New myxobacteria of the Myxococcaceae clade produce angiolams with antiparasitic activities.

In the past century, microbial natural products have proven themselves to be substantial and fruitful sources of anti-infectives. In addition to the well-studied Actinobacteria, understudied bacterial taxa like the Gram-negative myxobacteria have increasingly gained attention in the ongoing search for novel and biologically active natural products. In the course of a regional sampling campaign to source novel myxobacteria, we recently uncovered new myxobacterial strains MCy12716 and MCy12733 belonging to the Myxococcaceae clade. Early bioactivity screens of the bacterial extracts revealed the presence of bioactive natural products that were identified as angiolam A and several novel derivatives. Sequencing of the corresponding producer strains allowed the identification of the angiolam biosynthetic gene cluster, which was verified by targeted gene inactivation. Based on bioinformatic analysis of the biosynthetic gene cluster, a concise biosynthesis model was devised to explain angiolam biosynthesis. Importantly, novel angiolam derivatives uncovered in this study named angiolams B, C, and D were found to display promising antiparasitic activities against the malaria pathogen Plasmodium falciparum in the 0.3-0.8 µM range.IMPORTANCEThe COVID-19 pandemic and continuously emerging antimicrobial resistance (AMR) have recently raised awareness about limited treatment options against infectious diseases. However, the shortage of treatment options against protozoal parasitic infections, like malaria, is much more severe, especially for the treatment of so-called neglected tropical diseases. The detection of anti-parasitic bioactivities of angiolams produced by MCy12716 and MCy12733 displays the hidden potential of scarcely studied natural products to have promising biological activities in understudied indications. Furthermore, the improved biological activities of novel angiolam derivatives against Plasmodium falciparum and the evaluation of its biosynthesis display the opportunities of the angiolam scaffold on route to treat protozoal parasitic infections as well as possible ways to increase the production of derivatives with improved bioactivities.

Darobactins Exhibiting Superior Antibiotic Activity by Cryo-EM Structure Guided Biosynthetic Engineering.

Over recent decades, the pipeline of antibiotics acting against Gram-negative bacteria is running dry, as most discovered candidate antibiotics suffer from insufficient potency, pharmacokinetic properties, or toxicity. The darobactins, a promising new small peptide class of drug candidates, bind to novel antibiotic target BamA, an outer membrane protein. Previously, we reported that biosynthetic engineering in a heterologous host generated novel darobactins with enhanced antibacterial activity. Here we utilize an optimized purification method and present cryo-EM structures of the Bam complex with darobactin 9 (D9), which served as a blueprint for the biotechnological generation of twenty new darobactins including halogenated analogs. The newly engineered darobactin 22 binds more tightly to BamA and outperforms the favorable activity profile of D9 against clinically relevant pathogens such as carbapenem-resistant Acinetobacter baumannii up to 32-fold, without observing toxic effects.

Myxobacteria of the Cystobacterineae Suborder Are Producers of New Vitamin K2 Derived Myxoquinones.

Vitamin K is an essential, lipid soluble vitamin that plays an important role in the human blood coagulation cascade as well as in the life cycle of bacteria and plants. In this study, we report the isolation and structure elucidation of unprecedented polyhydroxylated menaquinone variants named myxoquinones that are produced by myxobacteria and structurally belong to the Vitamin K family. We analyze the occurrence of myxoquinones across an LC-MS data collection from myxobacterial extracts and shed light on the distribution of myxoquinone-type biosynthetic gene clusters among publicly available myxobacterial genomes. Our findings indicate that myxoquinones are specifically produced by strains of the Cystobacterineae suborder within myxobacteria. Furthermore, bioinformatic analysis of the matching gene clusters allowed us to propose a biosynthetic model for myxoquinone formation. Due to their increased water-solubility, the myxoquinones could be a suitable starting point for the development of a better bioavailable treatment of vitamin K deficiency.

Sandacrabins - Structurally Unique Antiviral RNA Polymerase Inhibitors from a Rare Myxobacterium.

