Megalochelin, a Tridecapeptide Siderophore from a Talented Streptomycete.

Streptomycetes are bacteria known for their extraordinary biosynthetic capabilities. Herein, we describe the genome and metabolome of a particularly talented strain, Streptomyces ID71268. Its 8.4-Mbp genome harbors 32 bioinformatically predicted biosynthetic gene clusters (BGCs), out of which 10 are expressed under a single experimental condition. In addition to five families of known metabolites with previously assigned BGCs (nigericin, azalomycin F, ectoine, SF2766, and piericidin), we were able to predict BGCs for three additional metabolites: streptochlorin, serpetene, and marinomycin. The strain also produced two families of presumably novel metabolites, one of which was associated with growth inhibitory activity against the human opportunistic pathogen Acinetobacter baumannii in an iron-dependent manner. Bioassay-guided fractionation, followed by extensive liquid chromatography-mass spectrometry (LC-MS) and NMR analyses, established that the molecule responsible for the observed antibacterial activity is an unusual tridecapeptide siderophore with a ring-and-tail structure: the heptapeptide ring is formed through a C-C bond between a 2,3-dihydroxybenzoate (DHB) cap on Gly1 and the imidazole moiety of His7, while the hexapeptide tail is sufficient for binding iron. This molecule, named megalochelin, is the largest known siderophore. The megalochelin BGC encodes a 13-module nonribosomal peptide synthetase for the synthesis of the tridecapeptide, and a copper-dependent oxidase, likely responsible for the DHB-imidazole cross-link, whereas the genes for synthesis of the DHB starter unit are apparently specified in trans by a different BGC. Our results suggest that prolific producers of specialized metabolites may conceal hidden treasures within a background of known compounds.

Allopeptimicins: unique antibacterial metabolites generated by hybrid PKS-NRPS, with original self-defense mechanism in Actinoallomurus.

In the search for structurally novel metabolites with antibacterial activity, innovative approaches must be implemented to increase the probability of discovering novel chemistry from microbial sources. Here we report on the application of metabolomic tools to the genus Actinoallomurus, a poorly explored member of the Actinobacteria. From examining extracts derived from 88 isolates belonging to this genus, we identified a family of cyclodepsipeptides acylated with a C20 polyketide chain, which we named allopeptimicins. These molecules possess unusual structural features, including several double bonds in the amino-polyketide chain and four non-proteinogenic amino acids in the octapeptide. Remarkably, allopeptimicins are produced as a complex of active and inactive congeners, the latter carrying a sulfate group on the polyketide amine. This modification is also a mechanism of self-protection in the producer strain. The structural uniqueness of allopeptimicins is reflected in a biosynthetic gene cluster showing a mosaic structure, with dedicated gene cassettes devoted to formation of specialized precursors and modular assembly lines related to those from different pathways.

N-Acetyl-Cysteinylated Streptophenazines from Streptomyces.

Here, we describe two N-acetyl-cysteinylated streptophenazines (1 and 2) produced by the soil-derived Streptomyces sp. ID63040 and identified through a metabolomic approach. These metabolites attracted our interest due to their low occurrence frequency in a large library of fermentation broth extracts and their consistent presence in biological replicates of the producer strain. The compounds were found to possess broad-spectrum antibacterial activity while exhibiting low cytotoxicity. The biosynthetic gene cluster from Streptomyces sp. ID63040 was found to be highly similar to the streptophenazine reference cluster in the MIBiG database, which originates from the marine Streptomyces sp. CNB-091. Compounds 1 and 2 were the main streptophenazine products from Streptomyces sp. ID63040 at all cultivation times but were not detected in Streptomyces sp. CNB-091. The lack of obvious candidates for cysteinylation in the Streptomyces sp. ID63040 biosynthetic gene cluster suggests that the N-acetyl-cysteine moiety derives from cellular functions, most likely from mycothiol. Overall, our data represent an interesting example of how to leverage metabolomics for the discovery of new natural products and point out the often-neglected contribution of house-keeping cellular functions to natural product diversification.

Biosynthesis of C-nucleoside antibiotics in actinobacteria: recent advances and future developments.

Epidemic diseases and antibiotic resistance are urgent threats to global health, and human is confronted with an unprecedented dilemma to conquer them by expediting development of new natural product related drugs. C-nucleoside antibiotics, a remarkable group of microbial natural products with diverse biological activities, feature a heterocycle base linked with a ribosyl moiety via an unusual C-glycosidic bond, and have played significant roles in healthcare and for plant protection. Elucidating how nature biosynthesizes such a group of antibiotics has provided the basis for engineered biosynthesis as well as targeted genome mining of more C-nucleoside antibiotics towards improved properties. In this review, we mainly summarize the recent advances on the biosynthesis of C-nucleoside antibiotics, and we also tentatively discuss the future developments on rationally accessing C-nucleoside diversities in a more efficient and economical way via synthetic biology strategies.

