The bacterium Streptomyces rimosus is mainly known for the production of broad-spectrum antibiotic oxytetracycline (OTC). To gain deeper understanding of the biosynthesis of OTC and other natural tetracyclines (TC) we initialy performed a bioinformatic comparison of the biosynthetic gene clusters (BGCs) encoding the biosynthesis of OTC, chlortetracycline, chelocardin, dactylocycline and SF2575. In the second step, we focused on selected genes involved in the early and late stages of OTC biosynthesis whose roles is not yet understood. We deleted selected genes from otc BGC (oxyI, oxyH, oxyG, oxyM, oxyO) in the S. rimosus high-producer strain. We then evaluated the titer of OTC produced by engineered strains and we attempted to identify potential new intermediates or side products by analyzing the non-targeted metabolome. We showed that the oxyI, oxyG, oxyM and oxyO genes are not essential for the OTC biosynthesis. However, when the oxyI, oxyG, and oxyM genes were deleted the OTC titer decreased suggesting that these genes play an auxiliary role. OTC production was entirely disrupted in the strain with deletion of oxyH gene, instead a new metabolite was detected. In the second part of the thesis, we focused on the analysis of all BGCs encoded in the S. rimosus genom by applying bioinformatics tools. After cultivation of S. rimosus in selected media and followed by LC-MS analysis, we detected the production of nine secondary metabolites. Finally, we aimed to identify BGCs in the S. rimosus genome encoding the biosynthesis of a red-colored metabolite, which only appears in co-cultivation with Bacillus subtilis. After deletion of selected BGCs, engineered S. rimosus strains in co-cultivation with B. subtilis still produced red-colored metabolite.
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