Structure elucidation and total synthesis of five unprecedented terpenoid-alkaloids, the sandacrabins, are reported, alongside with the first description of their producing organism Sandaracinus defensii MSr10575, which expands the Sandaracineae family by only its second member. The genome sequence of S. defensii as presented in this study was utilized to identify enzymes responsible for sandacrabin formation, whereby dimethylbenzimidazol, deriving from cobalamin biosynthesis, was identified as key intermediate. Biological activity profiling revealed that all sandacrabins except congener A exhibit potent antiviral activity against the human pathogenic coronavirus HCoV229E in the three digit nanomolar range. Investigation of the underlying mode of action discloses that the sandacrabins inhibit the SARS-CoV-2 RNA-dependent RNA polymerase complex, highlighting them as structurally distinct non-nucleoside RNA synthesis inhibitors. The observed segregation between cell toxicity at higher concentrations and viral inhibition opens the possibility for their medicinal chemistry optimization towards selective inhibitors.

Genome-Guided Discovery of the First Myxobacterial Biarylitide Myxarylin Reveals Distinct C-N Biaryl Crosslinking in RiPP Biosynthesis.

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.

Improved broad-spectrum antibiotics against Gram-negative pathogens via darobactin biosynthetic pathway engineering.

The development of new antibiotics is imperative to fight increasing mortality rates connected to infections caused by multidrug-resistant (MDR) bacteria. In this context, Gram-negative pathogens listed in the WHO priority list are particularly problematic. Darobactin is a ribosomally produced and post-translationally modified bicyclic heptapeptide antibiotic selectively killing Gram-negative bacteria by targeting the outer membrane protein BamA. The native darobactin A producer Photorhabdus khanii HGB1456 shows very limited production under laboratory cultivation conditions. Herein, we present the design and heterologous expression of a synthetically engineered darobactin biosynthetic gene cluster (BGC) in Escherichia coli to reach an average darobactin A production titre of 13.4 mg L-1. Rational design of darA variants, encoding the darobactin precursor peptide with altered core sequences, resulted in the production of 13 new 'non-natural' darobactin derivatives and 4 previously hypothetical natural darobactins. One of the non-natural compounds, darobactin 9, was more potent than darobactin A, and showed significantly improved activity especially against Pseudomonas aeruginosa (0.125 µg mL-1) and Acinetobacter baumannii (1-2 µg mL-1). Importantly, it also displayed superior activity against MDR clinical isolates of E. coli (1-2 µg mL-1) and Klebsiella pneumoniae (1-4 µg mL-1). Independent deletions of genes from the darobactin BGC showed that only darA and darE, encoding a radical forming S-adenosyl-l-methionine-dependent enzyme, are required for darobactin formation. Co-expression of two additional genes associated with the BGCs in hypothetical producer strains identified a proteolytic detoxification mechanism as a potential self-resistance strategy in native producers. Taken together, we describe a versatile heterologous darobactin platform allowing the production of unprecedented active derivatives in good yields, and we provide first experimental evidence for darobactin biosynthesis processes.

Synergizing the potential of bacterial genomics and metabolomics to find novel antibiotics.

Antibiotic development based on natural products has faced a long lasting decline since the 1970s, while both the speed and the extent of antimicrobial resistance (AMR) development have been severely underestimated. The discovery of antimicrobial natural products of bacterial and fungal origin featuring new chemistry and previously unknown mode of actions is increasingly challenged by rediscovery issues. Natural products that are abundantly produced by the corresponding wild type organisms often featuring strong UV signals have been extensively characterized, especially the ones produced by extensively screened microbial genera such as streptomycetes. Purely synthetic chemistry approaches aiming to replace the declining supply from natural products as starting materials to develop novel antibiotics largely failed to provide significant numbers of antibiotic drug leads. To cope with this fundamental issue, microbial natural products science is being transformed from a 'grind-and-find' study to an integrated approach based on bacterial genomics and metabolomics. Novel technologies in instrumental analytics are increasingly employed to lower detection limits and expand the space of detectable substance classes, while broadening the scope of accessible and potentially bioactive natural products. Furthermore, the almost exponential increase in publicly available bacterial genome data has shown that the biosynthetic potential of the investigated strains by far exceeds the amount of detected metabolites. This can be judged by the discrepancy between the number of biosynthetic gene clusters (BGC) encoded in the genome of each microbial strain and the number of secondary metabolites actually detected, even when considering the increased sensitivity provided by novel analytical instrumentation. In silico annotation tools for biosynthetic gene cluster classification and analysis allow fast prioritization in BGC-to-compound workflows, which is highly important to be able to process the enormous underlying data volumes. BGC prioritization is currently accompanied by novel molecular biology-based approaches to access the so-called orphan BGCs not yet correlated with a secondary metabolite. Integration of metabolomics, in silico genomics and molecular biology approaches into the mainstream of natural product research will critically influence future success and impact the natural product field in pharmaceutical, nutritional and agrochemical applications and especially in anti-infective research.

