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Chrysogine Biosynthesis Is Mediated by a Two-Module Nonribosomal Peptide Synthetase Rasmus Dam Wollenberg,†,⊥ Wagma Saei,†,⊥ Klaus Ringsborg Westphal,† Carina Sloth Klitgaard,† Kåre Lehmann Nielsen,† Erik Lysøe,‡ Donald Max Gardiner,§ Reinhard Wimmer,† Teis Esben Sondergaard,† and Jens Laurids Sørensen*,†,# †
Department of Chemistry and Bioscience, Aalborg University, Fredrik Bajers Vej 7H, 9220 Aalborg Ø, Denmark Department of Biotechnology and Plant Health, NIBIONorwegian Institute of Bioeconomy Research, Høgskoleveien 7, 1430 Ås, Norway § Commonwealth Scientific and Industrial Research Organization (CSIRO) Agriculture and Food, Queensland Bioscience Precinct, Brisbane, Australia # Department of Chemistry and Bioscience, Aalborg University, Niels Bohrs Vej 8, 6700 Esbjerg, Denmark ‡
S Supporting Information *
ABSTRACT: Production of chrysogine has been reported from several fungal genera including Penicillium, Aspergillus, and Fusarium. Anthranilic acid and pyruvic acid, which are expected precursors of chrysogine, enhance production of this compound. A possible route for the biosynthesis using these substrates is via a nonribosomal peptide synthetase (NRPS). Through comparative analysis of the NRPSs from genome-sequenced producers of chrysogine we identified a candidate NRPS cluster comprising five additional genes named chry2−6. Deletion of the two-module NRPS (NRPS14 = chry1) abolished chrysogine production in Fusarium graminearum, indicating that the gene cluster is responsible for chrysogine biosynthesis. Overexpression of NRPS14 enhanced chrysogine production, suggesting that the NRPS is the bottleneck in the biosynthetic pathway.
C
the same biosynthetic pathway, with 3 as the earliest entry. Compound 3 consists of an anthranilic acid moiety linked to pyruvic acid, and a fermentation study of P. chrysogenum with these substrates significantly increased production of 1.1 This indicates that the biosynthetic pathway could be mediated by a nonribosomal peptide synthetase (NRPS), which is a huge multidomain enzyme capable of recruiting amino acids and hydroxy acids via substrate-specific adenylation domains (A).17 The adenylated substrate is then transferred by a peptide acyl carrier domain (T or PCP) through a thioester bond to a condensation domain (C), where a peptide bond is formed.18 The compound can then be released by cyclization, reduction, or hydrolysis and subsequently modified by additional tailoring enzymes. In fungal genomes, these modifying enzymes are often encoded by genes located in close proximity to the NRPS, constituting a biosynthetic gene cluster.19 We have observed production of chrysogine in the genomesequenced strains of Fusarium graminearum (PH-1) and Fusarium pseudograminearum (CS3096) (Supplementary Figure 1), and chrysogine has furthermore been identified in three sequenced Fusarium avenaceum strains20 and one F. langsethiae strain.21 The responsible NRPS should therefore be present in
hrysogine (1; 2-(1-hydroxyetyl)-4(3H)-quinazolinone) is an alkaloid natural product first isolated from the filamentous fungus Penicillium chrysogenum.1 The compound has subsequently been identified in several other Penicillium species2−8 as well as members of the genera Aspergillus9 and Fusarium.10−15 The biological role of chrysogine has only been briefly examined and has shown no toxicity toward human cancer cell lines and various microorganisms.2 Two similar compounds [2acetyl-4(3H)-quinazolidinone (2) and 2-pyruvoylaminobenzamide (3)], together with chrysogine, have been identified in Penicillium and Fusarium.1,11,15 Compound 3 has furthermore been isolated from Colletotrichum lagenarium, where it was shown to inhibit the activity of the plant hormone auxin.16
Despite this relatively extensive list of chrysogine-producing genera, the biosynthetic pathway has not yet been identified. The related structures of compounds 1−3 suggest that they are part of © 2017 American Chemical Society and American Society of Pharmacognosy
Received: September 8, 2016 Published: July 14, 2017 2131
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Figure 1. Comparison of chrysogine clusters. (A) Overview of the hypothetical chrysogine clusters in Fusarium graminearum, F. culmorum, F. pseudograminearum, F. avenaceum, Microsporum canis, Colletotrichum orbiculare, Aspergillus nomius, and P. chrysogenum showing the predicted coding regions of the six cluster genes. (B) Similarity matrices (% identity) of the amino acid sequences of the six proteins based on clustalW alignments.
