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Identification of 6‑Hydroxymellein Synthase and Accessory Genes in the Lichen Cladonia uncialis Mona Abdel-Hameed,†,§ Robert L. Bertrand,†,§ Michele D. Piercey-Normore,‡ and John L. Sorensen*,† †
Department of Chemistry and ‡Department of Biological Sciences, University of Manitoba, Winnipeg, Manitoba, Canada, R3T 2N2 S Supporting Information *
ABSTRACT: A transcribed polyketide synthase (PKS) gene has been identified in the lichen Cladonia uncialis. The complete nucleotide sequence of this PKS was determined from the amplified cDNA, and an assignment of individual domains was accomplished by homology searching using AntiSMASH. A scan of the complete genome sequence of C. uncialis revealed the accessory genes associated with this PKS gene. A homology search has identified that several genes in this cluster are similar to genes responsible for the biosynthesis of terrein in Aspergillus terreus. This permitted assignment of putative function to each of the genes in this new C. uncialis cluster. It is proposed that this gene cluster is responsible for the biosynthesis of a halogenated iscoumarin. This is the first report linking a gene cluster to a halogenated metabolite in lichen.
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only two examples of lichen secondary metabolite gene clusters with putative functions assigned. The challenges in lichen fungal metabolic and genomic profiling, as compared to nonlichen fungi and bacteria, are the result of the slow growth rates for the wild-type lichen and difficulties in subculturing the fungal partner.14 This makes many of the assignment of function experiments, such as gene deletions, difficult to achieve in lichen fungi. In the absence of functional heterologous expression, the assignment of function by homology searching may serve as a substitute for gene deletion experiments. The gene cluster reported here, the third with an assigned function, provides further insight into lichen polyketide biosynthesis. In particular this is the first demonstration that a biosynthetic pathway is shared between lichenized and nonlichenized fungi.
ichens are associations between symbionts of fungi and algae (or cyanobacteria) that thrive in a wide range of terrestrial habitats.1 Lichens have extensive uses among traditional peoples as medicines, dyes, cosmetics, aphrodisiacs, and food.2 Polyketides are one of the most prevalent classes of secondary metabolites that are produced by lichens3 and are closely associated with the use of lichens as traditional medicines.4 The presence of polyketide natural products suggests that lichen may serve as a source of lead compounds in the search for new therapeutics5 as well as other potential applications such as new materials.6 Polyketide natural products are produced by polyketide synthases (PKSs), which have been well characterized from nonlichen fungi and bacteria7 but much less so from lichen fungi. Polyketide synthases add structural diversity to the polyketide products through variations in extent of reduction, number of carbon chain extensions, type of carbon extension unit used, and modes of aromatization.8 In addition, the presence of postsynthetic tailoring enzymes can further expand the structural diversity of the polyketide natural products.9 Halogenation is one such post-PKS modification of interest to pharmacology and industry because halogenated natural products often display a broad range of potent biological activities.10 We report the characterization of a complete biosynthetic gene cluster from the lichen Cladonia uncialis that appears similar to genes involved in the terrein (1) biosynthetic pathway recently elucidated in Aspergillus terreus.11 The completion of a de novo draft assembly of the C. uncialis genome12 enabled putative functional assignment of this pathway based on genetic, architectural, and phylogenetic lines of evidence. On the basis of assignment of function by homology with the terrein (1) pathway the newly identified gene cluster in C. uncialis is proposed to produce a halogenated isocoumarin derived from 2,3-dehydromellein (2). The usnic acid12 and grayanic acid13 biosynthetic gene clusters remain the © XXXX American Chemical Society and American Society of Pharmacognosy
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RESULTS AND DISCUSSION The initial experiments on C. uncialis were aimed at identifying the number of potential PKS genes that may be present in the genome of this lichen. To this end, four separate sets of degenerate primer pairs (Table S1 in the Supporting Information) that had been designed to amplify the ketosynthase (KS) domain of a putative PKS gene were used. The ketosynthase domain is responsible for the key carbon− carbon bond forming step in polyketide biosynthesis and therefore retains a high degree of homology across several species. The ketosynthase domain can be potentially used in a predictive manner to assign function.15 In separate experiments these degenerate primer pairs were used to produce four unique amplicons with sizes of 749, 644, 674, and 2683 bp, respectively (Figures S2−S4 in the Supporting Information). Received: March 21, 2016
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Figure 1. Architecture of the CU-PKS-4 gene cluster as suggested by AntiSMASH (V. 3.0). Functional assignments of each gene are based on consensus assignment of homologous genes of known function deposited in GenBank (see also Table 1).
