Previous studies have reported the functional characterization of 9 out of 11 genes found in the gene cluster responsible for biosynthesis of the polyketide pigment aurofusarin in Fusarium graminearum. Here we reanalyze the function of a putative aurofusarin pump (AurT) and the two remaining orphan genes, aurZ and aurS. Targeted gene replacement of aurZ resulted in the discovery that the compound YWA1, rather than nor-rubrofusarin, is the primary product of F. graminearum polyketide synthase 12 (FgPKS12). AurZ is the first representative of a novel class of dehydratases that act on hydroxylated γ-pyrones. Replacement of the aurS gene resulted in accumulation of rubrofusarin, an intermediate that also accumulates when the GIP1, aurF, or aurOgenes in the aurofusarin cluster are deleted. Based on the shared phenotype and predicted subcellular localization, we propose that AurS is a member of an extracellular enzyme complex (GIP1-AurF-AurO-AurS) responsible for converting rubrofusarin into aurofusarin. This implies that rubrofusarin, rather than aurofusarin, is pumped across the plasma membrane. Replacement of the putative aurofusarin pump aurT increased the rubrofusarin-to- aurofusarin ratio, supporting that rubrofusarin is normally pumped across the plasma membrane. These results provide functional information on two novel classes of proteins and their contribution to polyketide pigment biosynthesis. © 2011 by The American Society for Biochemistry and Molecular Biology, Inc.