Abstract:
The continuous emergence of antibiotic-resistant pathogens and the urgent need for novel antibacterial agents require innovative strategies for the discovery of new natural products. Actinobacteria have long been recognized as prolific producers of bioactive secondary metabolites; however, their vast chemical diversity remains far from fully explored. Expanding and facilitating the discovery of novel natural products from actinobacterial sources therefore represents an important challenge in contemporary drug discovery research.
In this thesis, novel natural products from actinobacteria of the DSMZ and Tübingen strain collections were investigated with the objective of expanding accessible chemical diversity for antibacterial drug discovery. A genome mining approach based on the phosphoenolpyruvate mutase (pepM) gene was applied to 940 actinomycete genomes, allowing the identification of putative phosphonate producers and the assessment of their phylogenetic distribution. Subsequently, 28 pepM-positive strains were cultivated under diverse growth conditions, and their metabolite profiles were analyzed by NMR spectroscopy. By combining bioinformatic analyses, targeted gene deletion, heterologous expression, and NMR-based structure elucidation, the minimal biosynthetic gene clusters required for phosphonate production were identified in Kitasatospora fiedleri DSM 114396 and Streptomyces iranensis DSM 41954. To enable the isolation and characterization of phosphonate compounds, several enrichment and purification strategies were developed, including chemical labeling reactions as well as size exclusion chromatography, hydrophilic interaction liquid chromatography (HILIC), and ion-exchange techniques. In addition, a multiplexed chemical metabolomics workflow (MCheM) employing post-column derivatization reactions was established for non-targeted LC-MS/MS analysis. Application of this workflow resulted in the isolation and structural characterization of 7-glycosyl oxazolomycin D from Streptomyces libani subsp. rufus DSM 41230, representing the first reported glycosylated member of the oxazolomycin family.
Furthermore, the metabolic potential of Streptomyces aureocirculatus DSM 40386 was explored, leading to the discovery of piperazic acid-containing peptides. Their mode of action was investigated using a bioreporter-based assay. Moreover, the presence of a brominated compound was detected, indicating additional unexplored chemical diversity, although its full characterization remains pending.
In summary, the results presented in this thesis demonstrate that the integration of genome mining, advanced metabolomics workflows and targeted biosynthetic investigations constitutes a robust strategy for the discovery and characterization of previously inaccessible natural products from actinobacteria, thereby providing a strong basis for future antibacterial lead discovery.
