Evolution Of Nonribosomal Peptide Synthetase Proteins Involved In Secondary Metabolism In Fungi

Nonribosomal peptide synthetases (NRPSs) are multimodular enzymes which biosynthesize peptides (NRPs) independently of ribosomes. Three core domains (adenylation (A), thiolation (T), condensation (C)) comprise a functional module for NRP biosynthesis. Although NRPSs produce a diversity of bioactive compounds, little is known about the evolutionary relationships of genes encoding NRPSs and the mechanisms by which they evolve. The objectives of this research were to perform phylogenomic analyses to identify major NRPS subclasses and determine evolutionary relationships and to elucidate fine-scale evolutionary mechanisms giving rise to the diverse NRPS domain structures in fungi. Chapter 2 is a published manuscript on ferrichrome synthetases tracking the evolution of domain architectures of these relatively conserved enzymes across fungi. Results supported the hypothesis that ferrichrome synthetases evolved by tandem duplication of complete modules (A-T-C) (single or double units) and loss of single A domains or complete A-T-C modules. A mechanism for evolution of iterative biosynthesis is proposed. Protein modeling of the A domain substrate binding pockets refined characterization of key residues involved in substrate specificity, by identifying novel sites. Chapter 3 reports a fungal kingdom-wide phylogenomic study of NRPSs, with the objective of identifying subclasses. Nine were identified which fell into two major groups. One consisted of primarily mono/bi-modular NRPSs with conserved domain architectures which group with bacterial NRPSs and whose products are associated with conserved metabolic roles. The other consisted of primarily multimodular and exclusively fungal NRPSs with variable domain architectures whose products perform niche-specific functions. All groups of NRPSs were much more common in Euascomycetes than in any other fungal taxonomic group. Although NRPSs are discontinuously distributed across fungal taxa, little evidence was found for horizontal gene transfer from bacteria to fungi. Overall, this study showed that both tandem duplication and loss, as well as recombination and rearrangement, of modular units (either complete A-T-C modules or single A domains) are mechanisms by which NRPSs and their chemical products evolve. Phylogenomic analysis identified subgroups of NRPSs possibly reflecting common function and suggested an older evolutionary origin of several mono/bimodular groups while multimodular fungal NRPSs are more recently derived and highly expanded in Euascomycetes

Phylogenomics reveals subfamilies of fungal nonribosomal peptide synthetases and their evolutionary relationships

<p><b>Abstract</b></p> <p>Background</p> <p>Nonribosomal peptide synthetases (NRPSs) are multimodular enzymes, found in fungi and bacteria, which biosynthesize peptides without the aid of ribosomes. Although their metabolite products have been the subject of intense investigation due to their life-saving roles as medicinals and injurious roles as mycotoxins and virulence factors, little is known of the phylogenetic relationships of the corresponding NRPSs or whether they can be ranked into subgroups of common function. We identified genes (<it>NPS</it>) encoding NRPS and NRPS-like proteins in 38 fungal genomes and undertook phylogenomic analyses in order to identify fungal NRPS subfamilies, assess taxonomic distribution, evaluate levels of conservation across subfamilies, and address mechanisms of evolution of multimodular NRPSs. We also characterized relationships of fungal NRPSs, a representative sampling of bacterial NRPSs, and related adenylating enzymes, including α-aminoadipate reductases (AARs) involved in lysine biosynthesis in fungi.</p> <p>Results</p> <p>Phylogenomic analysis identified nine major subfamilies of fungal NRPSs which fell into two main groups: one corresponds to <it>NPS </it>genes encoding primarily mono/bi-modular enzymes which grouped with bacterial NRPSs and the other includes genes encoding primarily multimodular and exclusively fungal NRPSs. AARs shared a closer phylogenetic relationship to NRPSs than to other acyl-adenylating enzymes. Phylogenetic analyses and taxonomic distribution suggest that several mono/bi-modular subfamilies arose either prior to, or early in, the evolution of fungi, while two multimodular groups appear restricted to and expanded in fungi. The older mono/bi-modular subfamilies show conserved domain architectures suggestive of functional conservation, while multimodular NRPSs, particularly those unique to euascomycetes, show a diversity of architectures and of genetic mechanisms generating this diversity.</p> <p>Conclusions</p> <p>This work is the first to characterize subfamilies of fungal NRPSs. Our analyses suggest that mono/bi-modular NRPSs have more ancient origins and more conserved domain architectures than most multimodular NRPSs. It also demonstrates that the α-aminoadipate reductases involved in lysine biosynthesis in fungi are closely related to mono/bi-modular NRPSs. Several groups of mono/bi-modular NRPS metabolites are predicted to play more pivotal roles in cellular metabolism than products of multimodular NRPSs. In contrast, multimodular subfamilies of NRPSs are of more recent origin, are restricted to fungi, show less stable domain architectures, and biosynthesize metabolites which perform more niche-specific functions than mono/bi-modular NRPS products. The euascomycete-only NRPS subfamily, in particular, shows evidence for extensive gain and loss of domains suggestive of the contribution of domain duplication and loss in responding to niche-specific pressures.</p

