This proposal requests four years of non-competing support for a study of the biochemistry and genetics of erythromycin A production by Streptomyces erythreus and midecamycin production by Streptomyces mycarofaciens. These macrolide antibiotics are widely used antiinfective agents; thus information about the genetic and biochemical mechanisms controlling their biosynthesis would have important benefits to the applied sciences. Their formation involves over twenty enzymatically catalyzed steps and perhaps as many structural genes. This suggests that a study of the organization and expression of the ery and mid production genes should uncover complex and interesting genetic and biochemical control mechanisms. We propose to establish the organization of the genes that control deoxysugar biosynthesis and the hydroxylation of a macrolactone intermediate in the erythromycin pathway, and to analyze the factors controlling their expression. In this work, we will investigate two interesting research leads found in the previous grant period which point to the existence of novel transcriptional or translational control mechanisms. The experiments will involve gentic complementation, DNA sequencing, promoter identification, and the analysis of transcriptional organization by low and high resolution S1 mapping and Northern hybridization. Expression of these genes as a function of time and growth conditions will be measured by dot-blot experiments. The characterization three enzymes encoded by these genes also will be pursued. The genes governing the assembly of platenolide I, an early intermediate of the midecamycin pathway, also will be studied. We will isolate a collection of Mid mutants of S. mycarofaciens and use them to locate mid genes by complementation of midA mutations and mutational cloning or transposon mutagenesis. A new method for characterizing the biochemical deficiency in MidA mutants also will be evaluated.
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