Table 1. Cladonia uncialis CU-PKS-4 Gene Cluster Accession Numbers and BLAST Results gene in PKS-4 cluster
accession no.
putative function
species highest homology (accession no.)
identity/query coverage
terrein pathway homologue (accession no.)
identity/query coverage
(1) (2) (3) (4) (5)
KU740324 KU740325 KU740326 KU740327 KU740328
monooxygenase KR-DH peptide type I PKS halogenase O-methyltransferase
C. metacorallifera (ADR00967) P. scopiformis (KUJ09199) C. grayi (ADX36086) S. borealis (ESZ96411) P. scopiformis (KUJ06291)
79/99 67/99 73/99 56/98 77/99
TerD (XP_001210228) TerB (XP_001210230) TerA (XP_001210231) N/A N/A
68/99 46/79 59/99 N/A N/A
Figure 2. Key biosynthetic genes associated with terrein (1) biosynthesis in Aspergillus terreus. These genes are as described in ref 11, and the regulatory genes have not been included. The polyketide synthase terA has a domain architecture identical to cu-pks-4. Some of the accessory genes terB−terF have also had their function assigned (see text).
Subsequent sequencing of each amplicon demonstrated that the nucleotide sequences of each gene fragment were unique with respect to each other, suggesting the presence of at least four different PKS genes in C. uncialis. These putative PKS genes were named cu-pks-1 to cu-pks-4, and the raw nucleotide sequences of these gene fragments are available in the Supporting Information. In an effort to narrow the search for genes related to secondary metabolite production it was determined whether any of the genes cu-pks-1 to cu-pks-4 were transcriptionally active. The total mRNA from a sample of C. uncialis was extracted and reverse transcribed into a complete cDNA library. The same four pairs of degenerate primers, designed to amplify the KS domain, were used to screen the cDNA for transcriptionally active PKS genes. Amplification from only one gene fragment was observed, and this corresponded to the cu-pks-4 gene (Figure S5 in the Supporting Information). Control experiments demonstrated that the RNA extracts were free of contaminating DNA (Figure S1 in the Supporting Information). This suggested that cu-pks-4 was, at the very least, transcriptionally active and may potentially have a role in natural product biosynthesis. In order to generate a complete sequence of cu-pks-4, the rapid amplification of cDNA ends (RACE) technique was used as a method to identify both the 3′ and 5′ ends of the gene. This technique takes advantage of the poly-A tail to identify the 3′ end of the cDNA (using primer RACER in Table S1 in the Supporting Information) and a gene-specific primer (KSATF in Table S1 in the Supporting Information) to amplify the 3′ terminal portion of the cDNA. The identification of the 5′ end of the cDNA was accomplished by using the primer pair RACEF and KSATR in Table S1 in the Supporting Information. These experiments generated a short fragment of DNA, which in turn was sequenced. This nucleotide sequence was used to design a new set of primers that allowed
for the amplification of the adjacent fragment of the cDNA. The process was continued in an iterative fashion until the fulllength gene of 6150 bp was sequenced. An initial homology search by BLAST determined that this gene had not been previously deposited in GenBank. However, consensus homology with deposited genes suggested that this gene was a polyketide synthase gene. This led to the conclusion that a novel lichen PKS had been identified. To determine the domain architecture of this newly identified gene, an early version of AntiSMASH (V. 1.0) was used.16 This analysis determined that cu-pks-4 is an iterative type I PKS that possesses a ketosynthase (KS), an acyl transferase (AT), two acyl carrier proteins (ACP), and a terminal thioesterase (TE) domain. A further manual BLAST17 search of cu-pks-4 also suggested the presence of a starter acyl transferase (SAT)18 and product template (PT) domain2 (Figure 1). Subsequent to this preliminary assignment of the domain architecture the sequencing of the complete genome of C. uncialis was carried out, the details of which are described in an earlier publication. 12 This information enabled the expansion of this preliminary work by identifying cu-pks-4 in the genome of C. uncialis. By searching genomic contigs for the cu-pks-4 sequence, a contig 54157 bp in length was found containing the gene. The annotation of post-PKS tailoring genes associated with cu-pks-4 also became possible. This was accomplished by using the latest version of AntiSMASH (V. 3.0),19 which includes the ability to assign putative function by homology with known genes in related biosynthetic clusters. This process identified four tailoring genes that appear to be associated with cu-pks-4, and their functions were assigned based on consensus assignment of characterized homologous genes deposited in GenBank. These genes include an upstream FAD-binding monooxygenase, an upstream PKS-like motif with ketoreductase and dehydratase (KR-DH) domains, a downB
