Activation of a cryptic polyketide synthase gene cluster hkn from Aspergillus hancockii via overexpression of the gene-cluster-specific transcription factor HknR led to the discovery of a novel polycyclic metabolite, which we named hancockinone A. The compound features an unprecedented prenylated 6/6/6/5 tetracarbocyclic skeleton and shows moderate antibacterial activity. Heterologous expression, substrate feeding, and in vitro assays confirmed the role of cytochrome P450 HknE in constructing the five-membered ring in hancockinone A from the precursor neosartoricin B.