Metabolome-guided genome mining of RiPP natural products.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a chemically diverse class of metabolites. Many RiPPs show potent biological activities that make them attractive starting points for drug development. A promising approach for the discovery of new classes of RiPPs is genome mining. However, the accuracy of genome mining is hampered by the lack of signature genes shared across different RiPP classes. One way to reduce false-positive predictions is by complementing genomic information with metabolomics data. In recent years, several new approaches addressing such integrative genomics and metabolomics analyses have been developed. In this review, we provide a detailed discussion of RiPP-compatible software tools that integrate paired genomics and metabolomics data. We highlight current challenges in data integration and identify opportunities for further developments targeting new classes of bioactive RiPPs.

Feature-Based Molecular Networking for the Targeted Identification of Gq-Inhibiting FR900359 Derivatives.

Both the soil bacterium Chromobacterium vaccinii and the bacterial endosymbiont Candidatus Burkholderia crenata of the plant Ardisia crenata are producers of FR900359 (FR). This cyclic depsipeptide is a potent and selective Gq protein inhibitor used extensively to investigate the intracellular signaling of G protein coupled receptors (GPCRs). In this study, the metabolomes of both FR producers were investigated and compared using feature-based molecular networking (FBMN). As a result, 30 previously unknown FR derivatives were identified, one-third being unique to C. vaccinii. Guided by MS, a novel FR derivative, FR-6 (compound 1), was isolated, and its structure unambiguously established. In a whole-cell biosensing assay based on detection of dynamic mass redistribution (DMR) as readout for Gq inhibition, FR-6 suppressed Gq signaling with micromolar potency (pIC50 = 5.56). This functional activity was confirmed in radioligand binding assays (pKi = 7.50). This work demonstrates the power of molecular networking, guiding the way to a novel Gq-inhibiting FR derivative and underlining the potency of FR as a Gq inhibitor.

Blocks in the pseudouridimycin pathway unlock hidden metabolites in the Streptomyces producer strain.

We report a metabolomic analysis of Streptomyces sp. ID38640, a soil isolate that produces the bacterial RNA polymerase inhibitor pseudouridimycin. The analysis was performed on the wild type, on three newly constructed and seven previously reported mutant strains disabled in different genes required for pseudouridimycin biosynthesis. The results indicate that Streptomyces sp. ID38640 is able to produce, in addition to lydicamycins and deferroxiamines, as previously reported, also the lassopeptide ulleungdin, the non-ribosomal peptide antipain and the osmoprotectant ectoine. The corresponding biosynthetic gene clusters were readily identified in the strain genome. We also detected the known compound pyridindolol, for which we propose a previously unreported biosynthetic gene cluster, as well as three families of unknown metabolites. Remarkably, the levels of most metabolites varied strongly in the different mutant strains, an observation that enabled detection of metabolites unnoticed in the wild type. Systematic investigation of the accumulated metabolites in the ten different pum mutants identified shed further light on pseudouridimycin biosynthesis. We also show that several Streptomyces strains, able to produce pseudouridimycin, have distinct genetic relationship and metabolic profile with ID38640.

Effective approaches to discover new microbial metabolites in a large strain library.

Natural products have provided many molecules to treat and prevent illnesses in humans, animals and plants. While only a small fraction of the existing microbial diversity has been explored for bioactive metabolites, tens of thousands of molecules have been reported in the literature over the past 80 years. Thus, the main challenge in microbial metabolite screening is to avoid the re-discovery of known metabolites in a cost-effective manner. In this perspective, we report and discuss different approaches used in our laboratory over the past few years, ranging from bioactivity-based screening to looking for metabolic rarity in different datasets to deeply investigating a single Streptomyces strain. Our results show that it is possible to find novel chemistry through a limited screening effort, provided that appropriate selection criteria are in place.

Planomonospora: A Metabolomics Perspective on an Underexplored Actinobacteria Genus.

Despite an excellent track record, microbial drug discovery suffers from high rates of rediscovery. Better workflows for the rapid investigation of complex extracts are needed to increase throughput and to allow early prioritization of samples. In addition, systematic characterization of poorly explored strains is seldomly performed. Here, we report a metabolomic study of 72 isolates belonging to the rare actinomycete genus Planomonospora, using a workflow of commonly used open access tools to investigate its secondary metabolites. The results reveal a correlation of chemical diversity and strain phylogeny, with classes of metabolites exclusive to certain phylogroups. We were able to identify previously reported Planomonospora metabolites, including the ureylene-containing oligopeptide antipain, the thiopeptide siomycin including new congeners, and the ribosomally synthesized peptides sphaericin and lantibiotic 97518. In addition, we found that Planomonospora strains can produce the siderophore desferrioxamine or a salinichelin-like peptide. Analysis of the genomes of three newly sequenced strains led to the detection of 59 gene cluster families, of which three were connected to products found by LC-MS/MS profiling. This study demonstrates the value of metabolomic studies to investigate poorly explored taxa and provides a first picture of the biosynthetic capabilities of the genus Planomonospora.

A biaryl-linked tripeptide from Planomonospora reveals a widespread class of minimal RiPP gene clusters.

Microbial natural products impress by their bioactivity, structural diversity, and ingenious biosynthesis. While screening the less exploited actinobacterial genus Planomonospora, two cyclopeptides were discovered, featuring an unusual Tyr-His biaryl bridging across a tripeptide scaffold, with the sequences N-acetyl-Tyr-Tyr-His and N-acetyl-Tyr-Phe-His. Planomonospora genomes pointed toward a ribosomal synthesis of the cyclopeptide from a pentapeptide precursor encoded by 18-bp bytA, to our knowledge the smallest coding gene ever reported. Closely linked to bytA is bytO, encoding a cytochrome P450 monooxygenase likely responsible for biaryl installment. In Streptomyces, the bytAO segment was sufficient to direct production of the crosslinked N-acetylated Tyr-Tyr-His tripeptide. Bioinformatic analysis of related cytochrome P450 monooxygenases indicated that they constitute a widespread family of enzymes, and the corresponding genes are closely linked to 5-amino acid coding sequences in approximately 200 (actino)bacterial genomes, all with potential for biaryl linkage between amino acids 1 and 3. We propose the named biarylitides this family of RiPPs.