The genome of the truffle-parasite Tolypocladium ophioglossoides and the evolution of antifungal peptaibiotics

Abstract Background Two major mycoparasitic lineages, the family Hypocreaceae and the genus Tolypocladium, exist within the fungal order, Hypocreales. Peptaibiotics are a group of secondary metabolites almost exclusively described from Trichoderma species of Hypocreaceae. Peptaibiotics are produced by nonribosomal peptide synthetases (NRPSs) and have antibiotic and antifungal activities. Tolypocladium species are mainly truffle parasites, but a few species are insect pathogens. Results The draft genome sequence of the truffle parasite Tolypocladium ophioglossoides was generated and numerous secondary metabolite clusters were discovered, many of which have no known putative product. However, three large peptaibiotic gene clusters were identified using phylogenetic analyses. Peptaibiotic genes are absent from the predominantly plant and insect pathogenic lineages of Hypocreales, and are therefore exclusive to the largely mycoparasitic lineages. Using NRPS adenylation domain phylogenies and reconciliation of the domain tree with the organismal phylogeny, it is demonstrated that the distribution of these domains is likely not the product of horizontal gene transfer between mycoparasitic lineages, but represents independent losses in insect pathogenic lineages. Peptaibiotic genes are less conserved between species of Tolypocladium and are the product of complex patterns of lineage sorting and module duplication. In contrast, these genes are more conserved within the genus Trichoderma and consistent with diversification through speciation. Conclusions Peptaibiotic NRPS genes are restricted to mycoparasitic lineages of Hypocreales, based on current sampling. Phylogenomics and comparative genomics can provide insights into the evolution of secondary metabolite genes, their distribution across a broader range of taxa, and their possible function related to host specificity.http://deepblue.lib.umich.edu/bitstream/2027.42/112062/1/12864_2015_Article_1777.pd

Cyclosporine Biosynthesis in Tolypocladium inflatum Benefits Fungal Adaptation to the Environment

