Secondary Metabolite Niches: Whole Genome Functional Analysis of Non-Ribosomal Peptide Synthetases

Cornell Univ - State: Awds Made Prior May 2010, Ithaca NY

Investigators

Abstract

The goal of this project is to undertake an exhaustive characterization of the set of Cochliobolus heterostrophus non-ribosomal peptide synthetase (NPS) genes, and the small molecule peptides produced by the enzymes they encode, to ascertain what peptide secondary metabolite products are doing in (and for) the fungal cell. Non-ribosomal peptide synthetases (NRPSs) are multimodular enzymes that make small non-ribosomal peptides (NRPs) through a mechanism, independent of organelles best known for protein synthesis, the ribosomes. To date, the NRPS method of peptide biosynthesis has been described for filamentous ascomycete fungi (and to a limited extent, for basidiomycete fungi) and for bacteria, only. The diversity of possible NRP products is potentially limitless as NRPs can be composed of D- and L-amino acids, protein and non-protein amino acids, hydroxy acids, ornithine, and other unusual constituents. Furthermore, NRPs can be linear, cyclic, or branched cyclic, and may be modified by glycosylation, N-methylation or acylation. In addition to structural diversity, NRPs have an astonishingly broad spectrum of biological activities. However, despite the fact that activities of the peptide products, with respect to interactions with other organisms, are well documented, and certainly remarkable, to suggest that this is their primary function is likely incorrect. For example, only one of the 12 C. heterostrophus NPS genes is required for virulence on the host, maize. In fact, the physiological significance of these small peptides to the producing fungi is largely unknown. The foundation of this project is the hypothesis that NRPS enzymes are purveyors of small molecules for both basal metabolism and for specialized environmental niches. The goals of this project are to create a library of C. heterostrophus NPS- deletion strains that will be used to resolve roles that small molecule, non-ribosomal peptides play in the biology of the fungal cell. Specifically, experiments are designed to: A. Create multiple NPS gene-deletion strains. B. Create strains in which each NPS gene is overexpressed. C. Perform a series of phenotypic assays to explore function. D. Determine conditions under which NPS genes are expressed. E. Perform chemical analyses on wild-type and selected mutant strains with interesting phenotypes, to identify NRP structure. This research will allow the development of an optional research module for a popular undergraduate biology course. This outreach collaboration program will expose and help move students into the science pipeline by interweaving and disseminating excitement about the second largest group of organisms on earth, the fungi. Interested students will be invited to one or two sessions where they will be introduced to the concept of NRPSs. Then, students will be assigned to assist with and eventually take charge of phenotypic assays. Students will also be challenged to use contemporary molecular genetics/genomics tools to answer questions about the genesis and function of important fungal metabolites including their applications in agriculture and their social implications.