Actinobacteria are Gram-positive bacteria that have a complex multicellular life cycle and are well known for their ability to produce a wide range of bioactive natural products (NPs). High... Show moreActinobacteria are Gram-positive bacteria that have a complex multicellular life cycle and are well known for their ability to produce a wide range of bioactive natural products (NPs). High throughput screening has failed to deliver the new antibiotics we so desperately need to combat multidrug-resistant pathogens. Therefore, new systematic approaches are needed to further explore the rich potential of Actinobacteria. The work described in this thesis entails systems biology approaches consisting of technologies such as proteomics, genomics, metabolomics and DNA binding studies. These were then applied to identify the biosynthetic gene clusters (BGCs) that are responsible for the production of novel antibiotics. Small molecules were thereby used as elicitors to activate the expression of cryptic BGCs in Streptomyces roseifaciens. Furthermore, S. coelicolor M1152 that was optimized for heterologous expression of antibiotics, was analysed for changes in protein expression, to understand which changes correlate to optimal antibiotic production. Finally, the role of the nucleoid associated protein SCO1839 in development and antibiotic production was studied. Chip-seq technology showed that it binds to thousands of DNA sequences on the S. coelicolor chromosome, which contain the motif GATC. I hope that this thesis contributes to utilizing multi-dimensional ‘omics approaches to answer major biological questions. Show less
Sulheim, S.; Kumelj, T.; Dissel, D. van; Salehzadeh-Yazdi, A.; Du, C.; Wezel, G.P. van; ... ; Kerkhoven, E.J. 2020
Angucyclines are a structurally diverse class of actinobacterial natural products defined by their varied polycyclic ring systems, which display a wide range of biological activities. We recently... Show moreAngucyclines are a structurally diverse class of actinobacterial natural products defined by their varied polycyclic ring systems, which display a wide range of biological activities. We recently discovered lugdunomycin (1), a highly rearranged polyketide antibiotic derived from the angucycline backbone thatis synthesized via several yet unexplained enzymatic reactions. Here, we show via in vivo, in vitro, and structural analysis that the promiscuous reductase LugOII catalyzes both a C6 and an unprecedented C1 ketoreduction. This then sets the stage for the subsequent C-ring cleavage that is key to the rearranged scaffolds of 1. The 1.1 Å structures of LugOII in complex with either ligand 8-O-Methylrabelomycin (4) or 8-O-Methyltetrangomycin (5) and of apoenzyme were resolved, which revealed a canonical Rossman fold and a remarkable conformational change during substrate capture and release. Mutational analysis uncovered key residues for substrate access, position, and catalysis as well as specific determinants that control its dual functionality. The insights obtained in this work hold promise for the discovery and engineering of other promiscuous reductases that may be harnessed for the generationof novel biocatalysts for chemoenzymatic applications. Show less
Xiao, X.; Elsayed, S.S.M.A.; Wu, C.; Heul, H.U. van der; Metsä-Ketelä, M.; Du, C.; ... ; Wezel, G.P. van 2020
One of the hallmark behaviors of social groups is division of labor, where different group members become specialized to carry out complementary tasks. By dividing labor, cooperative groups... Show moreOne of the hallmark behaviors of social groups is division of labor, where different group members become specialized to carry out complementary tasks. By dividing labor, cooperative groups increase efficiency, thereby raising group fitness even if these behaviors reduce individual fitness. We find that antibiotic production in colonies of Streptomyces coelicolor is coordinated by a division of labor. We show that S. coelicolor colonies are genetically heterogeneous because of amplifications and deletions to the chromosome. Cells with chromosomal changes produce diversified secondary metabolites and secrete more antibiotics; however, these changes reduced individual fitness, providing evidence for a trade-off between antibiotic production and fitness. Last, we show that colonies containing mixtures of mutants and their parents produce significantly more antibiotics, while colony-wide spore production remains unchanged. By generating specialized mutants that hyper-produce antibiotics, streptomycetes reduce the fitness costs of secreted secondary metabolites while maximizing the yield and diversity of these products. Show less
Wu, C.; Du, C.; Gubbens, J.; Choi, Y.H.; Wezel, G.P. van 2015