The Sandarazols are Cryptic and Structurally Unique Plasmid-Encoded Toxins from a Rare Myxobacterium*.

Herein, we describe a new plasmid found in Sandaracinus sp. MSr10575 named pSa001 spanning 209.7 kbp that harbors a cryptic secondary metabolite biosynthesis gene cluster (BGC). Activation of this BGC by homologous-recombination-mediated exchange of the native promoter sequence against a vanillate inducible system led to the production and subsequent isolation and structure elucidation of novel secondary metabolites, the sandarazols A-G. The sandarazols contain intriguing structural features and very reactive functional groups such as an α-chlorinated ketone, an epoxyketone, and a (2R)-2-amino-3-(N,N-dimethylamino)-propionic acid building block. In-depth investigation of the underlying biosynthetic machinery led to a concise biosynthetic model for the new compound family, including several uncommon biosynthetic steps. The chlorinated congener sandarazol C shows an IC50  value of 0.5 µm against HCT 116 cells and a MIC of 14 µm against Mycobacterium smegmatis, which points at the sandarazols' potential function as defensive secondary metabolites or toxins.

Expanding the Scope of Detectable Microbial Natural Products by Complementary Analytical Methods and Cultivation Systems.

Recent advances in genome sequencing have unveiled a large discrepancy between the genome-encoded capacity of microorganisms to produce secondary metabolites and the number detected. In this work, a two-platform mass spectrometry analysis for the comprehensive secondary metabolomics characterization of nine myxobacterial strains, focusing on extending the range of detectable secondary metabolites by diversifying analytical methods and cultivation conditions, is presented. Direct infusion measurements of crude extracts on a Fourier transform ion cyclotron resonance mass spectrometer are compared to a time-of-flight device coupled to liquid chromatography measurements. Both methods are successful in detecting known metabolites, whereas statistical analysis of unknowns highlights their complementarity: Strikingly, 82-99% of molecular features detected with one setup were not detectable with the other. Metabolite profile differences from our set of strains grown in liquid culture versus their swarming colonies on agar plates were evaluated. The detection of up to 96% more molecular features when both liquid and plate cultures were analyzed translates into increased chances to identify new secondary metabolites. Discrimination between primary and secondary metabolism in combination with GNPS molecular networking revealed strain Mx3 as particularly promising for the isolation of novel secondary metabolites among the nine strains investigated in this study.

Supercritical Fluid Extraction Enhances Discovery of Secondary Metabolites from Myxobacteria.

Supercritical fluid extraction (SFE) is widely used for the isolation of natural products from plants, but its application in efforts to identify structurally and physicochemically often dissimilar microbial natural products is limited to date. In this study, we evaluated the impact of SFE on the extractability of myxobacterial secondary metabolites, aiming to improve the prospects of discovering novel natural products. We investigated the influence of different co-solvents on the extraction efficiency of secondary metabolites from three myxobacterial strains and the antimicrobial activity profiles of the corresponding extracts. For each known secondary metabolite, we found extraction conditions using SFE leading to superior yields in the extracts compared to conventional solvent extraction. Compounds with a logP higher than 3 showed the best extraction efficiency using 20% EtOAc as a co-solvent, whereas compounds with logP values lower than 3 were better extractable using more polar co-solvents such as MeOH. Extracts generated with SFE showed increased antimicrobial activities including the presence of activities not explained by known myxobacterial secondary metabolites, highlighting the advantage of SFE for bioactivity-guided isolation. Moreover, non-targeted metabolomics analysis revealed a group of chlorinated metabolites produced by the well-studied model myxobacterium Myxococcus xanthus DK1622, which were not accessible previously due to their low concentration in conventional extracts. The enriched SF extracts were used for isolation and subsequent structure elucidation of chloroxanthic acid A as the founding member of a novel secondary metabolite family. Our findings encourage the increased utilization of SFE as a part of future screening workflows of microbial natural products.

