Secondary metabolites are a remarkably rich source of medically useful compounds. It is, consequently, of great value to identify new secondary metabolites and to develop methods to increase secondary metabolite production. In fungi, the genes of particular secondary metabolite biosynthetic pathways are usually clustered together, and sequencing of the genomes of several species of the filamentous fungus Aspergillus has revealed that there are many secondary metabolism gene clusters (approximately 46 in Aspergillus nidulans alone, a much larger number than would have been predicted prior to the sequencing of the genome). The products of the great majority of these pathways are unknown and A. nidulans is, thus, a potentially rich source of useful secondary metabolites as well as an excellent model organism for developing procedures to mine the secondary metabolomes of fungi. This proposal takes advantage of recent progress in gene targeting in A. nidulans to create a set of molecular genetic tools and strains that will allow the efficient exploitation of the A. nidulans secondary metabolome. Our first specific aim is to create a set of transformants that will allow the products of all A. nidulans secondary metabolism gene clusters to be identified. We will pursue two approaches. Approximately 15 unexplored gene clusters contain transcription factor genes that are predicted to control expression of the other genes in the cluster. We will replace the promoters of these transcription factor genes with inducible promoters. This will allow overexpression of the genes of target clusters and increased synthesis of the product(s) of the cluster. This will, in turn, allow our collaborator, Dr. Clay Wang, to identify and purify the products of particular clusters.
Our second aim will be to delete genes of selected secondary metabolism pathways to allow the steps of the pathways to be clarified and shunt products isolated. In some cases, the shunt products may be as useful as, or more useful than, the normal products of the pathway.
Our third aim will be to develop promoter replacement strains that will allow increased expression of secondary metabolism pathways. This will be particularly important in gene clusters that do not contain transcription factor genes. A portion of this aim will be to develop a serial promoter replacement procedure that will allow the promoters of all the genes in target pathways to be replaced. For all of these aims we will work closely with our collaborators, Dr. Wang and Dr. Nancy Keller.
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