TRNA-Dependent Chemoenzymatic Transformation of Aminoacyl Pendant Moieties of Streptothricin Antibiotics.

Macrolactamization of Glycosylated Peptide Thioesters by the Thioesterase Domain of Tyrocidine Synthetase

Cerium oxide nanoparticles as potential antibiotic adjuvant. Effects of CeO2 nanoparticles on bacterial outer membrane permeability

Nonribosomal peptide synthetases (NRPS) are essential for the biosynthesis of therapeutically valuable molecules, including antibiotics, immunosuppressants, and anticancer agents. The assembly-line mechanism of NRPS offers significant potential for engineering novel natural products through reprogramming. However, the challenging purification of NRPS proteins has impeded the investigation of their assembly and catalytic mechanisms. In this study, we employed homologous recombination to insert a purification tag at the C-terminus of the NRPS gene within the chromosome. This genetic modification enabled efficient purification of NRPS proteins from the tagged mutant strain using a one-step affinity chromatography approach. Additionally, we discovered that MbtH-like proteins (MLPs) form stable complexes with all pyoverdine (PVD) NRPS subunits, allowing for the purification of the entire NRPS assembly line via tagged MLP. Negative stain electron microscopy analysis revealed that the purified PVD NRPS proteins exist as dynamically linear monomers. Our in-situ tag-based purification method enhances NRPS research in both biochemical and structural biology, providing a robust platform for further investigations into NRPS mechanisms and applications.

Pathogens resistant to antimicrobials form a significant threat to public health worldwide. Tackling multidrug-resistant pathogens via screening metagenomic libraries has become a common approach for the discovery of new antibiotics from uncultured microorganisms. This study focuses on capturing nonribosomal peptide synthase (NRPS) gene clusters implicated in the synthesis of many natural compounds of industrial relevance. A NRPS PCR assay was used to screen 2976 Escherichia coli clones in a soil metagenomic library to target NRPS genes. DNA extracts from 4 clones were sequenced and subjected to bioinformatic analysis to identify NRPS domains, their phylogeny, and substrate specificity.Successfully, 17 NRPS-positive hits with a biosynthetic potential were identified. DNA sequencing and BLAST analysis confirmed that NRPS protein sequences shared similarities with members of the genus Delftia in the Proteobacteria taxonomic position. Multiple alignment and phylogenetic analysis demonstrated that clones no. 15cd35 and 15cd37 shared low bootstrap values (54%) and were distantly far from close phylogenetic neighbors. Additionally, NRPS domain substrate specificity has no hits with the known ones; hence, they are more likely to use different substrates to produce new diverse antimicrobials. Further analysis confirmed that the NRPS hits resemble several transposon elements from other bacterial taxa, confirming its diversity. We confirmed that the analyses of the soil metagenomic library revealed a diverse set of NRPS related to the genus Delftia. An in-depth understanding of those positive NRPS hits is a crucial step for genetic manipulation of NRPS, shedding light on alternative novel antimicrobial compounds that can be used in drug discovery and hence supports the pharmaceutical sector.

The growing threat of antimicrobial resistance necessitates the discovery of novel antibiotics with activity against drug-resistant pathogens. Members of the genus Paenibacillus are a rich source of nonribosomal peptides (NRPs), including well-known antibiotics such as polymyxins, paenibacterin, and tridecaptins. Here, we use a targeted mass spectrometry query language (MassQL)-based approach to identify the NRPs produced by a collection of 227 taxonomically diverse plant-associated Paenibacillus strains, providing detailed insights into their NRP-producing potential. Using MassQL to zoom in specifically on NRPs containing basic amino acids, we discovered a novel family of bacitracins, which we designated paenitracins. The paenitracins are the first bacitracin-type peptides reported in Paenibacillus and are distinguished from canonical bacitracins by three previously unseen amino acid substitutions. The paenitracins exhibit potent activity against gram-positive pathogens, including vancomycin-resistant Enterococcus faecium E155. Our work provides a novel metabolomics-guided and genomics-guided workflow for the discovery of bioactive NRPs as a strategy to prioritize natural product chemical space and accelerate antibiotic discovery.IMPORTANCEMembers of the genus Paenibacillus play an important role in soil ecology, producing a range of important nonribosomal peptides (NRPs). A collection of plant-associated Paenibacillus spp. were analyzed for their phylogenetic and metabolic diversity. We developed a novel discovery pipeline that combines feature-based molecular networking with mass spectrometry query language queries to systematically prioritize bioactive NRPs containing basic amino acids. Thus, we provide a comprehensive genus-wide inventory of NRPs produced by Paenibacillus spp. We thereby identified the paenitracins, a new sub-family of bacitracins active against multidrug-resistant gram-positive pathogens. Our pipeline enables the discovery of novel peptidic natural products to accelerate the prioritization of chemical space for antibiotics.

