An overlooked cyclase plays a central role in the biosynthesis of indole diterpenes.

Indole diterpenes (IDTs) are a large class of highly complex fungal natural products that possess a wide array of intriguing bioactivities. While IDTs are structurally diverse, the first four steps of IDT biosynthesis are highly conserved and result typically in the formation of a tetrahydropyran (THP)-ring containing structure, most commonly paspaline. The biosynthetic genes responsible for these steps are the most extensively studied of all IDT genes and collectively define the core biosynthetic pathway. Here we show that the fourth fundamental step, formation of the THP ring, is catalysed by a terpene cyclase encoded by an overlooked and uncharacterised fifth gene, . All previously delineated biosynthetic routes have incorrectly attributed this step to the terpene cyclase IdtB, leading to imprecise pathway reconstructions and ignoring the fully evolved biosynthetic solution for core IDT generation. Moreover, while IdtA terpene cyclases are found in Eurotiomycetes fungi, in Sordariomycetes fungi this step is catalysed by the unrelated protein IdtS, demonstrating that two distinct solutions to this chemistry exist. All biosynthetic gene clusters known to specify production of THP-containing IDTs include an or gene. These findings reset the paradigm for core IDT biosynthesis and support accurate heterologous biosynthesis of these complex natural products.