the genome-sequenced F. avenaceum, F. graminearum, and F. pseudograminearum strains, and the structure of chrysogine suggests that the NRPS has two functional A domains. Revisiting the comparative study of NRPSs in Fusarium revealed that NRPS14, which has an ATC-ATC domain structure, is the only two-module NRPS present in these strains.22,23 A conserved gene cluster containing five additional genes (chry2−6) was furthermore identified surrounding NRPS14 (=chry1) in F. avenaceum (FAVG1_11315−FAVG1_11320), F. graminearum (FGSG_11394−FGSG_11399), and F. pseudograminearum (FPSE_11015−FPSE_11020; Figure 1 and Supplementary Table 1). In addition to the two-module NRPS (chry1), the genes in the cluster are predicted to encode a two-module enzyme capable of transferring an amino group from glutamine (chry2): a desaturase (chry3), a dehydrogenase (chry4), oxidoreductase (chry5), and an amidotransferase (chry6). The gene cluster was also identified in Microsporum canis (MCYG_08601− MCYG_08606) and Colletotrichum orbiculare (Cob_01941− Cob_01946), while an incomplete version was also found in Aspergillus nomius (ANOM_009756−ANOM_009760), where chry4 was absent. A previous study has shown that A. nomius does produce chrysogine, in contrast to closely related species, which could be due to the incomplete gene cluster.24
In Fusarium culmorum the complete gene cluster is also present (BN852_0094320−BN852_0094350), although chry6 has not been annotated. Similarly, the genes are also located on two contigs in F. langsethiae with a gap between chry2 and 3 (data not shown). The gene cluster in P. chrysogenum differed from the other species by the presence of a transcription factor (Pc21g12640) and a rearrangement of chry6, which had relocated to the other side of the cluster, after a gene with unknown function. This transcription factor is not present in the proximity of the other clusters, suggesting that the transcription factor is either not involved in chrysogine biosynthesis in P. chrysogenum or a different regulation in Fusarium, independent of the transcription factor. Alignment of the amino acid sequences of the six genes furthermore showed that chry1−5 had a high sequence similarity within the Fusarium genus (61−99%) and among the different genera (48−85%) (Figure 2B). chry6 displayed, however, a higher level of intergenus variance, which was specifically noticeable for P. chrysogenum (22−50%). Recently, Driessen and co-workers reported that the chrysogine cluster had been tentatively identified in P. chrysogenum.25,26 The cluster was proposed to consist of chry1−6, the transcription factor, and a gene with unknown function (Pc21g12570).25−27 Transcriptional analysis of secondary metabolite genes in F. graminearum has previously shown that NRPS14 was active 2132
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The sequence coverages were 1.7 in the deletion and 14.8 in the overexpression mutant, respectively (data not shown). The strains were subsequently analyzed for production of secondary metabolites by reversed-phase high-performance liquid chromatography (RP-HPLC) coupled to a high-resolution mass spectrometer (HRMS). The base peak chromatogram showed that the metabolite profile of the wild type was comparable to ΔNRPS14 and OE::NRPS14 (Figure 3A). The extracted ion
Figure 2. Expression of the chrysogine cluster and neighboring genes in Fusarium graminearum and Fusarium pseudograminearum. (A) Hierarchical clustering of the proposed chrysogine gene cluster in F. graminearum based on the expression profiles from wheat and barley infection studies after 24, 48, 72, 96, 144, and 192 h after inoculation.28,29 (B) Expression in Fusarium pseudograminearum during infection of barley and Brachypodium in comparison to the trichodiene synthase encoding gene (TRI5). FPKM: fragments per kilobase of transcript per million mapped reads. The bars represent the average of four biological replicates, with error bars representing the standard deviation.
Figure 3. Production of secondary metabolites. (A) Partial base peak chromatogram (BPC) of F. graminearum wild type (Wt), OE::NRPS14, and ΔNRPS14. (B) Partial extracted ion chromatogram (EIC) for chrysogine ([M + H]+: 191.0815 ± 0.005) from a chrysogine standard, Wt, OE::NRPS14, and ΔNRPS14. (C) Mean peak area of chrysogine in Wt, OE:: NRPS14, and Δ NRPS14 (n = 4; error bars represent standard error of the mean).
