Fungi produce biologically active molecules such as antibiotics and immune system suppressants through complex biosynthetic pathways. This research investigates the evolutionary processes that operate in the diversification of these pathways, and thus the metabolites they produce, using the soil dwelling fungus Tolypocladium. Species of Tolypocladium are known to be particularly rich in biologically active metabolites with the best-known example being Cyclosporin A, which is used medicinally to suppress the immune system in organ transplant patients. Recent advancements in genomics and computational biology show significantly greater diversity of metabolic pathways than previously detected by traditional chemical analyses. As such, science has only observed the "tip of the iceberg" of fungal biochemical diversity, while the majority await discovery as promising sources of pharmaceuticals, alternatives for the control of insect pests and bioremediation of contaminated soils, and essential in the development of biofuels. A more robust understanding of patterns and processes that have led to the diversification of fungal genomes, and subsequently their biologically active molecules, will enhance the ability to exploit fungi for the betterment of society. This research establishes a case study methodology by which other fungi can be studied and exploited for scientific advancements. In achieving these advancements, students and young researchers are trained in an interdisciplinary manner and equipped to study and develop biological solutions to the challenges of the modern World.
Genomes of 20 fungal isolates will be sequenced, identifying and characterizing secondary metabolite gene clusters. These genomic analyses will be coupled with manipulative growth experiments and chemical analyses, linking the genetic factors, environment and ecology, with the metabolites produced, and resulting in a metabolite census of this group of fungi. Evolutionary processes and patterns of diversification (e.g., gene duplication-divergence, horizontal gene transfer, gene fusion, diversifying selection) will be tested using phylogenomic methodologies. The analyses will be coupled with liquid chromatography-tandem mass spectrometry metabolite profiling, selective growth experiments, and RNA sequencing to link secondary metabolite gene clusters with their metabolites. The specific aims and experimental plan are designed to compare two major hypotheses. The first is that diversification of secondary metabolites results from complex processes incongruent with phylogeny. The second is that homologous domains produce chemically distinct metabolites not previously suspected of having common evolutionary origins. The research determines if the complex patterns of evolution shown by genetically distinct non-ribosomal peptide synthetases and polyketide synthases represent homologous domains and genes, respectively, produced by evolutionary processes such as duplication-divergence, horizontal genet transfer, gene fusion, and diversifying selection. The integration of liquid chromatography-tandem mass spectrometry metabolite profiling, selective growth experiments, RNA sequencing and phylogenomic analyses will link secondary metabolite gene clusters and the metabolites they produce, showing the evolutionary connection between genotypes and chemical phenotypes.
