Resistance Gene-Guided Discovery of a Fungal Spirotetramate as an Acetolactate Synthase Inhibitor

Biosynthetic gene clusters (BGCs) of bioactive natural products occasionally encode resistant versions of the proteins they inhibit, offering opportunities for resistance gene-guided genome mining to uncover natural products with predictable modes of action. In this study, we developed a genome mining tool designed to identify fungal BGCs harboring putative resistance genes. Applying this tool to approximately 2,500 fungal genomes, we identified a BGC designated as the pts cluster, which encodes an acetolactate synthase (ALS) homologue. Functional characterization of the pts cluster resulted in the identification of pterrespiramide A (1), featuring unique spirotetramate and cis-decalin moieties. Consistent with the predicted activity, 1 was confirmed as an ALS inhibitor and exhibited both antifungal and herbicidal activities. This study illuminates the potential of resistance gene-guided genome mining as a powerful strategy for accelerating the discovery of previously undescribed bioactive natural products.