Multi-omics Study of Planobispora rosea, Producer of the Thiopeptide Antibiotic GE2270A.

Planobispora rosea is the natural producer of the potent thiopeptide antibiotic GE2270A. Here, we present the results of a metabolomics and transcriptomics analysis of P. rosea during production of GE2270A. The data generated provides useful insights into the biology of this genetically intractable bacterium. We characterize the details of the shutdown of protein biosynthesis and the respiratory chain associated with the end of the exponential growth phase. We also provide the first description of the phosphate regulon in P. rosea. Based on the transcriptomics data, we show that both phosphate and iron are limiting P. rosea growth in our experimental conditions. Additionally, we identified and validated a new biosynthetic gene cluster associated with the production of the siderophores benarthin and dibenarthin in P. rosea. Together, the metabolomics and transcriptomics data are used to inform and refine the very first genome-scale metabolic model for P. rosea, which will be a valuable framework for the interpretation of future studies of the biology of this interesting but poorly characterized species. IMPORTANCE Planobispora rosea is a genetically intractable bacterium used for the production of GE2270A on an industrial scale. GE2270A is a potent thiopeptide antibiotic currently used as a precursor for the synthesis of two compounds under clinical studies for the treatment of Clostridium difficile infection and acne. Here, we present the very first systematic multi-omics investigation of this important bacterium, which provides a much-needed detailed picture of the dynamics of metabolism of P. rosea while producing GE2270A.

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.

A Two-Component regulatory system with opposite effects on glycopeptide antibiotic biosynthesis and resistance.

The glycopeptide A40926, produced by the actinomycete Nonomuraea gerenzanensis, is the precursor of dalbavancin, a second-generation glycopeptide antibiotic approved for clinical use in the USA and Europe in 2014 and 2015, respectively. The final product of the biosynthetic pathway is an O-acetylated form of A40926 (acA40926). Glycopeptide biosynthesis in N. gerenzanensis is dependent upon the dbv gene cluster that encodes, in addition to the two essential positive regulators Dbv3 and Dbv4, the putative members of a two-component signal transduction system, specifically the response regulator Dbv6 and the sensor kinase Dbv22. The aim of this work was to assign a role to these two genes. Our results demonstrate that deletion of dbv22 leads to an increased antibiotic production with a concomitant reduction in glycopeptide resistance. Deletion of dbv6 results in a similar phenotype, although the effects are not as strong as in the Δdbv22 mutant. Consistently, quantitative RT-PCR analysis showed that Dbv6 and Dbv22 negatively regulate the regulatory genes (dbv3 and dbv4), as well as some dbv biosynthetic genes (dbv23 and dbv24), whereas Dbv6 and Dbv22 positively regulate transcription of the single, cluster-associated resistance gene. Finally, we demonstrate that exogenously added acA40926 and its precursor A40926 can modulate transcription of dbv genes but with an opposite extent: A40926 strongly stimulates transcription of the Dbv6/Dbv22 target genes while acA40926 has a neutral or negative effect on transcription of those genes. We propose a model in which glycopeptide biosynthesis in N. gerenzanensis is modulated through a positive feedback by the biosynthetic precursor A40926 and a negative feedback by the final product acA40926. In addition to previously reported control systems, this sophisticated control loop might help the producing strain cope with the toxicity of its own product. This work, besides leading to improved glycopeptide producing strains, enlarges our knowledge on the regulation of glycopeptide biosynthesis in actinomycetes, setting N. gerenzanensis and its two-component system Dbv6-Dbv22 apart from other glycopeptide producers.

Challenges and advances in genetic manipulation of filamentous actinomycetes - the remarkable producers of specialized metabolites.

Covering: up to February 2019Actinomycetes are Gram positive bacteria of the phylum Actinobacteria. These organisms are one of the most important sources of structurally diverse, clinically used antibiotics and other valuable bioactive products, as well as biotechnologically relevant enzymes. Most strains were discovered by their ability to produce a given molecule and were often poorly characterized, physiologically and genetically. The development of genetic methods for Streptomyces and related filamentous actinomycetes has led to the successful manipulation of antibiotic biosynthesis to attain structural modification of microbial metabolites that would have been inaccessible by chemical means and improved production yields. Moreover, genome mining reveals that actinomycete genomes contain multiple biosynthetic gene clusters (BGCs), however only a few of them are expressed under standard laboratory conditions, leading to the production of the respective compound(s). Thus, to access and activate the so-called "silent" BGCs, to improve their biosynthetic potential and to discover novel natural products methodologies for genetic manipulation are required. Although different methods have been applied for many actinomycete strains, genetic engineering is still remaining very challenging for some "underexplored" and poorly characterized actinomycetes. This review summarizes the strategies developed to overcome the obstacles to genetic manipulation of actinomycetes and allowing thereby rational genetic engineering of this industrially relevant group of microorganisms. At the end of this review we give some tips to researchers with limited or no previous experience in genetic manipulation of actinomycetes. The article covers the most relevant literature published until February 2019.

