Traditionally, natural products have been isolated by bioactivity-guided fractionation. This strategy, however, led to the frequent re-isolation of known metabolites. An alternative strategy for the discovery of novel specialized metabolites is genome mining. Genome mining is an in-silico natural product discovery strategy which uses genetic information to identify natural product biosynthetic gene clusters (BGCs). The identified BGCs are annotated and the information about the BGC constitution is used to predict the structure of the corresponding secondary metabolite. Based on these predictions the corresponding natural product can be isolated in a targeted fashion. Trans-acyltransferase polyketide synthases (trans-AT PKSs) are giant modular multi-domain enzymes that are responsible for the biosynthesis of complex polyketides. These bacterial assembly-lines, ubiquitously distributed across the bacterial branch of life, were shown to produce bioactive polyketides of pharmaceutical and agricultural value. Phylogenetic analysis of ketosynthase (KS) domains from characterized PKSs, responsible for polyketide chain elongation, revealed a correlation between KS domains that phylogenetically cluster together and the substrates they recognize. Based on the correlation between phylogenetic origin and substrate specificity, referred to as the trans-AT PKS correlation rule, we developed the trans-AT PKS-derived polyketide predictor (TransATor) web application for the precise predictions of trans-AT PKS-derived polyketide structures. The TransATor web-application can be used to conduct in-silico dereplication studies. The TransATor-based in-silico annotation of a BGC to a known metabolite prevents the re-isolation of known metabolites, as exemplified by the isolation of tartrolon D from Gynuella sunshinyii. Moreover, TransATor predictions were employed to prioritize isolation efforts and, most importantly, to guide the purification and structure elucidation workflow. This has led to the isolation of a novel polyketide scaffold from Brevibacillus sp. Leaf182 and a new phormidolide-like polyketide, leptolyngbialide, from Leptolyngbia sp. PCC 7375. The prediction of KS substrate specificities furthermore resulted in the revision of the biosynthetic model for oocydin from Serratia plymuthica, the proposal of a model for sesbanimide biosynthesis in Azorhizobium doebereinerae and the reinvestigation and revision of the absolute stereochemistry of phormidolide isolated from a Leptolyngbia sp. ISBN3-Nov-94-8. A previous study on misakinolide-like polyketides suggested that the corresponding PKS family has evolved through the exchange, deletion or insertion of conserved sequential arrangements of PKS modules (PKS motifs). Driven by the hypothesis that trans-AT PKSs evolved in a mosaic-like manner, through combinatorial recombination of conserved motifs from different pathways, we analyzed all 1724 trans-AT PKSs deposited in GenBank for the presence of motifs shared between different PKSs. In total, 327 conserved sequential arrangements of PKS modules were identified. Based on these conserved motifs, also reflected in the polyketide structure, we were able to isolate new polyketides with conserved substructures. These included the novel glutarimide-containing polyketide secimide from the plant pathogen Pseudomonas syringae pv. syringae and the new phormidolide, gynuellalide, from G. sunshinyii. Moreover, the identification of a conserved PKS gene fragment encoding part of the PKS responsible for the biosynthesis of the cytotoxic spliceostatin in the plant pathogen Xanthomonas campestris pv. cannabis resulted in the identification of the pathogenicity factor of the bacterium. In addition, PKSs that share conserved motifs with other orphan PKSs but do not show any conserved motifs with known polyketides are good candidates for the discovery of structural novelty in the corresponding polyketides. The analysis of conserved PKS motifs shared between different PKSs furthermore paves the way towards evolutionary inspired engineering of non-natural hybrid PKSs to produce novel polyketides. 8 A complementary strategy to genome mining is the activation of silent BGCs. Here, we studied the potential of the actinobacterial signaling metabolite hormaomycin to activate silent BGCs in the producing organism and the model strain Streptomyces coelicolor. The effect of hormaomycin on the metabolome of the producing organism, Streptomyces griseoflavus W-384 was analyzed, and compared to the metabolome of hormaomycin production deficient- and hormaomycin overproducing mutants. Two novel metabolites, xanthoductin and xanthotryptin were only produced in the hormaomycin overproducing mutant. Moreover, the production of pharmaceutically important natural products was either strongly up- or downregulated in the presence of hormaomycin. In order to study its effect on other Streptomyces, we subjected S. coelicolor, either challenged with the hormaomcyin overproducer or purified hormaomycin to matrix-assisted laser-desorption ionization time-of-flight (MALDI-TOF) imaging mass spectrometry and nanospray desorption ionization mass spectrometry (nanoDESI) coupled to molecular network analysis. These analyses revealed that hormaomycin induces the production of siderophores, antibiotics, morphogens and pigments belonging to different classes of natural products. In addition, hormaomycin was able to trigger the production of unknown metabolites. These studies show that hormaomycin can be used as a tool to activate silent BGCs and to increase the production of pharmaceutically relevant specialized metabolites. Intrigued by the chemotype triggered by hormaomycin, we were interested in identifying its molecular target. Using a mutasynthesis strategy, hormaomycin analogs with either alkyne or azide handles were generated. These analogs were coupled to different tags and the obtained probes used for pull-down assays to identify the target of hormaomcyin. These assays revealed the the F-ATP synthase as the putative target molecule. Quantification of intracellular ATP levels in the presence or absence of hormaomycin and the phenotypic comparison of the effect of hormaomycin with the decoupling agent carbonylcyanide m-chlorophenylhydrazone supported our initial hypothesis that hormaomycin exerts its effect by interacting with the F-ATP synthase. Moreover, we conducted a high-throughput bipartite interaction screen of all strains of the At-LSPHERE strain collection (50,000 interactions) of bacterial leaf isolates to identify talented antibiotic producers. Linking these data with genome mining studies (AntiSMASH and BigScape analysis), resulted in the identification of antibiotic producers harboring BGCs that encode for putative biosynthetic and structural novelty. The most promising inhibiting strain, Brevibacillus sp. Leaf182 that contains a dozen BGCs was selected for bioactivity-guided fractionation to identify the antibiotic(s) responsible for the observed inhibition of one third of all strains of the strain collection. Isolation of the metabolites responsible for the majority of the observed antibiotic activity yielded tyrocidin analogs and marthiapeptide A. To our surprise, marthiapeptide A is putatively produced via the non-ribosomal route of peptide biosynthesis, which is remarkable given that two structurally close homologs were previously reported to be ribosomally-synthesized and post-translationally modified peptides. The biosynthesis of the same class of polythiazole/polyoxazole-containing 7-8 membered cyclopeptides from two biorthogonal pathways displays an extreme case of convergent evolution. Moreover, the isolation of a novel trans-AT PKS scaffold from the same organism highlights the huge untapped biosynthetic potential of bacterial isolates from the plant phyllosphere.