation and nutrient competition. Lichen evolution has responded to these dangers by producing protective secondary metabolites, for example, antimicrobials and antifeedants. More than 1000 secondary metabolites have been isolated from lichens, some possessing medicinal properties.3 By convention, the name of the fungal partner is also the name of the lichen symbiosis.1 Little information about the genetic programming of lichen secondary metabolite biosynthesis is available because of the challenges of working with lichenizing fungi. Lichens have life spans sometimes extending thousands of years and grow slowly even under controlled conditions.4 The slow rate of growth presents unique challenges, for example, collecting enough DNA for sequencing.4 The fungal partner produces the majority of secondary metabolites; thus, it is necessary to separate and maintain axenic cultures of the fungal partner for study, a challenging feat for obligate symbionts.5 The lichen metabolome is sensitive to environmental stimuli, and its incubatory conditions must be carefully monitored to replicate findings.6 © XXXX American Chemical Society and American Society of Pharmacognosy

Received: September 8, 2017

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DOI: 10.1021/acs.jnatprod.7b00769 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

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Figure 1. Type III polyketide synthase (PKS) gene clusters in the C. uncialis mycobiont genome. Abbreviations: KS, ketosynthase; AT, acyltransferase; DH, dehydratase; MT, C-methyltransferase; ER, enoylreductase; KR, ketoreductase; ACP, acetyl carrier protein.

Figure 2. Multimodule nonribosomal peptide synthetase (NRPS) gene cluster in the C. uncialis mycobiont genome. Abbreviations: A, adenylation; PCP, peptidyl carrier protein; C, condensation.

species of lichenizing fungi. C. uncialis was chosen for study because it is an easily accessible and identifiable species that produces only usnic acid. We were originally interested in identifying the usnic acid biosynthetic gene cluster, requiring genome sequencing, and a preliminary description of polyketide synthase genes.8 Using the secondary metabolite gene cluster annotation software, fungiSMASH (version 4.0),18 we describe up to 48 biosynthetic gene clusters containing type I PKS, type III PKS, NRPS, PKS-NRPS, and terpene synthase genes. We expand upon the preliminary report on type I PKS in C. uncialis8 with a more detailed description of these clusters. The functionality of these gene products (if any) has yet to be experimentally demonstrated. Herein, we will describe a homology mapping approach that was used to provide putative biosynthetic assignments for some of these gene clusters.19 Profiling work in nonlichenized fungi and bacteria demonstrates that more biosynthetic clusters are present in these organisms than metabolites known to be produced by them.20 As this chemotype of C. uncialis is known to produce only usnic acid,8 a similar observation is now made in C. uncialis, and perhaps similar results may extend across lichenizing fungi in general.

chalcone synthase genes deposited in GenBank. Chalcone synthases make up a subgroup of type III PKS that use pcoumaroyl-CoA or cinnamoyl-CoA as starter units and typically perform three condensations with malonyl-CoA.23 If functional, these PKS likely produce small aromatic polyketides. The Cut3pks-1 gene is conspicuous for its proximity to a type I reducing PKS (named Cu-r-pks-1), perhaps suggesting CUT3PKS-1 and CU-R-PKS-1 cooperate in a single biosynthetic pathway. A cytochrome P-450 is observed next to Cu-t3-pks-3, suggesting an oxidative tailoring function. Multimodule NRPS. Nonribosomal peptide synthetases (NRPS) operate independently of mRNA but assemble polypeptides through an assembly line of catalytic domains using proteinogenic and nonproteinogenic amino acids.24 These enzymes minimally require adenylation (A), condensation (C), and peptidyl carrier protein (PCP) domains. Two multimodular NRPS have been previously reported in lichens. One possesses canonical architecture of five modules of A-PCPE-C (where E is an epimerization domain), but with an unusual PCP-C terminal module (F481_01122).16 The second known NRPS bears noncanonical (A-C-A-C-C-A-C-C-C) architecture (F481_04482).16 We now report a multimodule NRPS in the C. uncialis mycobiont as well as what appear to be several postsynthetic tailoring genes. This NRPS gene, named Cu-nrps1, contains four (A-PCP-C) modules with a terminal condensation (C) domain (Figure 2). No terminal thioesterase was found. The absence of a TE domain is an alternative architectural style that is common among fungal NRPS when macrocyclization is used to release the peptide chain.25 The presence of four modules and a terminal condensation domain therefore suggests that this NRPS may produce a cyclized tetrapeptide. FungiSMASH18 identified the amino acids as leucine, isovaline, homoalanine, and glutamine; however, these predictions would need to be confirmed experimentally. This NRPS was located with two cytochrome P-450 oxidases, an aminotransferase, and two fatty acid synthases (Figure 2). Dedicated fatty acid synthases (FAS) can participate in secondary metabolite biosynthesis in cooperation with PKS

