This application proposes to investigate the genetic and biochemical regulation of doxorubicin (DXR) production in Streptomyces peucetius. DXR is an important cancer chemotherapy drug produced commercially by fermentation. Information about the regulation of DXR biosynthesis could be used to increase DXR production, which could have definite economic benefits because FXR is a very costly drug and unimproved strains typically produce less than ug/ml of it. The principal investigator will study how the dnrl and dnrN genes, identified as key regulators of DXR production in our previous work, regulate expression of the dnr structural ad resistance genes temporally and in response to environmental factors. He also will initiate a search for loci outside the cluster of DXR production genes that control expression of the dnrN response regulator gene or the activity of the transcription factors DnrN and DnrI. This work eventually will uncover the signal transduction network that directs when and how much DXR is made in response to the growth environment. It will also help to define the regulons and stimulon of S. peucetius that govern its global responses to environmental stimuli, an important but largely unexplored aspect of Streptomyces biology.
Specific Aims : 1. The applicants will purify and characterize the DnrI and DnrN proteins that have been overproduced in Escherichia coli and peucetius. 2. The applicants will determine whether the purified DnrL protein binds to promoter regions of the dpsABCD polyketide synthase genes, dnrUV deoxysugar biosynthesis genes, dnrDKPQS late-acting and sugar biosynthesis genes, and the drrAB and drrC resistance genes by DNA footprinting and gel mobility shift experiments. DnrN binding to the dnrN and Dnrl promoters will also be investigated. 3. The applicants will determine if phosphorylation is required for activity of the DnrN and possibly DnrI proteins by P32-labeling experiments in vitro coupled with the DNA binding studies above. If phosphorylation occurs, then we will determine the site of protein phosphorylation by limited proteolysis and amino acid sequence analysis of labeled oligopeptides made from DnrN and DnrI. 4. If phosphorylation of DnrN is required then we will attempt to purify the relevant kinase from S. peucetius so that its gene can be cloned. As an alternative approach to cloning this gene, we will use hybridization analysis with selected Streptomyces regulatory genes or mutagenesis coupled with its effect on the expression of a dnrI:melC gene fusion to seek loci outside the dnr gene cluster that control the posttranslational activation of DnrN. 5. They will address whether the DnrO repressor protein controls dnrN expression temporally or in response to DXR or intermediates of its biosynthesis by determining of dnrO inactivation deregulates or derepress dnrN expression. They also will investigate whether DnrO negatively regulates expression of the drrAB DNR/DXR resistance genes. 6. They will assess whether the expression of dnrN and dnrl is influenced by the phosphate level in the culture at the time of DXR production, since DXR production is known to be phosphate but not carbon or nitrogen catabolite repressed. If not, then the applicants will determine if the expression of selected dnr structural genes is controlled by phosphate level, with or without dnrN or dnrI involvement.
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