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

Abstract

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.

Evolution of Chemical Diversity in Echinocandin Lipopeptide Antifungal Metabolites

“…This topology was supported by strong bootstrap values and indicated that the A domains for each amino acid position from each distantly related fungus were more similar to each other than to the individual A domains from a single given echinocandin-producing strain. This pattern of a one-to-one correspondence between modules and the amino acid specificity of different species is consistent with models of tandem duplication and subfunctionalization prior to divergence from a hypothetical ancestor, as has been hypothesized to occur during the evolution of cyclosporine and peptaibol NRPSs (24,63). Furthermore, within each of these A-domain clades, the branchings of terminal leaves were congruent with the phylogenetic species trees.…”

Atlas of nonribosomal peptide and polyketide biosynthetic pathways reveals common occurrence of nonmodular enzymes

“…The bulk of natural products in clinical use today come from a handful of bacterial and fungal lineages (17)(18)(19)(20)(21). However, genomics studies imply that the ability to make these compounds is much more widespread (22)(23)(24).…”

Unique amalgamation of primary and secondary structural elements transform peptaibols into potent bioactive cell-penetrating peptides

“…Analogous 21-module NRPS clusters have been reported from the genomes of Tolypocladium sp. T1 (NP T1 ) (41) and a recently sequenced Tolypocladium inflatum (NP Ti ) (46) (Fig. 4 and SI Appendix, Tables S16 and S18).…”