Toward Single-Peak Dalbavancin Analogs through Biology and Chemistry.

Glycopeptide antibiotics are used to treat severe multidrug resistant infections caused by Gram-positive bacteria. Dalbavancin is a second generation glycopeptide approved for human use, which is obtained from A40926, a lipoglycopeptide produced by Nonomuraea sp. ATCC39727 as a mixture of biologically active congeners mainly differing in the fatty acid chains present on the glucuronic moiety. In this study, we constructed a double mutant of the A40926 producer strain lacking dbv23, and thus defective in mannose acetylation, a feature that increases A40926 production, and lacking the acyltransferases Dbv8, and thus incapable of installing the fatty acid chains. The double mutant afforded the desired deacyl, deacetyl A40926 intermediates, which could be converted by chemical reacylation yielding A40926 analogs with a greatly reduced number of congeners. The newly acylated analogs could then be transformed into dalbavancin analogs possessing the same in vitro properties as the approved drug.

Antibacterial Aromatic Polyketides Incorporating the Unusual Amino Acid Enduracididine.

The increasing incidence of infections caused by drug-resistant pathogens requires new efforts for the discovery of novel antibiotics. By screening microbial extracts in an assay aimed at identifying compounds interfering with cell wall biosynthesis, based on differential activity against a Staphylococcus aureus strain and its isogenic l-form, the potent enduracyclinones (1, 2), containing the uncommon amino acid enduracididine linked to a six-ring aromatic skeleton, were discovered from different Nonomuraea strains. The structures of 1 and 2 were established through a combination of derivatizations, oxidative cleavages, and NMR analyses of natural and 13C-15N-labeled compounds. Analysis of the biosynthetic cluster provides the combination of genes for the synthesis of enduracididine and type II polyketide synthases. Enduracyclinones are active against Gram-positive pathogens (especially Staphylococcus spp.), including multi-drug-resistant strains, with minimal inhibitory concentrations in the range of 0.0005 to 4 µg mL-1 and with limited toxicity toward eukaryotic cells. The combined results from assays and macromolecular syntheses suggest a possible dual mechanism of action in which both peptidoglycan and DNA syntheses are inhibited by these molecules.

Discovery, properties, and biosynthesis of pseudouridimycin, an antibacterial nucleoside-analog inhibitor of bacterial RNA polymerase.

Pseudouridimycin (PUM) is a novel pseudouridine-containing peptidyl-nucleoside antibiotic that inhibits bacterial RNA polymerase (RNAP) through a binding site and mechanism different from those of clinically approved RNAP inhibitors of the rifamycin and lipiarmycin (fidaxomicin) classes. PUM was discovered by screening microbial fermentation extracts for RNAP inhibitors. In this review, we describe the discovery and characterization of PUM. We also describe the RNAP-inhibitory and antibacterial properties of PUM. Finally, we review available information on the gene cluster and pathway for PUM biosynthesis and on the potential for discovering additional novel pseudouridine-containing nucleoside antibiotics by searching bacterial genome and metagenome sequences for sequences similar to pumJ, the pseudouridine-synthase gene of the PUM biosynthesis gene cluster.

Novel Polyethers from Screening Actinoallomurus spp.

In screening for novel antibiotics, an attractive element of novelty can be represented by screening previously underexplored groups of microorganisms. We report the results of screening 200 strains belonging to the actinobacterial genus Actinoallomurus for their production of antibacterial compounds. When grown under just one condition, about half of the strains produced an extract that was able to inhibit growth of Staphylococcus aureus. We report here on the metabolites produced by 37 strains. In addition to previously reported aminocoumarins, lantibiotics and aromatic polyketides, we described two novel and structurally unrelated polyethers, designated α-770 and α-823. While we identified only one producer strain of the former polyether, 10 independent Actinoallomurus isolates were found to produce α-823, with the same molecule as main congener. Remarkably, production of α-823 was associated with a common lineage within Actinoallomurus, which includes A.fulvus and A.amamiensis. All polyether producers were isolated from soil samples collected in tropical parts of the world.