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RESULTS AND DISCUSSION Type III Polyketide Synthases. Type III PKS are distinct from type I and II PKS because they lack distinct catalytic domains. A single active site instead performs priming, extension, and cyclization reactions through iterative condensation cycles.21 Type III PKS are known to appear in fungi; however, relatively little is known compared to what is known about fungal type I PKS.22 Experiments using degenerate primers have demonstrated that type III PKS exist in lichens, though complete gene cluster sequence information is unavailable.17 We describe for the first time the presence of type III PKS gene clusters in lichens. Two type III PKS genes, named Cut3pks-1 and Cu-t3pks-2, are displayed (Figure 1). BLAST alignment of both type III PKS revealed consensus similarity to B



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Figure 3. Single-module nonribosomal peptide synthetase (NRPS) gene clusters in the C. uncialis mycobiont genome. Abbreviations: A, adenylation; PCP, peptidyl carrier protein; TE, thioesterase; R, reductase.

Figure 4. Nonribosomal peptide synthetase-polyketide synthase (PKS-NRPS) gene clusters identified in the C. uncialis mycobiont genome. Abbreviations: KS, ketosynthase; AT, acyltransferase; MT, C-methyltransferase; KR, ketoreductase; DH, dehydratase; ER, enoylreductase; ACP, acetyl carrier protein; TE, thioesterase; R, reductase; C, condensation; H, HxxPF domain; A, adenylation; PCP, peptidyl carrier protein.

and NRPS by producing highly reduced acyl products.26 A dedicated FAS producing hexanoic acid for aflatoxin biosynthesis is a well-known example in Fungi.27 The architecture of the NRPS and the types of accessory tailoring genes suggest that the cluster could be responsible for the biosynthesis of a macrocyclized tetrapeptide bound to a fatty acyl chain. Single-Module NRPS. Eight single-module NRPS genes were identified, named Cu-nrps-2 to Cu-nrps-9 (Figure 3). These NRPS are conspicuous because they lack condensation (C) domains. Single-module NRPS lacking condensation domains are known to appear in fungi but are poorly understood.28 Two examples in bacteria include IndC, responsible for the conversion of glutamine to the blue pigment indigoidine in Photorhabdus luminescens,29 and an εpoly-L-lysine synthetase, responsible for the iterative polymerization of L-lysine in Streptomyces albulus.30 Six of the eight single-module NRPS genes found in C. uncialis appear to be associated with postsynthetic tailoring genes. We speculate that the encoded NRPS produce small peptides that are then altered

by one or more distinct chemical steps by the accessory gene products. FungiSMASH18 was unable to predict amino acid usage for any of these atypical NRPS. Hybrid PKS-NRPS. Hybrid PKS-NRPS combine acetatederived polyketide chains from the PKS with amino acids from the NRPS to produce structurally complex natural products.31 Two PKS-NRPS genes have been reported in lichenizing fungi, bearing [A-PCP-KS-AT-KR-ACP-TD] (Gene ID F481_05757) and [KS-AT-DH-ER-KR-ACP-C-A-PCP-TD] (Gene ID F481_00492) architecture.16 We now report three more PKS-NRPS in lichenizing fungi as well as the accompanying accessory genes for each. These are named Cu-pks-nrps-1 to Cupks-nrps-3, and their domain architecture and tailoring genes are displayed (Figure 4). The PKS component of all hybrid PKSNRPS contain KR and DH reducing domains, suggesting that the polyketide is reduced prior to peptidylation. The presence of cis-ER with Cu-pks-nrps-2 and trans-ER with Cu-pks-nrps-3 suggests that the polyacyl moieties would be highly reduced. A trans-TE was observed near Cu-pks-nrps-2, whereas terminal C