Myxobacteria-Derived Outer Membrane Vesicles: Potential Applicability Against Intracellular Infections.

In 2019, it was estimated that 2.5 million people die from lower tract respiratory infections annually. One of the main causes of these infections is Staphylococcus aureus, a bacterium that can invade and survive within mammalian cells. S. aureus intracellular infections are difficult to treat because several classes of antibiotics are unable to permeate through the cell wall and reach the pathogen. This condition increases the need for new therapeutic avenues, able to deliver antibiotics efficiently. In this work, we obtained outer membrane vesicles (OMVs) derived from the myxobacteria Cystobacter velatus strain Cbv34 and Cystobacter ferrugineus strain Cbfe23, that are naturally antimicrobial, to target intracellular infections, and investigated how they can affect the viability of epithelial and macrophage cell lines. We evaluated by cytometric bead array whether they induce the expression of proinflammatory cytokines in blood immune cells. Using confocal laser scanning microscopy and flow cytometry, we also investigated their interaction and uptake into mammalian cells. Finally, we studied the effect of OMVs on planktonic and intracellular S. aureus. We found that while Cbv34 OMVs were not cytotoxic to cells at any concentration tested, Cbfe23 OMVs affected the viability of macrophages, leading to a 50% decrease at a concentration of 125,000 OMVs/cell. We observed only little to moderate stimulation of release of TNF-alpha, IL-8, IL-6 and IL-1beta by both OMVs. Cbfe23 OMVs have better interaction with the cells than Cbv34 OMVs, being taken up faster by them, but both seem to remain mostly on the cell surface after 24 h of incubation. This, however, did not impair their bacteriostatic activity against intracellular S. aureus. In this study, we provide an important basis for implementing OMVs in the treatment of intracellular infections.

In depth natural product discovery - Myxobacterial strains that provided multiple secondary metabolites.

In recognition of many microorganisms ability to produce a variety of secondary metabolites in parallel, Zeeck and coworkers introduced the term "OSMAC" (one strain many compounds) around the turn of the century. Since then, additional efforts focused on the systematic characterization of a single bacterial species ability to form multiple secondary metabolite scaffolds. With the beginning of the genomic era mainly initiated by a dramatic reduction of sequencing costs, investigations of the genome encoded biosynthetic potential and especially the exploitation of biosynthetic gene clusters of undefined function gained attention. This was seen as a novel means to extend range and diversity of bacterial secondary metabolites. Genome analyses showed that even for well-studied bacterial strains, like the myxobacterium Myxococcus xanthus DK1622, many biosynthetic gene clusters are not yet assigned to their corresponding hypothetical secondary metabolites. In contrast to the results from emerging genome and metabolome mining techniques that show the large untapped biosynthetic potential per strain, many newly isolated bacterial species are still used for the isolation of only one target compound class and successively abandoned in the sense that no follow up studies are published from the same species. This work provides an overview about myxobacterial bacterial strains, from which not just one but multiple different secondary metabolite classes were successfully isolated. The underlying methods used for strain prioritization and natural product discovery such as biological characterization of crude extracts against a panel of pathogens, in-silico prediction of secondary metabolite abundance from genome data and state of the art instrumental analytics required for new natural product scaffold discovery in comparative settings are summarized and classified according to their output. Furthermore, for each approach selected studies performed with actinobacteria are shown to underline especially innovative methods used for natural product discovery.

Production of a Dibrominated Aromatic Secondary Metabolite by a Planctomycete Implies Complex Interaction with a Macroalgal Host.

The roles of the majority of bacterial secondary metabolites, especially those from uncommon sources, are still elusive even though many of these compounds show striking biological activities. To further investigate the secondary metabolite repertoire of underexploited bacterial families, we chose to analyze a novel representative of the yet untapped bacterial phylum Planctomycetes for the production of secondary metabolites under laboratory culture conditions. Development of a planctomycetal high density cultivation technique in combination with high resolution mass spectrometric analysis revealed Planctomycetales strain 10988 to produce the plant toxin 3,5-dibromo-p-anisic acid. This molecule represents the first secondary metabolite reported from any planctomycete. Genome mining revealed the biosynthetic origin of this doubly brominated secondary metabolite, and a biosynthesis model for the compound was devised. Comparison of the biosynthetic route to biosynthetic gene clusters responsible for formation of polybrominated small aromatic compounds reveals evidence of an evolutionary link, while the compound's herbicidal activity points toward a complex interaction of planctomycetes with their macroalgal host.