The cycloundecapeptide cyclosporin A (CsA) was first isolated from the insect-pathogenic fungus Tolypocladium inflatum for its antifungal activity and later developed as an immunosuppressant drug. However, the full biosynthetic mechanism of CsA remains unknown and has puzzled researchers for decades. In this study, the biosynthetic gene cluster is suggested to include 12 genes encoding enzymes, including the nonribosomal peptide synthetase (NRPS) (SimA) responsible for assembling the 11 amino acid substrates of cyclosporine and a polyketide synthase (PKS) (SimG) to mediate the production of the unusual amino acid (4R)-4-[(E)-2butenyl]-4-methyl-L-threonine (Bmt). Individual deletion of 10 genes, isolation of intermediates, and substrate feeding experiments show that Bmt is biosynthesized by three enzymes, including SimG, SimI, and SimJ. The substrate D-alanine is catalyzed from L-alanine by alanine racemase SimB. Gene cluster transcription is regulated by a putative basic leucine zipper (bZIP)-type protein encoded by the cluster gene SimL. We also found that the cluster cyclophilin (SimC) and transporter (SimD) genes contribute to the tolerance of CsA in the CsA-producing fungus. We also found that cyclosporine production could enable the fungus to outcompete other fungi during cocultivation tests. Deletion of the CsA biosynthetic genes also impaired fungal virulence against insect hosts. Taking all the data together, in addition to proposing a biosynthetic pathway of cyclosporines, the results of this study suggest that CsA produced by this fungus might play important ecological roles in fungal environment interactions. IMPORTANCE The cyclopeptide cyclosporin A was first isolated from the filamentous fungus Tolypocladium inflatum showing antifungal activity and was later developed as an immunosuppressant drug. We report the biosynthetic mechanism of cyclosporines that are mediated by a cluster of genes encoding NRPS and PKS controlled by a bZIP-type transcriptional regulator. The two unusual amino acids Bmt and D-Ala are produced by the PKS pathway and alanine racemase, respectively. The cyclophilin and transporter genes jointly contribute to fungal self-protection against cyclosporines. Cyclosporine confers on T. inflatum the abilities to outcompete other fungi in competitive interactions and to facilitate fungal infection of insect hosts, which therefore benefits fungal adaptations to different environments

The Genome of Tolypocladium inflatum: Evolution, Organization, and Expression of the Cyclosporin Biosynthetic Gene Cluster

The ascomycete fungus Tolypocladium inflatum, a pathogen of beetle larvae, is best known as the producer of the immunosuppressant drug cyclosporin. The draft genome of T. inflatum strain NRRL 8044 (ATCC 34921), the isolate from which cyclosporin was first isolated, is presented along with comparative analyses of the biosynthesis of cyclosporin and other secondary metabolites in T. inflatum and related taxa. Phylogenomic analyses reveal previously undetected and complex patterns of homology between the nonribosomal peptide synthetase (NRPS) that encodes for cyclosporin synthetase (simA) and those of other secondary metabolites with activities against insects (e.g., beauvericin, destruxins, etc.), and demonstrate the roles of module duplication and gene fusion in diversification of NRPSs. The secondary metabolite gene cluster responsible for cyclosporin biosynthesis is described. In addition to genes necessary for cyclosporin biosynthesis, it harbors a gene for a cyclophilin, which is a member of a family of immunophilins known to bind cyclosporin. Comparative analyses support a lineage specific origin of the cyclosporin gene cluster rather than horizontal gene transfer from bacteria or other fungi. RNA-Seq transcriptome analyses in a cyclosporin-inducing medium delineate the boundaries of the cyclosporin cluster and reveal high levels of expression of the gene cluster cyclophilin. In medium containing insect hemolymph, weaker but significant upregulation of several genes within the cyclosporin cluster, including the highly expressed cyclophilin gene, was observed. T. inflatum also represents the first reference draft genome of Ophiocordycipitaceae, a third family of insect pathogenic fungi within the fungal order Hypocreales, and supports parallel and qualitatively distinct radiations of insect pathogens. The T. inflatum genome provides additional insight into the evolution and biosynthesis of cyclosporin and lays a foundation for further investigations of the role of secondary metabolite gene clusters and their metabolites in fungal biology

Comparative Genome Structure, Secondary Metabolite, and Effector Coding Capacity across Cochliobolus Pathogens

The genomes of five Cochliobolus heterostrophus strains, two Cochliobolus sativus strains, three additional Cochliobolus species (Cochliobolus victoriae, Cochliobolus carbonum, Cochliobolus miyabeanus), and closely related Setosphaeria turcica were sequenced at the Joint Genome Institute (JGI). The datasets were used to identify SNPs between strains and species, unique genomic regions, core secondary metabolism genes, and small secreted protein (SSP) candidate effector encoding genes with a view towards pinpointing structural elements and gene content associated with specificity of these closely related fungi to different cereal hosts. Whole-genome alignment shows that three to five percent of each genome differs between strains of the same species, while a quarter of each genome differs between species. On average, SNP counts among field isolates of the same C. heterostrophus species are more than 25x higher than those between inbred lines and 50x lower than SNPs between Cochliobolus species. The suites of nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS), and SSP-encoding genes are astoundingly diverse among species but remarkably conserved among isolates of the same species, whether inbred or field strains, except for defining examples that map to unique genomic regions. Functional analysis of several strain-unique PKSs and NRPSs reveal a strong correlation with a role in virulence