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Figure 5. Terpene synthase gene clusters in the C. uncialis mycobiont genome. Abbreviations: KS, ketosynthase; AT, acyltransferase; DH, dehydratase; MT, C-methyltransferase; ER, enoylreductase; KR, ketoreductase; ACP, acetyl carrier protein; GNAT, Gcn5-related Nacetyltransferase.

5) is proposed to be a farnesyl-diphosphate farnesyl transferase (squalene synthase). This enzyme dimerizes two units of farnesyl pyrophosphate into squalene, a 30-carbon triterpene that is an intermediate in sterol biosynthesis. 38 Two aristolochene synthases, Cu-terp-7 and Cu-terp-9 (Figure 5), were also suggested to be encoded in the C. uncialis genome. Aristolochene is a bicyclic sesquiterpene that is derivatized to form a range of mycotoxins, for example, PR toxin from the cheese mold Penicillium roqueforti.39 Type I Polyketide Synthases. Type I polyketide synthases (PKS) are multidomain synthases functionally analogous to fatty acid synthases that vary in their extent of reductions performed on the β-keto group during assembly of acyl monomers.40 The absence or presence of reducing domains subcategorizes the PKS as “reducing” or “nonreducing”. Degenerate primer and genome sequencing studies have observed that PKS genes are ubiquitous among lichenizing fungi and that dozens of PKS genes could be present in a single lichen genome.16,41 We have previously reported on the discovery of 32 type I PKS genes in C. uncialis.8 We now expand these findings by detailing PKS domain architecture, postsynthetic tailoring genes encoded next to PKS genes, and unusual features. We describe 14 nonreducing PKS genes named Cu-nr-pks-1 to Cu-nr-pks-14 and 18 reducing PKS genes named Cu-r-pks-1 to Cu-r-pks-18. Nonreducing PKS genes are shown in Figure 6. Reducing PKS Cu-nr-pks-1 to Cu-nr-pks-4 are shown in Figures 1, 4, and 6, and the remaining 14 are shown in Figure 7. The function of the cluster containing Cunr-pks-2 has been previously proposed to involve usnic acid biosynthesis,8 and the function of the cluster containing Cu-nrpks-8 has been proposed to involve 6-hydroxymellein biosynthesis.9 Some PKS in fungi and bacteria are known to lack essential domains in their architecture. These domains are instead transencoded, and the gene products act as distinct enzymes.42 An ACP-R-like gene near Cu-nr-pks-10 and a DH-KR-like gene near Cu-nr-pks-8 are two genes that were found to be genetically similar to PKS domains (Figure 6). Experimental demonstration of function for these gene products would confirm a role of trans-acting domains in lichen polyketide biosynthesis. We have previously proposed that the DH-KR-