Production optimization and biosynthesis revision of corallopyronin A, a potent anti-filarial antibiotic.

Corallopyronins (COR) are α-pyrone antibiotics from myxobacteria representing highly promising lead structures for the development of antibacterial therapeutic agents. Their ability to inhibit RNA polymerase through interaction with the "switch region", a novel target, distant from binding sites of previously characterized RNA polymerase inhibitors (e.g. rifampicin), makes them particularly promising as antibiotic candidates. Corallopyronin A is currently also investigated as a lead compound for the treatment of lymphatic filariasis because of its superb activity against the nematode symbiont Wolbachia. As total synthesis is not a valid production option biotechnological optimization of compound supply is of utmost importance to further develop this highly potent compound class. Here we describe decisive improvements of the previously reported heterologous COR production and engineering platform yielding production of ~100 mg/L COR A. Furthermore, we provide a revised model of COR biosynthesis shedding light on the function of several biosynthetic proteins, including an unusual ECH-like enzyme providing dehydration functionality in trans and an uncharacterized protein conferring COR self-resistance in the myxobacterial heterologous host Myxococcus xanthus DK1622. We also report two new COR derivatives, COR D and oxyCOR A discovered in genetically engineered strains.

Novel Methoxymethacrylate Natural Products Uncovered by Statistics-Based Mining of the Myxococcus fulvus Secondary Metabolome.

This study reports the uncovering of new myxobacterial natural products through comprehensive analysis of the Myxococcus fulvus secondary metabolome. Statistics-based mining of mass spectrometry data paved the way for full structure elucidation of two new secondary metabolites named fulvuthiacene A and B, and investigation of the underlying biosynthetic pathway revealed an evolutionary link between the fulvuthiacene hybrid polyketide synthase (PKS) and nonribosomal peptide synthetase (NRPS) gene cluster and the related myxothiazol and melithiazol assembly lines. Detailed characterization of the post-PKS modification enzyme cascade responsible for the fulvuthiacenes' terminal ß-methoxy-methyl acrylate moiety was pursued by heterologous expression of these enzymes in the myxothiazol producer Stigmatella aurantiaca DW4/3-1. The discovery of fulvuthiacenes provides new insights into the overall structure-activity relationship picture for the ß-methoxyacrylate class of respiratory chain inhibitors and might thus serve as starting point for the development of next-generation ß-methoxymethacrylate fungicides.

Genome mining reveals uncommon alkylpyrones as type III PKS products from myxobacteria.

Type III polyketide synthases (PKSs) are comparatively small homodimeric enzymes affording natural products with diverse structures and functions. While type III PKS biosynthetic pathways have been studied thoroughly in plants, their counterparts from bacteria and fungi are to date scarcely characterized. This gap is exemplified by myxobacteria from which no type III PKS-derived small molecule has previously been isolated. In this study, we conducted a genomic survey of myxobacterial type III PKSs and report the identification of uncommon alkylpyrones as the products of type III PKS biosynthesis from the myxobacterial model strain Myxococcus xanthus DK1622 through a self-resistance-guided screening approach focusing on genes encoding pentapetide repeat proteins, proficient to confer resistance to topoisomerase inhibitors. Using promoter-induced gene expression in the native host as well as heterologous expression of biosynthetic type III PKS genes, sufficient amounts of material could be obtained for structural elucidation and bioactivity testing, revealing potent topoisomerase activity in vitro.

Biocompatible bacteria-derived vesicles show inherent antimicrobial activity.

