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 2500 fungal genomes, we identified a BGC designated as the <i>pts</i> cluster, which encodes an acetolactate synthase (ALS) homologue. Functional characterization of the <i>pts</i> cluster resulted in the identification of pterrespiramide A (<b>1</b>), featuring unique spirotetramate and <i>cis</i>-decalin moieties. Consistent with the predicted activity, <b>1</b> 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.