Comparative genome structure, secondary metabolite, and effector coding capacity across Cochliobolus pathogens.

The genomes of five Cochliobolus heterostrophus strains, two Cochliobolus sativus strains, three additional Cochliobolus species (Cochliobolus victoriae, Cochliobolus carbonum, Cochliobolus miyabeanus), and closely related Setosphaeria turcica were sequenced at the Joint Genome Institute (JGI). The datasets were used to identify SNPs between strains and species, unique genomic regions, core secondary metabolism genes, and small secreted protein (SSP) candidate effector encoding genes with a view towards pinpointing structural elements and gene content associated with specificity of these closely related fungi to different cereal hosts. Whole-genome alignment shows that three to five percent of each genome differs between strains of the same species, while a quarter of each genome differs between species. On average, SNP counts among field isolates of the same C. heterostrophus species are more than 25× higher than those between inbred lines and 50× lower than SNPs between Cochliobolus species. The suites of nonribosomal peptide synthetase (NRPS), polyketide synthase (PKS), and SSP-encoding genes are astoundingly diverse among species but remarkably conserved among isolates of the same species, whether inbred or field strains, except for defining examples that map to unique genomic regions. Functional analysis of several strain-unique PKSs and NRPSs reveal a strong correlation with a role in virulence

Module evolution and substrate specificity of fungal nonribosomal peptide synthetases involved in siderophore biosynthesis

Abstract Background Most filamentous ascomycete fungi produce high affinity iron chelators called siderophores, biosynthesized nonribosomally by multimodular adenylating enzymes called nonribosomal peptide synthetases (NRPSs). While genes encoding the majority of NRPSs are intermittently distributed across the fungal kingdom, those encoding ferrichrome synthetase NRPSs, responsible for biosynthesis of ferrichrome siderophores, are conserved, which offers an opportunity to trace their evolution and the genesis of their multimodular domain architecture. Furthermore, since the chemistry of many ferrichromes is known, the biochemical and structural 'rules' guiding NRPS substrate choice can be addressed using protein structural modeling and evolutionary approaches. Results A search of forty-nine complete fungal genome sequences revealed that, with the exception of Schizosaccharomyces pombe, none of the yeast, chytrid, or zygomycete genomes contained a candidate ferrichrome synthetase. In contrast, all filamentous ascomycetes queried contained at least one, while presence and numbers in basidiomycetes varied. Genes encoding ferrichrome synthetases were monophyletic when analyzed with other NRPSs. Phylogenetic analyses provided support for an ancestral duplication event resulting in two main lineages. They also supported the proposed hypothesis that ferrichrome synthetases derive from an ancestral hexamodular gene, likely created by tandem duplication of complete NRPS modules. Recurrent losses of individual domains or complete modules from this ancestral gene best explain the diversity of extant domain architectures observed. Key residues and regions in the adenylation domain pocket involved in substrate choice and for binding the amino and carboxy termini of the substrate were identified. Conclusion Iron-chelating ferrichrome synthetases appear restricted to fission yeast, filamentous ascomycetes, and basidiomycetes and fall into two main lineages. Phylogenetic analyses suggest that loss of domains or modules led to evolution of iterative biosynthetic mechanisms that allow flexibility in biosynthesis of the ferrichrome product. The 10 amino acid NRPS code, proposed earlier, failed when we tried to infer substrate preference. Instead, our analyses point to several regions of the binding pocket important in substrate choice and suggest that two positions of the code are involved in substrate anchoring, not substrate choice.</p