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the terA gene from A. terreus was observed, suggesting that the product of CU-PKS-4 is indeed 2,3-dehydro-6-hydroxymellein (2) (Table 1). In addition, high homology between the separately encoded KR-DH in C. uncialis (gene 2 in Figure 2) and terB in A. terreus was also observed. High homology was also observed for the monooxygenase in C. uncialis (gene 1 in Figure 1) and terD in A. terreus. There are no counterparts for the C. uncialis O-methyltransferase (gene 5 in Figure 1) or the halogenase (gene 4 in Figure 1) in the terrein gene cluster of A. terreus (Table 1). However, the AntiSMASH and manual BLAST searches strongly suggest that the assigned functions for these genes are indeed correct. On the basis of the similarities in the two pathways it is proposed that the natural product produced by this gene cluster in C. uncialis is an oxidized, methylated, and halogenated derivative of 6hydroxymellein, shown as compound 5 in Figure 4. It is suggested that the PKS and KR-DH genes in C. uncialis have a similar function to that in A. terreus, resulting in the biosynthesis of 6-hydroxymellein (3). The monooxygenase suggests that 3 would be hydroxylated, potentially at C-7 or likely at C-5, to give 4. An O-methylation followed by a halogenation would result in the production of compound 5. Although compound 5 does not appear to have been previously reported in the literature, there have been several related natural products described. For example, both compounds 6 and 7 were reported from the ascomycete Lachnum papyraceum.20 In addition, compound 8 was reported from a species of the lichen genus Graphis.21 The HPLC analysis of both wild and cultured C. uncialis have been previously reported, and no metabolite was discovered that was consistent with this pathway.12 However, the fact that transcription of cupks-4 was observed does suggest that the pathway may be functional. The final confirmation of function for this pathway will be functional expression in a nonlichen fungus or yeast. To place this gene cluster in evolutionary context and to further lend support to the putative assignment of function, phylogenetic analyses on cu-pks-4, gene 2 (KR-DH), and gene 1 (monooxygenase) from the C. uncialis gene cluster were performed. The KS domain of cu-pks-4 was used for phylogenetic analysis of the PKS genes, as this domain catalyzes the carbon−carbon bond forming reactions common to all PKSs and many KS domain sequences have been deposited in GenBank, further facilitating the analysis. As PKSs with recent evolutionary origin often retain similar function, it was anticipated that cu-pks-4 could cluster with other PKSs of known function, which would lend support to our assignment
stream halogenase, and a downstream O-methyltransferase (Figure 1). This is the first report of a lichen PKS gene with an associated halogenase post-PKS tailoring gene. The gene cluster is flanked by 7.2 and 26.1 Kb of DNA in which no other accessory genes could be detected, suggesting that the complete biosynthetic gene cluster was identified. These genes have been deposited in GenBank, and the accession numbers with BLAST homology data are displayed in Table 1. The most recent version of AntiSMASH (V. 3.0) includes an algorithm for predicting the function of biosynthetic gene clusters based on homology to characterized pathways. In this case, the CU-PKS-4 gene cluster displayed a 27% similarity to a PKS that is responsible for terrein (1) biosynthesis in Aspergillus terreus.11 Terrein (1) biosynthesis requires six enzymes, encoded by genes TerA to TerF as shown in Figure 2. Terrein (1) biosynthesis proceeds with the assembly of the nonreduced pentaketide 2,3-dehydro-6-hydroxymellein (2) by the PKS TerA (Figure 3). Reduction by a separately encoded
Figure 3. Biosynthesis of terrein in Aspergillus terreus.