Up to 25,000 people die each year from resistant infections in Europe alone, with increasing incidence. It is estimated that a continued rise in bacterial resistance by 2050 would lead up to 10 million annual deaths worldwide, exceeding the incidence of cancer deaths. Although the design of new antibiotics is still one way to tackle the problem, pharmaceutical companies investigate far less into new drugs than 30 years ago. Incorporation of antibiotics into nanoparticle drug carriers ("nanoantibiotics") is currently investigated as a promising strategy to make existing antibiotics regain antimicrobial strength and overcome certain types of microbial drug resistance. Many of these synthetic systems enhance the antimicrobial effect of drugs by protecting antibiotics from degradation and reducing their side effects. Nevertheless, they often cannot selectively target pathogenic bacteria and - due to their synthetic origin - may induce side-effects themselves. In this work, we present the characterisation of naturally derived outer membrane vesicles (OMVs) as biocompatible and inherently antibiotic drug carriers. We isolated OMVs from two representative strains of myxobacteria, Cystobacter velatus Cbv34 and Sorangiineae species strain SBSr073, a bacterial order with the ability of lysing other bacterial strains and currently investigated as sources of new secondary metabolites. We investigated the myxobacterias' inherent antibacterial properties after isolation by differential centrifugation and purification by size-exclusion chromatography. OMVs have an average size range of 145-194 nm. We characterised their morphology by electron cryomicroscopy and found that OMVs are biocompatible with epithelial cells and differentiated macrophages. They showed a low endotoxin activity comparable to those of control samples, indicating a low acute inflammatory potential. In addition, OMVs showed inherent stability under different storage conditions, including 4 °C, -20 °C, -80 °C and freeze-drying. OMV uptake in Gram-negative model bacterium Escherichia coli (E. coli) showed similar to better incorporation than liposome controls, indicating the OMVs may interact with model bacteria via membrane fusion. Bacterial uptake correlated with antimicrobial activity of OMVs as measured by growth inhibition of E. coli. OMVs from Cbv34 inhibited growth of E. coli to a comparable extent as the clinically established antibiotic gentamicin. Liquid-chromatography coupled mass spectrometry analyses revealed the presence of cystobactamids in OMVs, inhibitors of bacterial topoisomerase currently studied to treat different Gram-negative and Gram-positive pathogens. This work, may serve as an important basis for further evaluation of OMVs derived from myxobacteria as novel therapeutic delivery systems against bacterial infections.

Structure, Total Synthesis, and Biosynthesis of Chloromyxamides: Myxobacterial Tetrapeptides Featuring an Uncommon 6-Chloromethyl-5-methoxypipecolic Acid Building Block.

Soil-living microbes are an important resource for the discovery of new natural products featuring great structural diversity that are reflective of the underlying biosynthetic pathways as well as incorporating a wide range of intriguing small-molecule building blocks. We report here the full structural elucidation, total synthesis, and biosynthesis of chloromyxamides, a new class of tetrapeptides that display an unprecedented 6-chloromethyl-5-methoxypipecolic acid (CMPA) substructure. Chemical synthesis-including an approach to access the CMPA unit-was pursued to confirm the structure of the chloromyxamides and enabled determination of the absolute configuration in the CMPA ring. A model for the nonribosomal assembly of chloromyxamides was devised on the basis of the combined evaluation of the biosynthetic gene cluster sequence and the feeding of stable isotope-labeled precursors. This provided insight into the formation of the various chloromyxamide derivatives and the biogenesis of the CMPA unit.

Self-resistance guided genome mining uncovers new topoisomerase inhibitors from myxobacteria.

There is astounding discrepancy between the genome-inscribed production capacity and the set of known secondary metabolite classes from many microorganisms as detected under laboratory cultivation conditions. Genome-mining techniques are meant to fill this gap, but in order to favor discovery of structurally novel as well as bioactive compounds it is crucial to amend genomics-based strategies with selective filtering principles. In this study, we followed a self-resistance guided approach aiming at the discovery of inhibitors of topoisomerase, known as valid target in both cancer and antibiotic therapy. A common host self-defense mechanism against such inhibitors in bacteria is mediated by so-called pentapeptide repeat proteins (PRP). Genes encoding the biosynthetic machinery for production of an alleged topoisomerase inhibitor were found on the basis of their collocation adjacent to a predicted PRP in the genome of the myxobacterium Pyxidicoccus fallax An d48, but to date no matching compound has been reported from this bacterium. Activation of this peculiar polyketide synthase type-II gene cluster in the native host as well as its heterologous expression led to the structure elucidation of new natural products that were named pyxidicyclines and provided an insight into their biosynthesis. Subsequent topoisomerase inhibition assays showed strong affinity to - and inhibition of - unwinding topoisomerases such as E. coli topoisomerase IV and human topoisomerase I by pyxidicyclines as well as precise selectivity, since E. coli topoisomerase II (gyrase) was not inhibited at concentrations up to 50 µg ml-1.