Project Summary. A new functional metabologenomics approach is proposed for the development of a second generation of fernene-based antifungal terpenes. Our team led by Mead (Varigen), Keller (University of Wisconsin) and Kelleher (Northwestern University) explore the diversity of this unique family of triterpenes using a fusion of technologies that unites genomics with metabolomic understanding of fungal terpene synthesis. We begin by applying a genome mining-driven approach to find new natural analogs of enfumafungin. The resulting data are then integrated with metabolomic analysis and antifungal bioactivity assay data to establish the putative enfumafungin-like metabolites encoded by diverse strains and ultimately chart the structure-activity relationship space within this potent class of antifungals. In parallel, our team is constructing a modular platform capable of generating hundreds of different analogs in functional amounts. Here, we will heterologously express the enfumafungin-like pathways from fungal strains in an engineered Aspergillus nidulans host. The sequence- specific cloning and heterologous expression of lead molecules discovered in this program will establish methods to conduct targeted swapping of cyclase genes across multiple species to generate new antifungal fernene-like scaffolds. The long-term goal, requiring additional resources, is to advance a second generation of triterpene antifungal analogs into the research and clinical market place. Overall, this program provides an important next step towards establishing functional metabologenomics as a viable tool to go from genome to functional therapeutic. As a testbed, we turn to a natural product family with a recent history of clinical utility for the development of new analogs and technologies to access them.

Project Narrative. Fungal infections pose a serious threat to patients and our healthcare system, and it is of the utmost urgency that new antifungal agents be discovered and developed for the ongoing clinical battle against multi-drug resistant and emerging fungal pathogens. Using computational genomics, metabolomics, and synthetic biology-enabled manipulation of triterpene pathways we propose to develop a second generation of enfumafungin-like antifungal analogs.