KR-DH peptide (TerB) yields 6-hydroxymellein (3). The enzymes TerC to TerF are all FAD-dependent monooxygenases or multicopper oxidases that are required for the conversion of 3 to 1.11 As the functions of TerC to TerF remain unassigned, the order of each oxidation step has yet to be fully elucidated. The genes in the cluster identified in C. uncialis with the genes in A. terreus that are responsible for the biosynthesis of terrein were then compared for similarity. A manual homology search using BLAST was performed to determine the similarity between the individual genes in the clusters. As expected, a high degree of homology between the cu-pks-4 from C. uncialis and
Figure 4. Proposed new natural product, compound 5, that is produced by the newly identified gene cluster in C. uncialis. The substituents shown in red on 5 can be variable; however their positions are based on precedent from known lichen metabolites (6−8). C
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of function.22 It was expected that cu-pks-4 and genes 1 and 2 from C. uncialis would all form a unique phylogenetic cluster with the terA, terB, and terD counterparts from A. terreus, thereby implying similar roles in secondary metabolite production. This prediction is consistent with the observations shown in the condensed phylogenetic trees (Figure 4; for complete trees see Figures S6−S8 in the Supporting Information). The C. uncialis monooxygenase (gene 1) clustered with A. terreus terD and four other genes, a distinct evolutionary history from the remaining 30 genes studied, which instead formed separate clusters and subclusters (Figure 5; Figure S6 in the Supporting Information). Gene 2 (KR-DH)
evidence that the three C. uncialis enzymes are true homologues of their respective A. terreus counterparts and that putative functional assignments could be reasonably proposed based on this relationship. In conclusion the sequencing of a gene encoding a type I iterative nonreducing polyketide synthase with SAT-KS-ATPT-ACP-ACP-TE architecture from the lichen C. uncialis is reported. The assembly of a draft genome sequence of C. uncialis12 enabled the annotation of the entire gene cluster and the proposition of a metabolite arising from the pathway. The gene cluster encodes a halogenase, an O-methyltransferase, a monooxygenase, and a peptide with ketoreductase-dehydratase-like features. Gene cluster architecture, genetic homology, and phylogenetic analyses suggest that the PKS, monooxygenase, and KR-DH peptide are homologous to characterized genes of the terrein pathway. It is proposed that this gene cluster encodes biosynthetic machinery for the production of a halogenated isocoumarin with a structure similar to that proposed for compound 5 in Figure 3. This is the first report linking a gene cluster to the production of a halogenated natural product in a lichen. A final confirmation of function will require functional heterologous expression of this gene cluster in a nonlichen fungus or yeast.
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EXPERIMENTAL SECTION
Collection and Taxonomic Identification of Cladonia uncialis. C. uncialis (voucher number: Normore 8774, Herbarium, University of Manitoba, Winnipeg, Canada) was morphologically identified and collected in October 2008 from Northern Manitoba on a south-facing granite ridge (N 54°42′24.7″; W 101°33′53.1″). The specimen was air-dried and stored until needed. DNA isolation was performed following a previously published procedure.23 The nuclear ribosomal internal transcribed spacer (ITS) region24 was amplified from the total DNA extract using primers 1780F/ITS4,25 as described elsewhere.26 Primer sequences are listed in Table S1 in the Supporting Information. A single amplification product was observed on a 1% (w/ v) agarose gel. This band was purified using the Wizard SV gel and PCR clean-up system (Promega) in accordance with the manufacturer’s instructions. The DNA was sequenced using primers 1780F/ ITS4 and BigDye (V. 3.1; Applied Biosystems, Foster City, CA, USA): The amplified DNA was dried, dissolved in 20 μL of formamide, denatured at 95 °C for 5 min, placed on ice, loaded into a 96-well plate, and sequenced with an Applied Biosystems 3130 genetic analyzer. The DNA sequence matched other sequences deposited for C. uncialis with 95% identities and 0% gaps, thereby confirming