reductase domains are present in the other two genes. FungiSMASH18 was unable to provide confident predictions of amino acid incorporation among these PKS-NRPS. Two PKS-NRPS genes, Cu-pks-nrps-1 and Cu-pks-nrps-2, are noteworthy for their proximity to three terpene synthase genes, herein named Cu-terp-1 to Cu-terp-3. It is possible that these terpene synthases cooperate with the PKS-NRPS to produce chimeric natural products. Terpene Synthases. Terpenes are chains of activated (C5H8)n isoprene units and make up a diverse class of natural products with both structural and functional roles in nature.32 We report nine terpene synthase genes, named Cu-terp-1 to Cuterp-9. Three of these genes were found with PKS-NRPS genes and are displayed in Figure 4. The six remaining genes are shown in Figure 5. One of these terpene synthases, Cu-terp-9, was found next to a reducing type I PKS named Cu-r-pks-2. Putative functions of terpene synthase gene products (Figure 5) can be proposed on the basis of consensus similarity to characterized genes deposited in GenBank. The gene product of Cu-terp-1 is proposed to be pentalenene synthase, a sesquiterpene synthase that cyclizes farnesyl diphosphate into pentalenene.33 The gene product of Cu-terp-2 (Figure 4) was identified as a phytoene synthase. Phytoene is a 40-carbon tetraterpene formed from two molecules of geranylgeranyl pyrophosphate.34 Phytoene is the intermediate to the carotenoid class of natural products, which are highly conjugated terpenoids typically acting as pigments and photoprotectants. The identification of what appears to be a phytoene dehydrogenase proximal to phytoene synthase suggests that these two genes comprise a gene cluster responsible for the biosynthesis of carotenoids or carotenoid precursors. The gene product of Cu-terp-3 (Figure 4) is proposed to be an ent-kaurene synthase, an enzyme that converts copalyl diphosphate to the tetracyclic diterpene entkaurene, an intermediate in the biosynthesis of gibberellins.35 Two genes, Cu-terp-4 and Cu-terp-8 (Figure 5), are suggested to be squalene-hopene cyclases. This enzyme converts squalene into the pentacyclic triterpenoid hopene.36 Consensus similarity identified Cu-terp-5 (Figure 5) as a possible dimethylallyltryptophan synthase. This enzyme binds dimethylallyl pyrophosphate and L-tryptophan to form dimethylallyltryptophan, an intermediate in ergot biosynthesis.37 Cu-terp-6 (Figure D



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Figure 6. Type I nonreducing polyketide synthase (PKS) gene clusters in the C. uncialis mycobiont genome. Abbreviations: SAT, starter acyltransferase; KS, ketosynthase; AT, acyltransferase; PT, product template domain; MT, C-methyltransferase; ACP, acetyl carrier protein; KR, ketoreductase; DH, dehydratase; ER, enoylreductase; CYC, Claisen cyclase; TE, thioesterase; R, reductase; GNAT, Gcn5-related N-acetyltransferase.

Gcn5-related N-acetyltransferases (GNAT) use acetyl-CoA to acetylate substrates, including secondary metabolites. This

like protein functions as a reductase involved in the biosynthesis of 6-hydroxymellein.9 E



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Figure 7. Type I reducing polyketide synthase (PKS) gene clusters in the C. uncialis mycobiont genome. Abbreviations: KS, ketosynthase; AT, acyltransferase; DH, dehydratase; MT, C-methyltransferase; ER, enoylreductase; KR, ketoreductase; ACP, acetyl carrier protein; R, reductase.

have been observed in other fungal PKS.44 Inactivation studies have demonstrated that double ACP domains operate in parallel to increase the rate of catalytic turnover of the PKS.45 Fungal reducing PKS lack SAT, PT, and terminal domains.46 With the exception of Cu-r-pks-15 (Figure 7), this architecture is consistent with reducing PKS genes observed in this study and other documented examples of lichen reducing PKS genes.10,12,15,16,47 A terminal reductase domain found in Cu-rpks-15 instead suggests that the encoded PKS requires

enzyme may also prime ACP domains with acetyl-CoA in lieu of a SAT domain or loading module.43 Consensus similarity suggests a GNAT could be encoded near Cu-nr-pks-12 (Figure 6). A second putative GNAT was encoded next to Cu-terp-5 (Figure 5). Experimental demonstration of GNAT activity would confirm a role for the GNAT superfamily in lichen natural product biosynthesis. Half of the 14 nonreducing PKS genes were observed to possess two ACP domains (Figure 6). Tandem ACP domains F



