Microbial systems that produce secondary metabolites have been studied for many decades, often with a view to producing increased yields of structurally modified compounds with improved therapeutic activity. Investigations of primary metabolic processes, particularly fatty acid biosynthesis, in such systems have received less attention, yet are important in terms of the interaction and relationship with secondary metabolic processes. The first specific aim of this proposal is to purify, characterize, and subsequently, clone, sequence, and compare enoyl CoA reductases with different carbon-chain-length specificities from Streptomyces collinus. These enzymes are thought to be involved a novel malonyl CoA-independent fatty acid biosynthetic process. This possibility will be investigated by classical isotope incorporation studies and by the generation of S. Collinus mutants in which these enzymes are inactive. The stereochemical course of reduction catalyzed by all these enzymes and the level of homology between the genes which encode them will be elucidated. This will allow for a determination of which of the stereochemical features are subject to divergent evolution, an area that has seen considerable controversy over recent years. The second specific aim of this proposal is to purify and characterize enoyl CoA reductases putatively involved in a similar malonyl CoA independent pathway of fatty acid biosynthesis in Aspergillus parasiticus. In this case the enzymes are thought to be involved in the formation of hexanoyl CoA which is the putative starter unit for biosynthesis of the secondary metabolite averufin. Generation of A parasiticus mutants in which this enzyme is inactive will ultimately show its physiological role.
