This is an application for a competitive revision of Program Project grant (5P01GM084077) entitled "Mining the Aspergillus nidulans Secondary Metabolome," It is written in response to Notice Number NOT-OD-09-058, Notice Title: NIH Announces the Availability of Recovery Act Funds for Competitive Revision Applications. The goal of the Program Project grant as funded is to use a synergistic combination of genomics, molecular genetics and natural products chemistry to mine the Aspergillus nidulans secondary metabolome for useful natural products. Although the grant has been in effect for less than one year, we are making excellent progress and wish to extend this approach to the hundreds of other fungi which likely make medically useful secondary metabolites, but lack the excellent genetic and molecular genetics tools that have been developed for A. nidulans. Since developing molecular genetic systems for these fungi is likely to be difficult and extremely time consuming, we wish, instead, to develop A. nidulans as a system for heterologous expression of secondary metabolite gene clusters from other fungi. In the requested two year period of support, we propose to improve A. nidulans as a host organism for heterologous expression of secondary metabolites by deleting A. nidulans secondary metabolite gene clusters that is expressed at high levels under normal growth conditions. This may allow greater production of target secondary metabolites, and should certainly allow target secondary metabolites to be detected more easily by mass spectroscopy. We will then develop two cosmid-based systems for expression of gene clusters from other fungi. We will develop the system using gene clusters of Aspergillus terreus, an economically and medically important fungus with a sequenced genome, but with a much less well developed set of molecular genetics tools than A. nidulans. We will develop a system for integrating gene clusters from A. terreus into the A. nidulans genome and a system for maintenance of A. terreus gene clusters on autonomously replicating cosmid vectors. We will analyze production of secondary metabolites by liquid chromatography/mass spectroscopy and develop procedures that allow us to obtain production of adequate quantities of secondary metabolites for purification and structural analysis. PUBLIC HEALTH RELEVANCE: Fungal secondary metabolites are a rich source of medically valuable compounds. Molecular genetics approaches in Aspergillus nidulans are providing a very efficient system for identifying new secondary metabolites and deducing the steps of the biosynthetic pathways that produce them. This application will allow us to apply the same approach to other fungi that lack the molecular tools available for A. nidulans.