Targeted Genome Mining for the Discovery and Study of Sterol Pathway Fungal Natural Product Drugs

For millennia, humans have been utilizing plants, fungi, and microbes for their medicinal purposes or for commercial use as dietary supplements, cosmetics, etc that are derived from natural products. Natural products can be broadly defined as any chemical compound that can be found from living organisms in nature. Natural products have a wide variety of bioactivities and uses that have spurred efforts to discover and characterize novel natural products. The genesis of these efforts in the modern era began with the discovery of penicillin by Sir Alexander Fleming, which helped realize the power of utilizing chemical compounds from nature. Natural products are very much like superheroes; they all have a backstory and Nature draws the comic books panels that depict their tales in the form of genetic information found in living organisms. The study of natural product biosynthesis thus aims to decipher the narratives told through the ATCG nucleotides to explain the origins of these molecules through enzymatic pathways and their superpowers (bioactivities). The rise of next generation sequencing has opened a new world of genetic information and facilitated a renaissance in drug discovery based on genomics. Analysis of microbial genomes has revealed that we have only accessed <10% of the chemicals these organisms are capable of producing. This dissertation describes efforts to access this genomic space in fungi with targeted genome mining, which aims to search for natural products of desired bioactivity through the presence of self-resistant enzymes. Fungi have evolved to contain these self-resistant enzymes, which are copies of the target enzyme of a natural product that are resistant to the inhibitory effects of the produced molecule. We aimed to utilize targeted genome mining to discover natural product inhibitors of the sterol pathway, which already contains drug targets that many commercial anticholesteremic and antifungal drugs specifically inhibit. This search strategy allowed us to discover and elucidate the biosynthesis of zaragozic acid A (a cholesterol lowering natural product) and restricticin (an antifungal natural product). Discovery of the zaragozic acid A cluster in Curvularia lunata was done through targeted genome mining of squalene synthase. The elucidation of the zaragozic acid A biosynthetic pathway in the engineered Aspergillus nidulans heterologous host has described the unique steps that lead to the production of the alkylcitrate benzylic polyketide intermediate of the pathway. Restricticin is another example of successful targeted genome mining using Cyp51 as a query. Cyp51 is an important sterol pathway target that is inhibited by commercial azole drugs. Targeted searches for Cyp51 clusters led to the identification of the restricitin biosynthetic gene cluster in Aspergillus nomius. Biosynthetic elucidation of restricticin reveals the critical steps that lead up to attachment of the glycyl ester group that serve as the chemical warhead for its antifungal properties. Identification of the cluster allowed us to evaluate the rstn2 self resistance gene which exhibits azole resistance. Targeted genome mining has also helped to identify other possible novel Cyp51 inhibitors