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Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a structurally diverse group of natural products. They feature a wide range of intriguing post-translational modifications, as exemplified by the biarylitides. These are a family of cyclic tripeptides found in Planomonospora, carrying a biaryl linkage between two aromatic amino acids. Recent genomic analyses revealed that the minimal biosynthetic prerequisite of biarylitide biosynthesis consists of only one ribosomally synthesized pentapeptide precursor as the substrate and a modifying cytochrome-P450-dependent enzyme. In silico analyses revealed that minimal biarylitide RiPP clusters are widespread among natural product producers across phylogenetic borders, including myxobacteria. We report here the genome-guided discovery of the first myxobacterial biarylitide MeYLH, termed Myxarylin, from Pyxidicoccus fallax An d48. Myxarylin was found to be an N-methylated tripeptide that surprisingly exhibits a C–N biaryl crosslink. In contrast to Myxarylin, previously isolated biarylitides are N-acetylated tripeptides that feature a C–C biaryl crosslink. Furthermore, the formation of Myxarylin was confirmed by the heterologous expression of the identified biosynthetic genes in Myxococcus xanthus DK1622. These findings expand the structural and biosynthetic scope of biarylitide-type RiPPs and emphasize the distinct biochemistry found in the myxobacterial realm.

Myxobacteria represent a viable source of chemically diverse and biologically active secondary metabolites. The myxochelins are a well-studied family of catecholate-type siderophores produced by various myxobacterial strains. Here, we report the discovery, isolation and structure elucidation of three new myxochelins N1–N3 from the terrestrial myxobacterium Corallococcus sp. MCy9049, featuring an unusual nicotinic acid moiety. Precursor-directed biosynthesis (PDB) experiments and total synthesis were performed in order to confirm structures, improve access to pure compounds for bioactivity testing and to devise a biosynthesis proposal. The combined evaluation of metabolome and genome data covering myxobacteria supports the notion that the new myxochelin congeners reported here are in fact frequent side products of the known myxochelin A biosynthetic pathway in myxobacteria.

Asperphenamate is a small peptide natural product that has gained much interest due to its antitumor activity. In the recent years numerous bioactive synthetic asperphenamate analogs have been reported, whereas only a handful of natural analogs either of microbial or plant origin has been discovered. Herein we describe a UHPLC-HRMS/MS and amino acid supplement approach for discovery and design of novel asperphenamate analogs. Chemical analysis of Penicillium astrolabium, a prolific producer of asperphenamate, revealed three previously described and two novel asperphenamate analogs produced in significant amounts, suggesting a potential for biosynthesis of further asperphenamate analogs by varying the amino acid availability. Subsequent growth on proteogenic and non-proteogenic amino acid enriched media, revealed a series of novel asperphenamate analogs, including single or double amino acid exchange, as well as benzoic acid exchange for nicotinic acid, with the latter observed from a natural source for the first time. In total, 22 new asperphenamate analogs were characterized by HRMS/MS, with one additionally confirmed by isolation and NMR structure elucidation. This study indicates an extraordinary nonribosomal peptide synthetase (NRPS) flexibility based on substrate availability, and therefore the potential for manipulating and designing novel peptide natural products in filamentous fungi.

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.