the specimen as C. uncialis. Detection of PKS Genes from Cladonia uncialis gDNA. To detect possible polyketide synthase genes encoded within the C. uncialis genome, four pairs of degenerate PCR primers were employed: LC3/LC5c | PKS1F/PKS2R | PKSDA13F/PKSDA13R | PKS1F1/JSAT2R (Table S1 in the Supporting Information). The original use of the first three primer pairs is described elsewhere27 and is designed to target a segment of the ketosynthase domain. The fourth primer pair targets a longer stretch of DNA covering both the KS and AT domains and was designed based on conserved DNA regions found in lichen PKS genes deposited in GenBank (this study). Amplification of DNA by PCR was performed with the following reaction mixture: 1× Phusion HF buffer (New England Biolabs, Whitby, ON, Canada), 200 μM of each dNTP (Promega, Madison, WI, USA), 0.5 μM of each primer, 2 U/μL of Phusion DNA polymerase (New England Biolabs), 3% DMSO, 50 ng of template cDNA, and enough water to total 50 μL. The initial denaturation was at 98 °C for 60 s, followed by 30 cycles of denaturation at 98 °C for 10 s, annealing ranging from 52 to 56 °C for 30 s (depending on the primer pair used), extension at 72 °C for 60 s, final extension of 72 °C for 5 min, and subsequent cool down to 4 °C. Each primer pair produced a single amplification product, named cu-pks-1 to -4 (Figures
Figure 5. Condensed phylogenetic trees demonstrating clustering of Aspergillus terreus terrein biosynthetic genes with genes from the Cladonia uncialis PKS gene cluster: (A) gene 1 (monooxygenase); (B) gene 2 (KR-DH); (C) cu-pks-4. Evolutionary distances are drawn to scale and are in units of number of amino acid substitutions per site. Nodes with confidence probabilities of 70% or greater are displayed. Each tree was constructed with 37 amino acid sequences and were chosen based on greatest homology to the investigated C. uncialis gene as determined by BLAST analysis. The species and accession numbers are displayed. The C. uncialis genes of interest and the identified A. terreus homologues are highlighted in bold. A Saccharomyces cerevisiae monooxygenase (EDV10006), S. cerevisiae fatty acid synthase (CAA54218), and Magnaporthe oryzae polyketide synthase (ELQ39647) were chosen as out-groups for the C. uncialis monooxygenase, KR-DH peptide, and PKS, respectively. The complete phylogenetic trees are displayed in Figures S6−S8 in the Supporting Information.
also formed a cluster with the A. terreus terB and a gene identified in nonlichen fungi Oidiodendron maius that proved distinct from the evolutionary histories of 33 other genes studied (Figure 4B; Figure S7 in the Supporting Information). The C. uncialis PKS also proved to have a unique evolutionary history that was shared only with the A. terreus terA and three other PKSs (Figure 4C, Figure S8). In summary, BLAST analysis revealed that the C. uncialis gene cluster is highly homologous to A. terreus terD, terB, and terA. The construction of these trees also suggested that the evolutionary history of the C. uncialis genes are shared with their A. terreus counterpart in the terrein biosynthetic cluster. In addition both the C. uncialis and A. terreus gene clusters are distinct from other homologous genes deposited in GenBank. These findings are interpreted as D
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S2−S4 in the Supporting Information). The DNA was sequenced by a commercial service supplied by GeneWiz (South Plainfield, NJ, USA). Extraction of Total RNA. Dry lichen thallus was rehydrated with dH2O and incubated for 2 h in natural light at room temperature. Approximately 100 mg of thallus was lysed by freezing with liquid N2 and grinding with a mortar and pestle. Total RNA was extracted from whole lichen thallus using the RNeasy Plant Mini kit (Qiagen, Mississauga, ON, Canada) following the manufacturer’s directions. To remove DNA, the RNA extract was treated with DNase I (Invitrogen, Burlington, ON, Canada) according to the manufacturer’s instructions. Testing for DNA Contamination. To confirm that no contaminating DNA was present in RNA extracts following DNase application, two PCR reactions were attempted on both DNasetreated RNA and genomic DNA extracts from C. uncialis using primer pairs PKS1F/PKS2R and LC1/ISATc. The expected outcome was that amplification products would be produced from the gDNA