during infection of wheat and barley, but not expressed on various growth media.28 Hierarchical clustering analysis of the proposed chrysogine cluster and neighboring genes showed furthermore that chry2−5 were expressed in a similar pattern to NRPS14 (Figure 2A). However, chry6 and the neighboring genes were, however, expressed to a lower degree and did therefore not group together with the other members of the gene cluster. NRPS14 and chry2−5 were also expressed in F. pseudograminearum during infection of barley and Brachypodium distachyon (Figure 2B). chry6 was also expressed, but at a lower level than the remaining cluster genes. The orthology and transcription data suggests that chry6 is not involved in the chrysogine biosynthetic cluster. To verify that NRPS14 is responsible for chrysogine biosynthesis, a deletion and overexpression strategy was applied in F graminearum (Supplementary Figure 2A). Transformation of F. graminearum resulted in one deletion (ΔNRPS14) and one overexpression (OE::NRPS14) mutant, which were verified by diagnostic PCR (Supplementary Figure 2B). Single and correct integration events were verified by whole genome sequencing.
chromatogram for chrysogine showed that chrysogine could be detected in the wild type, while overexpression of NRPS14 enhanced the production more than 5-fold (Figure 3B and C). Chrysogine could not be detected in the deletion mutant, suggesting that NRPS14 is responsible for biosynthesis of this compound. Transcriptional analyses by RT-PCR showed that NRPS14 expression could be detected only in the OE::NRPS14 mutant. The enhanced production in the OE::NRPS14 mutant suggests that the NRPS is the limiting factor in the biosynthetic pathway or due to a feedback mechanism where the product of the NRPS induced transcription of the other genes. This could be due to the observed lower expression levels or due to slower substrate conversion. The structures of 2 and 3 suggest that they are likely intermediates of a pathway that has chrysogine as an end product. The initial compound is produced by NRPS14, which is capable of adenylating two substrates. Predicting the substrates of 2133
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(10 μM), 2 μL of reverse primer (10 μM), 1 μL of template (F. graminearum PH-1 genomic DNA), and 10.68 μL of DNA water. The flanking regions were cloned into a linearized pRF-HU2 vector42 as previously described.43 Overexpression mutants were generated by amplifying a region upstream of NRPS14 using O1+O2 and an additional region at the beginning of the gene with the primers NRPS14O3 and NRPS14O4. The PCR was performed with the same conditions as described above, and the amplified regions were cloned into a linearized pRFHU2E vector under control of the gpdA promoter as previously described.43 The resulting plasmids were introduced into F. graminearum (PH-1) by Agrobacterium tumefaciens-mediated transformation44,45 and verified by diagnostic PCR using primers V1+V3, V2+V3, and V2+V4 (Supplementary Figure 2 and Supplementary Table 2). Genome Sequencing. A deletion and overexpression mutant were inoculated in YPG media and incubated at 28 °C for 2 days in preparation of DNA extraction. Cultivated fungi were filtered through sterile MiraCloth (Calbiochem), and genomic DNA was extracted using the DNeasy plant mini kit (Qiagen). 550 bp paired-end libraries of genomic DNA were prepared in accordance with the TruSeq DNA PCR-Free Library Prep protocol provided by Illumina, and the indexed DNA libraries were normalized to 4 nM. The libraries were subsequently denatured and diluted in accordance with the MiSeq System, Denature and Dilute Libraries Guide, using the MiSeq reagent kit v3. Sequencing was performed on the Illumina MiSeq. Low-quality reads were filtered, and the remaining reads were mapped to the PH-1 reference genomic sequence using CLC Main Workbench (Qiagen). Secondary Metabolite Profiling and Transcription Analysis in F. graminearum. The F. graminearum strains were cultivated on yeast extract sucrose agar medium (Scharlau, Barcelona, Spain)46 for 14 days in the dark at 25 °C. Secondary metabolites were extracted according to Smedsgaard (1997)47 and analyzed by HPLC-HRMS. A 40 μL amount of extract was separated using a flow of 1 mL/min with a linear water− acetonitrile gradient, with both eluents buffered with 0.1% formic acid. The gradient started at 10% acetonitrile and reached 100% in 20 min, which was held for 5 min. A chrysogine standard was available from previous studies48 and used for verification of the production in F. graminearum. Transcriptional analysis of NRPS14 in F. graminearum, wild type, and deletion strains was performed on 2-week-old cultures growing on solid YES medium as previously described.49 Primers NRPS14-Fw and NRPS14-Rv yielding a theoretical product of 739 bp were used together with β-tubulin and translation elongation factor 1α as controls.49
NRPS14 was inconclusive, as the available tools (NRPSpredictor2,30 PKS/NRPS analysis,31 NRPS/PKS substrate predictor,32 SEQL-NRPS,33 and antiSMASH34) returned variable results for the two A domains. The study by Hikino et al. (1973) suggested, however, that pyruvic acid and anthranilic acid are likely substrates for NRPS14, with pyruvic acid adenylated by the first A domain and anthranilic acid by the second. Although anthranilic acid is a primary metabolite involved in tryptophan biosynthesis in prokaryotes, it is not a standard α-amino acid in fungi. Several anthranilic acid adenylating NRPSs have however been identified.35,36 If pyruvic acid and anthranilic acid are merged and released from NRPS14 by hydrolysis, a subsequent amidation would lead to compound 3. Such a process is catalyzed by an amidotransferase (e.g., Chry2) using glutamine as amino donor. Cyclization of compound 3 will result in compound 2, which could be catalyzed by the dehydrogenase chry5 that has a terminal berberine bridge domain for C−N cyclization.37 A final reduction of compound 2 catalyzed by the oxidoreductase chry4 would result in chrysogine.
