The aim of this project is to generate novel inhibitors of gyrase and topoisomerase IV by engineering the biosynthetic pathways of very potent natural inhibitors of these enzymes. The project will focus on antibiotics of the aminocoumarin type (novobiocin, clorobiocin, coumermycin A1, simocyclinones) and the cyclothialidine type (cyclothialidine and GR12222X). All these compounds are produced by Stneptomyces strains. The interaction of novobiocin, clorobiocin and the cyclothialidines with bacterial gyrase has been elucidated with biophysical and X-ray crystallographic techniques by members of this consortium. The interaction of the simocyclinones, and of newly generated inhibitors, with gyrase and topoisomerase IV will be investigated in this project. The results will be utilized for rational approaches for the development of new inhibitors by genetic engineering. The complete gene clusters involved in the biosynthesis of novobiocin, clorobiocin, coumermycin and simocyclinone have been cloned and sequenced by the members of this consortium. The biosynthetic gene cluster of cyclothialidine will be identified in this project. Genes and operons within the gene clusters will be modified by gene inactivation, gene expression or gene replacement experiments within the natural producers. Furthermore, vectors containing entire biosynthetic gene clusters will be modified in E. coli and subsequently introduced into suitable Streptomyces host strains for production of novel metabolites. New molecules will also be generated by feeding specific strains with synthetic analogues of precursors of biosynthesis. Accumulated compounds will be modified chemically in order to generate an even wider array of new molecules. The biological activity of the novel molecules will be assayed using enzyme assay systems with gyrase and topoisomerase IV and antibacterial assays with human pathogens. Promising drug candidates will be investigated in animal infection models...
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Funding SchemeSTREP - Specific Targeted Research Project