but not the RNA extract under identical conditions. The PCR reaction was performed using 1× Phusion HF buffer (New England Biolabs), 200 μM each dNTP (Promega), 0.5 μM each primer, 2U/μL of Phusion DNA polymerase (New England Biolabs), 3% DMSO, 50−100 ng of genetic material, and enough dH2O to total 50 μL. Initial denaturation was at 98 °C for 60 s followed by 30 cycles of denaturation at 98 °C for 10 s, annealing at 54 or 56 °C for 30 s (depending on primer pair), extension at 72 °C for 60 s, final extension of 72 °C for 5 min, and cool-down to 4 °C. As expected, a single amplification product was observed from the DNA template but not RNA extract, confirming that DNA was not present in the isolated RNA (Figure S1 in the Supporting Information). Sequencing of Transcribed PKS Gene. An aliquot of total RNA was converted to cDNA using oligo dT primers and the ThermoScript RT-PCR System (Invitrogen) following the manufacturer’s directions. The same primer pairs that were described in the Detection of PKS Genes from C. uncialis gDNA section were used in this step. A short DNA sequence corresponding to cu-pks-4 was amplified (Figure S5 in the Supporting Information). This suggested that cu-pks-4 was the only transcriptionally active gene of the four identified above. To sequence cu-pks-4, the gene was assembled using the rapid amplification of cDNA ends (RACE) technique.28 In separate reactions, 3′-RACE and 5′-RACE-cDNA synthesis was performed on RNA extracts of the lichen thallus using the FirstChoice RLM-RACE kit (Invitrogen) according to the manufacturer’s protocols. The 3′-RACE primer was used for reverse transcription of total RNA into cDNA using the ThermoScript RT-PCR System (Invitrogen) following the manufacturer’s directions. Gene-specific primers KSATF and KSATR (Table S1) were designed from the partial PKS sequence of cu-pks-4 obtained in earlier experiments. These primers were combined with RACE primers RACER and RACEF (Table S1 in the Supporting Information) to amplify in separate reactions fragments of cu-pks-4 cDNA that corresponded to the 3′ and 5′ ends of the gene. The PCR reaction mix contained 1× long amplification polymerase buffer, 200 μM each dNTP, 0.5 μM each primer, 2 U/μL long amplification polymerase (New England Biolab, Canada), 80 ng of cDNA, and enough dH2O to total 50 μL. Initial denaturation was at 95 °C for 2 min, followed by 30 cycles of denaturation at 94 °C for 30 s, annealing at 56 or 60 °C (depending on primer pair) for 15 s, extension at 65 °C for 4 min, final extension at 65 °C for 10 min, and cool-down to 4 °C. The gene was sequenced through primer walking (iterations of sequencing and primer design) until the entire cDNA transcript was sequenced (GeneWiz Services; South Plainfield, NJ, USA). Annotation of the PKS gene was performed using Antibiotics and Secondary Metabolites Analysis Shell (AntiSMASH version 3.0), freely available for academic use at http://www.secondarymetabolites.org/.16,19 Genes of interest were analyzed by BLAST.17 The 35 highest homologous returned sequences were used to construct a phylogenetic tree using the neighbor-joining method29 with MEGA6 software.30 Confidence probability was estimated using 1000 replicates of the interior branch test31 and is reported in percentage at each node. Evolutionary distances were computed using the Poisson correction method32 and are reported in units of amino acid substitutions per site.
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ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.6b00257. Gel images, nucleotide sequences, and complete phylogenetic trees (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. Tel: +1-204-4749504. Author Contributions §
M. Abdel-Hameed and R. L. Bertrand made equal contributions to this work.
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS National Sciences and Engineering Research Council of Canada (NSERC) Discovery Grants awarded to J.L.S. and to M.P.N. are gratefully acknowledged. An NSERC postgraduate scholarship awarded to R.L.B. is also gratefully acknowledged. All authors acknowledge additional financial support from the Department of Chemistry and the University of Manitoba.
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REFERENCES
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DOI: 10.1021/acs.jnatprod.6b00257 J. Nat. Prod. XXXX, XXX, XXX−XXX