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EXPERIMENTAL SECTION
General Experimental Procedures. Analyses of secondary metabolites were performed on a Hitachi Elite LaChrom HPLC system equipped with a 150 × 4.6 mm Ascentis Xpress 2.7 μm phenyl-hexyl (Sigma-Aldrich) and coupled to a high-resolution mass spectrometer (compact qTOF, Bruker, Germany) with an electrospray source using a 3:97 flowsplitter. Bioinformatic Analysis. The genomic DNA region of NRPS14 from F. graminearum was used to identify possible ortholog gene clusters using antiSMASH.34 Ortholog clusters from F. graminearum, F. culmorum, F. pseudograminearum, F. avenaceum, A. nomius, P. chrysogenum, M. canis, and C. orbiculare were visualized with CLC Main Work Bench (CLC Bio-Qiagen, Aarhus, Denmark). Pairwise comparison of the protein encoded by the cluster genes was performed in CLC using the clustalW algorithm. Microarray samples from F. graminearum-infected wheat and barley (FG1 and FG15 from plexdb.com) were used to study gene expression patterns.28,29 The Affymetrix CEL files were processed through the “affy” package in Bioconductor and further analyzed in CLC Main Workbench. The hierarchical clustering of features was performed with complete linkage using Euclidean distance matrix.38 Transcription of the chrysogine cluster and neighboring genes in F. pseudograminearum was analyzed during infection of barley and Brachypodium distachyon. Four-day-old barley seedlings from a susceptible breeding line were inoculated by immersion in F. pseudograminearum spores followed by transfer to soil as described previously.39 Seedlings of Brachypodium line Bd21 were also infected by immersion with an F. pseudograminearum spore suspension. The plants were grown on moist paper towels until infection prior to sampling 5 days postinoculation. The experiments were conducted with four biological replicates, and RNA was extracted using a QIAgen RNAeasy spin plant RNA extraction kit according to the manufacturer’s protocol. The RNA was analyzed at the Australian Genome Research Facility (Melbourne Australia) for Illumina TruSeq with 125 bp paired end sequencing. Transcripts were assembled using cufflinks for individual samples and then merged into a final transcriptome assembly using cuffmerge, both run with default parameters and F. pseudograminearum isolate CS3096 reference genome40 to align the reads. Generation of Deletion and Overexpression Mutants. Upstream and downstream regions of NRPS14 (chry1) were amplified by PCR from F. graminearum (PH-1)41 using primers NRPS14O1+NRPS14O2 and NRPS14KO3+NRPS14KO4 (Supplementary Table 1), respectively, for generation of deletion plasmids. The PCR reactions were performed in a 20 μL volume containing 2 μL of 10× reaction buffer (Agilent), 2 μL of 2.5 mM dNTP solution, 0.32 μL of Pfu Turbo Hotstart DNA polymerase (Agilent; 2.5 U/μL), 2 μL of forward primer
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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.6b00822. Production of chrysogine by F. pseudograminearum, transcription of the chrysogine gene cluster in F. pseudograminearum, and RT-PCR analyses of NRPS14 in F. graminearum (PDF)
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AUTHOR INFORMATION
Corresponding Author
*Tel: 0045 9940 7659. Fax: 0045 9814 1808. E-mail:
[email protected]. dk (J. L. Sørensen). ORCID
Jens Laurids Sørensen: 0000-0002-2392-5343 Author Contributions ⊥
Shared first author.
Notes
The authors declare no competing financial interest. 2134
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ACKNOWLEDGMENTS The study was supported by grants from The Danish Research Council, Technology and Production (12-132415) and the Novo Nordisk Foundation (NNF15OC0016186). The MS laboratory at Aalborg University is supported by the Obel and Spar Nord Foundations. The chrysogine standard was a kind gift from Dr. Kristian Fog Nielsen, Technical University of Denmark, for which we are very grateful.
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