Streptomyces rimosus is an important industrial microorganism producing broad-spectrum antibiotic oxytetracycline. A number of semi-synthetic tetracyclines, are currently used in human and veterinary medicine. Due to emerging bacterial resistance, there is rising interest for new natural tetracyclines and their biosynthetic intermediates, which could be used to develop novel and more effective antibiotics . The aim of this work was to inactivate methyltransferase oxyF gene within oxytetracycline gene cluster in S. rimosus and to potentially identify target product, 6-desmethyl oxytetracycline. We consteructed a plasmid containing DNA fragment with homology to the sequence adjacent to oxyF gene from S. rimosus genome, but missing the entire oxyF gene. This way constructed DNA fragment was inserted into »suicide« plasmid designated as pKC1139, containing E. coli origin of replication and suitable resistance markers for selection in E. coli and S. rimosus. We transformed the plasmid into S.rimosus, and by applying two-stage process of chromosomal integration, followed by a double cross-over deletion, we selected for S. rimosus transformants containing precise deletion of targeted oxyF gene. We then confirmed correct double-cross over deletion of oxyF gene in a number of S. rimosus colonies. After cultivation, we analyse metabolic profile of the selected engioneered strains by thin layer chromatography. Although at low yield, we confirmed production of 6-dsemethyl oxyteracycline by mass spectroscopy.
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