PROJECT SUMMARY/ABSTRACT Fungal natural products are an invaluable resource for drug discovery, containing numerous biosynthetic gene clusters (BGCs) that produce structurally unique compounds with the potential to treat infectious diseases, immune disorders, and many other conditions. Despite the plethora of bioactive chemicals contained within fungi, recent studies have suggested that less than 5% of the possible fungal secondary metabolites have yet been discovered. While advances in genomic sequencing have exposed this gap, the successful stimulation of unknown natural products from these silent BGCs represents a major bottleneck stymieing drug discovery efforts. To overcome this challenge, targeted approaches designed to understand and manipulate the genetic and environmental cues influencing natural product biosynthesis are required. Recent innovations in heterologous gene expression and comparative metabolomics have led to the development of the fungal artificial chromosome-metabolite scoring (FAC-MS) pipeline, which enables the insertion of fungal genomic DNA into a fungal host, identification of heterologously expressed metabolites, and elucidation of their biosynthetic pathways. Although many FACs have expressed otherwise cryptic metabolites, many BGCs have not yet been expressed with this approach. The proposal herein details plans for activating cryptic FAC-encoded BGCs, enabling targeted analysis of the factors controlling gene expression. In Aim 1, we will explore the impact of chromatin organization on gene expression by culturing FACs with epigenetic modifying agents and by inserting FACs into genetic chromatin mutants with altered levels of important regulatory proteins. With Aim 2, we will stimulate expression of BGC-encoded defense compounds through culturing FACs in the presence of fungal and bacterial signaling molecules and complex microbial extracts. Activated FAC-encoded compounds will be targeted for isolation and biosynthesis studies and will be evaluated for antimicrobial activity. In addition to uncovering novel metabolites, this approach is expected to provide insight into the numerous environmental and epigenetic cues that regulate gene expression. This platform aims to improve the FAC-MS pipeline to accelerate the discovery of novel drug leads by providing access to the untapped potential of fungi. These studies will also provide critical training in fungal genetics, chemical ecology, and analytical metabolomics, providing a foundation for an independent research career.