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lichen was genetically identified as C. uncialis by sequencing the internal transcribed spacer (ITS) region.49 Total genomic DNA was extracted by the described method,50 with the following modifications. Podetia were ground with a mortar and pestle. TES buffer (500 μL) containing 100 mM Tris-HCI and 10 mM EDTA (pH 8.0), and sodium dodecyl sulfate (2%), was added in separate steps and homogenized with the powder. A solution containing 1.4 M NaCl and 1% (v/v) cetyltrimethylammonium bromide (CTAB) was added, and the homogenate was incubated for 1 h at 65 °C. The mix was centrifuged for 5 min at 5000 rpm to pellet cellular debris, and the supernatant was twice extracted with an equal volume of a CHCl3/ isoamyl alcohol mixture [24:1 (v/v)]. Genomic DNA was precipitated by adding 0.2 volume of 5 M NaCl and 2.5 volumes of 100% EtOH to the extract. The DNA was pelleted by centrifugation for 10 min at 13000 rpm, washed with 80% EtOH, and left to air-dry. The dried pellet was resuspended in sterile water and stored at −20 °C until it was needed. The ITS region was amplified by PCR by the described method,51 using forward primer 1780F (5′-CTGCGGAAGGATCATTAATGAG-3′) and reverse primer ITS4 (5′-TCCTCCGCTTATTGATATGC-3′). A 32-cycle PCR was performed as follows: initial denaturation (94 °C for 5 min), denaturation (94 °C for 1 min), touchdown annealing phase (60 to 54 °C for 1 min), extension (72 °C for 1 min), and final extension (72 °C for 5 min). A single amplification product was observed via electrophoresis using 1% (w/v) agarose gel, which was excised and purified with the Wizard SV Gel and PCR Clean-Up System (Promega) in accordance with the manufacturer’s instructions. Primers 1780F and ITS4 were used for DNA sequencing via the BigDye Terminator V3.1 Cycle Sequencing Kit (Applied Biosystems). The DNA was prepared by dissolving the dried DNA sample in 20 μL of formamide, denatured by being incubated for 5 min at 95 °C, and sequenced with a 3130 Genetic Analyzer (Applied Biosystems). BLASTn analysis of this ITS sequence (accession number KY80361) aligned exclusively with deposited C. uncialis ITS sequences among the 20 highest-scoring entries, with identity scores of ≥93% and coverage scores of ≥96% (Table S50). The specimen was therefore morphologically and genetically identified as C. uncialis. Subculturing of the Fungal Partner of C. uncialis. The fungal partner was subcultured from the lichen using the described method,52 with the following modifications. An apothecium was washed and affixed to an inverted Petri dish with petroleum jelly. The apothecium was permitted to eject spores onto 1.5% (w/v) water/agar media. Spores germinating on the plate were then transferred to solid maltyeast extract and incubated at room temperature in the dark. The C. uncialis fungal partner was subcultured in fresh medium every two months for one year. To confirm that a contamination-free culture was created, degenerate primers 1780F and ITS4 were again used to amplify the ITS region. These primers are able to amplify the ITS regions of a broad range of fungal species, including the more than 200 species within the Cladonia genus.51 BLASTn analysis of the resultant ITS sequence (accession number KY80362) aligned exclusively with deposited C. uncialis ITS sequences among the 20 highest-scoring entries, with identity scores of ≥98% and coverage scores of ≥99% (Table S51). These results were interpreted as evidence that an axenic culture of the C. uncialis mycobiont was established. Whole-Genome Sequencing and Contig Assembly. Total genomic DNA was extracted from the cultured mycobiont using the Gen JET Genomic DNA Purification Kit (ThermoFisher Scientific). The genomic DNA was sequenced via MICB DNA sequencing services (Manitoba Institute of Cell Biology, CancerCare Manitoba, University of Manitoba) using an Illumina MiSeq sequencer with a MiSeq Micro V2 sequencing kit. A paired-end DNA library with 150 bp paired-end sequencing reads at 43× coverage was generated. The genome was estimated to be 30 Mb in length, an estimate based on studies of genome length of Cladonia grayi.53 Raw sequence reads were deposited in GenBank and are available under accession number SRR4418292. The raw reads were assembled using four DNA assembly programs: DNAstar, Geneious, SPAdes, and Velvet.54 Contigs produced by SPAdes presented the highest N50 value (34.7 kb) and were therefore used for further analysis. Contigs exceeding 1

