Proteasome Inhibitors from Actinobacteria Biosynthesis of the Cyclopropylalanine Moiety of Belactosin A and the Role of Proteasome Inhibitors from Nocardia in Pathogen Survival

Abstract

Diese Dissertation ist bis zum 4. August 2025 gesperrt !

Natural product research is a fruitful area of investigation for the discovery of new drugs and bioactive molecules. The biosynthesis of proteasome inhibitor associated natural products has gained attention in recent years, including the class of β-lactone-containing peptide natural product proteasome inhibitors. Belactosin A and hormaomycins are such peptide natural products containing 3-(2-aminocyclopropyl)alanine (Acpa) and 3-(2- nitrocyclopropyl)alanine (Ncpa) residues, respectively. In the first project of this thesis I shed light on the formation of the unique Acpa moiety of belactosin A. I was able to establish heterologous pathway expression of the biosynthetic gene cluster of the belactosin series from Streptomyces sp. UCK14, including border cluster knock-outs. We could thereby show that the biosynthetic gene cluster of belactosins reaches from genes belF to belV and the four gene operon belK-N is responsible for building the Acpa moiety of belactosin A. Construction of a set of gene deletion and feeding experiments for chemical complementation that include incorporation of stable isotope-labeled precursors clearly demonstrated that in the biosynthesis of the Acpa building block, a cryptic nitrocyclopropylalanine (Ncpa) intermediate is generated from L-lysine. Additionally, I could demonstrate that the subsequent reduction of the N-oxygenated precursor Ncpa to the corresponding amine compound Acpa is mediated by the molybdopterin-dependent enzyme BelN. Feeding of double deuterium labeled Acpa to Streptomyces sp. UCK14/ΔbelN fully restored belactosin A production. These results were further verified by genetic complementation of Streptomyces sp. UCK14/belN with pUWL_belN. Additionally feeding of 13C6-L-lysine to the Streptomyces sp. UCK14/belN mutant resulted in the accumulation of the Ncpa intermediate. The determination of the actual enzymatic mechanism for the reduction of Ncpa to Acpa will be an intriguing subject for future investigations and further studies are currently ongoing. In parallel to our studies, we started a close collaboration with the research group around Prof. Dr. Abe from the University of Tokyo. In agreement with our initial in vivo feeding studies, they could enlighten the function of heme oxygenase-like enzyme BelK and nonheme iron enzyme BelL in detail. The second half of this work focuses on natural products from Nocardia and their potential connection to the virulence of pathogenic strains. Nocardia spp. are filamentous actinobacteria and can cause localized and systemic infections in humans. However, the virulence mechanisms of human pathogenic Nocardia and the progression of nocardiosis are poorly understood. As more Nocardia strains are genome sequenced, genome analysis tools, such as genome mining approaches, provide access to the potential for the production of novel and unique natural products.