Abstract
Fungi are important sources for the discovery of natural products. During the last decades, technological progress and the increasing number of sequenced genomes facilitated the exploration of new secondary metabolites. Among those, polyketides represent a structurally diverse group with manifold biological activities. In this study, we successfully used genome mining and genetic manipulation for functional proof of a polyketide biosynthetic gene cluster from the filamentous fungus Penicillium crustosum. Gene activation in the native host and heterologous expression in Aspergillus nidulans led to the identification of the xil cluster, being responsible for the formation of the 6-methyl-2-pyrone derivative xylariolide D. Feeding with (13)C-labeled precursors supported the hypothesis of chain branching during the backbone formation catalyzed by a highly reducing fungal polyketide synthase. A cytochrome P450-catalyzed hydroxylation converts the PKS product to the final metabolite. This proved that just two enzymes are required for the biosynthesis of xylariolide D.