The hallmark trait of fungal secondary-metabolite gene clusters is well established, consisting of contiguous enzymatic and often regulatory gene(s) devoted to the production of a metabolite of a specific chemical class. Unexpectedly, we have found a deviation from this motif in a subtelomeric region of Aspergillus fumigatus. This region, under the control of the master regulator of secondary metabolism, LaeA, contains, in its entirety, the genetic machinery for three natural products (fumitremorgin, fumagillin, and pseurotin), where genes for fumagillin and pseurotin are physically intertwined in a single supercluster. Deletions of 29 adjoining genes revealed that fumagillin and pseurotin are coregulated by the supercluster-embedded regulatory gene with biosynthetic genes belonging to one of the two metabolic pathways in a noncontiguous manner. Comparative genomics indicates the fumagillin/pseurotin supercluster is maintained in a rapidly evolving region of diverse fungal genomes. This blended design confounds predictions from established secondary-metabolite cluster search algorithms and provides an expanded view of natural product evolution.gene regulation | Zn(II) 2 Cys 6 transcription factor | FapR | biosynthesis | cluster evolution F ilamentous fungi are well known for their ability to produce a variety of natural products, so-called secondary metabolites that are not essential for growth under laboratory conditions (reviewed in refs. 1 and 2). However, the maintenance of the genetic information allowing fungi to produce secondary metabolites suggests that these small molecules provide essential benefits in environmental niches ranging from protection from fungivory (reviewed in ref.3) to chemical shields from UV radiation (4). Apart from providing evolutionary fitness to the producing organism in their natural habitat, many secondary metabolites are of major importance to humans because of their beneficial and deleterious effects as drugs and toxins, respectively.Fungal secondary metabolism has been characterized by physical linkage of the genes required for synthesis of specific metabolites and the distinct enzymatic machinery encoded by these genes (1, 2, 5). For instance, most fungal secondary metabolites belong to one of four chemical classes that are characterized based on the key or backbone enzymes that consist of polyketide synthases (PKSs), nonribosomal peptide synthetases (NRPSs), terpene cyclases (TCs), and prenyltransferases (PTs). Typically, cluster genes adjacent to these backbone genes code for accessory enzymes involved in either modification of the chemical scaffold, transcriptional control of cluster genes, transport of substrates and/or products, and resistance mechanisms. The most common regulatory genes of clusters encode fungal-specific C6 zinc binuclear cluster (Zn(II) 2 Cys 6 ) transcription factors (6), which, in general, exert positive transcriptional regulation of most of the genes within a single cluster (7). In addition to cluster-specific transcription factors, a higher order...