Large inserts for big data: artificial chromosomes in the genomic era.

The exponential increase in available microbial genome sequences coupled with predictive bioinformatic tools is underscoring the genetic capacity of bacteria to produce an unexpected large number of specialized bioactive compounds. Since most of the biosynthetic gene clusters (BGCs) present in microbial genomes are cryptic, i.e. not expressed under laboratory conditions, a variety of cloning systems and vectors have been devised to harbor DNA fragments large enough to carry entire BGCs and to allow their transfer in suitable heterologous hosts. This minireview provides an overview of the vectors and approaches that have been developed for cloning large BGCs, and successful examples of heterologous expression.

Complex Regulatory Networks Governing Production of the Glycopeptide A40926.

Glycopeptides (GPAs) are an important class of antibiotics, with vancomycin and teicoplanin being used in the last 40 years as drugs of last resort to treat infections caused by Gram-positive pathogens, including methicillin-resistant Staphylococcus aureus. A few new GPAs have since reached the market. One of them is dalbavancin, a derivative of A40926 produced by the actinomycete Nonomuraea sp. ATCC 39727, recently classified as N. gerenzanensis. This review summarizes what we currently know on the multilevel regulatory processes governing production of the glycopeptide A40926 and the different approaches used to increase antibiotic yields. Some nutrients, e.g., valine, l-glutamine and maltodextrin, and some endogenous proteins, e.g., Dbv3, Dbv4 and RpoBR, have a positive role on A40926 biosynthesis, while other factors, e.g., phosphate, ammonium and Dbv23, have a negative effect. Overall, the results available so far point to a complex regulatory network controlling A40926 in the native producing strain.

Analysis of the Pseudouridimycin Biosynthetic Pathway Provides Insights into the Formation of C-nucleoside Antibiotics.

Pseudouridimycin (PUM) is a selective nucleoside-analog inhibitor of bacterial RNA polymerase with activity against Gram-positive and Gram-negative bacteria. PUM, produced by Streptomyces sp. ID38640, consists of a formamidinylated, N-hydroxylated Gly-Gln dipeptide conjugated to 5'-aminopseudouridine. We report the characterization of the PUM gene cluster. Bioinformatic analysis and mutational knockouts of pum genes with analysis of accumulated intermediates, define the PUM biosynthetic pathway. The work provides the first biosynthetic pathway of a C-nucleoside antibiotic and reveals three unexpected features: production of free pseudouridine by the dedicated pseudouridine synthase, PumJ; nucleoside activation by specialized oxidoreductases and aminotransferases; and peptide-bond formation by amide ligases. A central role in the PUM biosynthetic pathway is played by the PumJ, which represents a divergent branch within the TruD family of pseudouridine synthases. PumJ-like sequences are associated with diverse gene clusters likely to govern the biosynthesis of different classes of C-nucleoside antibiotics.

Expanding the potential of NAI-107 for treating serious ESKAPE pathogens: synergistic combinations against Gram-negatives and bactericidal activity against non-dividing cells.

Objectives: To characterize NAI-107 and related lantibiotics for their in vitro activity against Gram-negative pathogens, alone or in combination with polymyxin, and against non-dividing cells or biofilms of Staphylococcus aureus. NAI-107 was also evaluated for its propensity to select or induce self-resistance in Gram-positive bacteria. Methods: We used MIC determinations and chequerboard experiments to establish the antibacterial activity of the examined compounds against target microorganisms. Time-kill assays were used to evaluate killing of exponential and stationary-phase cells. The effects on biofilms (growth inhibition and biofilm eradication) were evaluated using biofilm-coated pegs. The frequency of spontaneous resistant mutants was evaluated by either direct plating or by continuous sub-culturing at 0.5 × MIC levels, followed by population analysis profiles. Results: The results showed that NAI-107 and its brominated variant are highly active against Neisseria gonorrhoeae and some other fastidious Gram-negative pathogens. Furthermore, all compounds strongly synergized with polymyxin against Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae and Pseudomonas aeruginosa, and showed bactericidal activity. Surprisingly, NAI-107 alone was bactericidal against non-dividing A. baumannii cells. Against S. aureus, NAI-107 and related lantibiotics showed strong bactericidal activity against dividing and non-dividing cells. Activity was also observed against S. aureus biofilms. As expected for a lipid II binder, no significant resistance to NAI-107 was observed by direct plating or serial passages. Conclusions: Overall, the results of the current work, along with previously published results on the efficacy of NAI-107 in experimental models of infection, indicate that this lantibiotic represents a promising option in addressing the serious threat of antibiotic resistance.