reductive termination. The domain architecture of Cu-nr-pks-13 is notable for its lack of a terminal domain (Figure 6). Caveats. The fungal partner of C. uncialis was separated from the algal partner and grown until enough biomass was grown for Illumina DNA sequencing. This process required one year and resulted in a modest genome coverage of 43× for a genome estimated to be 30 megabases in length. By assembling the raw reads with several programs, we were able to generate a contig N50 of 34.7 kb. This process allowed us to identify up to 48 contigs containing putative biosynthetic gene clusters. As a consequence of limited DNA bioavailability, some of the clusters shown in this study appear to be incompletely reported. It is for this reason that we illustrate the entire cluster-bearing contig so that the reader may judge the relative completeness of the cluster. This information will be useful when prioritizing clusters for experimental characterization. For example, Cu-t3pks-2 (Figure 1) is an example of a cluster flanked by large regions of DNA that contain only genes unrelated to secondary metabolite biosynthesis. This suggests that a complete cluster was likely captured. In this case, the cluster is proposed to comprise a cytochrome P-450 and a type III PKS. The cluster containing Cu-t3-pks-1 (Figure 1) is an example of a cluster that appears to have been completely captured at the downstream end; however, the completeness of the cluster upstream of the type I PKS present remains an open question. The cluster containing Cu-r-pks-18 (Figure 7) is an example of a cluster for which both upstream and downstream edges may not have been completely captured by the assembly. We would caution that smaller clusters could be fragments of a larger gene cluster that failed to unite despite efforts with the various assembly programs. It is therefore possible that the true number of biosynthetic gene clusters is fewer than 48. In summary, secondary metabolite biosynthesis in lichenizing fungi is poorly understood because of the challenges of studying lichens. To date, only a few type I PKS gene clusters have been reported, and no definitive evidence links any lichen metabolite to its genetic origin. Genome sequencing and contig assembly of the fungal partner of C. uncialis revealed up to 48 putative biosynthetic gene clusters comprising type I and III PKS, NRPS, PKS-NRPS, and terpene synthases. Our report of gene clusters of type III PKS, NRPS, hybrid PKS-NRPS, and terpene synthases could be particularly useful for functional characterization experiments because they have not been previously described among lichenizing fungi. The chemotype of C. uncialis used in this study is known to produce only usnic acid.8 This is striking considering the apparent number of biosynthetic gene clusters with no obvious function. Similar disparities between total biosynthetic gene clusters and characterized metabolites have been observed among nonlichenizing fungi and bacteria.20 Genome mining and successful activation of cryptic gene cluster fungi and bacteria suggest that these clusters in C. uncialis could also be functional and produce hitherto undiscovered secondary metabolites.48 The adaptation of functional heterologous expression methodologies to lichen gene clusters will be required to bridge the gap between gene surveying and metabolite discovery. We are exploring these methods and hope to report on our progress in the near future.

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EXPERIMENTAL SECTION

Identification of Lichen Sample. C. uncialis (voucher number Normore 8774, Herbarium, University of Manitoba, Winnipeg, MB) was morphologically identified and collected from northern Manitoba on a south-facing granite ridge (N54°42′24.7″, W101°33′53.1″). The G



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kb in length (2109 in total) were deposited in GenBank under accession number NAPT00000000. Annotation and Illustration of Lichen Gene Clusters. The draft contig library was uploaded to the Antibiotics and Secondary Metabolites Analysis Shell (FungiSMASH version 4.0)18 at http:// www.secondarymetabolites.org. The domain architectures of PKS and NRPS identified by FungiSMASH (version 4.0) were verified with BLAST.55 The broad functional role of all other genes (e.g., Omethyltransferase) was predicted with BLAST and is based on consensus assignment of genetically similar sequences deposited in GenBank. In some cases, it was necessary to suggest more than one putative function due to a lack of consensus. BLAST statistics, including the most closely similar gene for each annotated gene, are provided (Tables S1−S48). Any gene with coverage values of

Lichen Biosynthetic Gene Clusters. Part I. Genome ... - ACS Publications

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