L-2-amino-4-methoxy-trans-3-butenoic acid (methoxyvinylglycine, or short as AMB) is a γ-substituted vinylglycine that was identified in 1972 as an inhibitor of pyridoxal 5’-phosphate-dependent enzymes, resulting in antibiotic activity. Recent studies indicate that the gene cluster ambABCDE is responsible for AMB biosynthesis in Pseudomonas aeruginosa. Among the products of this gene cluster, AmbC and AmbD, two α-ketoglutarate-dependent oxygenases are involved in the modification of L-glutamate as substrate for AMB synthesis. AMB is then synthetized from one modified glutamate and two alanine moieties condensed to a tripeptide by the non-ribosomal peptide synthetases (NRPS) AmbB and AmbE. However, exact timing of the tailoring reactions remains unclear so far. The structural research was primarily focused on AmbC and AmbD. Additionally, the structure of unknown domain in NRPS AmbE needs to be determined to gain information on its function in AMB synthesis. X-ray crystallography was mainly applied to obtain structures of targeted proteins, combining with preliminary mass spectrometry (MS) experiments to provide a model for biosynthesis of AMB in Pseudomonas aeruginosa. The structures of AmbD and AmbC in the native state as well as in the ligand-bound state complex with the co-factor α-ketoglutarate and analog N-oxalylglycine (NOG) were determined. Additionally, the structure of an uncharacterized domain of AmbE was obtained and submitted to PDBeFold and Dali server, suggesting it to be related to NRPS condensation domains. However, there is no conserved motif HHxxxDG that would be expected for condensation domains. Further, the two condensation domains within AmbB and AmbE are sufficient to synthesize a tripeptide product, such that a third condensation domain seems unnecessary.

Harnessing the Natural Pool of Polyketide and Non-ribosomal Peptide Family: A Route Map towards Novel Drug Development.

Non-ribosomal peptide (NRP) natural products are one of the most promising resources for drug discovery and development because of their wide-ranging of therapeutic potential, and their behavior as virulence factors and signaling molecules. The NRPs are biosynthesized independently of the ribosome by enzyme assembly lines known as the non-ribosomal peptide synthetase (NRPS) machinery. Genetic, biochemical, and bioinformatics analyses have provided a detailed understanding of the mechanism of NRPS catalysis. However, proteomic techniques for natural product biosynthesis remain a developing field. New strategies are needed to investigate the proteomes of diverse producer organisms and directly analyze the endogenous NRPS machinery. Advanced platforms should verify protein expression, protein folding, and activities and also enable the profiling of the NRPS machinery in biological samples from wild-type, heterologous, and engineered bacterial systems. Here, we focus on activity-based protein profiling strategies that have been recently developed for studies aimed at visualizing and monitoring the NRPS machinery and also for rapid labeling, identification, and biochemical analysis of NRPS enzyme family members as required for proteomic chemistry in natural product sciences.

Research into new antibiotics is becoming increasingly important as antibiotic resistance increases worldwide. The genus Streptomyces in particular is able to produce a wide range of antimicrobial products due to the large number of biosynthetic gene clusters (BGCs) in its genome. However, not all BGCs are expressed under laboratory conditions. In this work, deletion of the gene wblA, encoding a global regulator of natural product biosynthesis and morphogenesis in Streptomyces, led to the production of a novel natural product, olikomycin A, in Streptomyces ghanaensis ATCC 14672. Complete structure elucidation revealed that olikomycin A belongs to a class of calcium-dependent antibiotics known as non-ribosomal peptide synthetase (NRPS)-encoded acidic lipopeptides. These compounds exhibit remarkable antimicrobial activity in the presence of calcium. Insights into olikomycin A biosynthesis were provided by whole genome sequencing and gene inactivation studies, while bioactivity assays showed strong inhibition of the growth of multidrug-resistant Gram-positive pathogens via disrupting cell membrane integrity. Olikomycin A shows an antibiotic profile similar to that of daptomycin, which is already in clinical use.

Natural macrocyclic peptides produced by microorganisms serve as valuable resources for therapeutic compounds, including antibiotics, anticancer agents, and immune suppressive agents. Nonribosomal peptide synthetases (NRPSs) are responsible for the biosynthesis of macrocyclic peptides. NRPSs are large multimodular enzymes, and each module recognizes and incorporates one specific amino acid into the polypeptide product. In the final biosynthetic step, the mature linear peptide precursor is subject to head-to-tail cyclization by the thioesterase (TE) domain in the C-terminal module. Since the TE domains can autonomously catalyze the cyclization of diverse linear peptide substrates, isolated TE domains can be used to produce natural product derivatives. To understand the mechanism of TE domains in NRPSs as a base for therapeutic applications, we investigated the TE domain (residues 6236-6486) of tyrocidine synthetase TycC by NMR. Tyrocidine is a cyclic decapeptide with antibiotic activity, and TycC-TE catalyzes the cyclization of the linear decapeptide precursor. Here, we report the backbone resonance assignments of TycC-TE. The assignments of TycC-TE provide the basis for NMR investigations of the structure and substrate-recognition mode of the TE domain in NRPS.

Covering: up to 2017.Natural products are important secondary metabolites produced by bacterial and fungal species that play important roles in cellular growth and signaling, nutrient acquisition, intra- and interspecies communication, and virulence. A subset of natural products is produced by nonribosomal peptide synthetases (NRPSs), a family of large, modular enzymes that function in an assembly line fashion. Because of the pharmaceutical activity of many NRPS products, much effort has gone into the exploration of their biosynthetic pathways and the diverse products they make. Many interesting NRPS pathways have been identified and characterized from both terrestrial and marine bacterial sources. Recently, several NRPS pathways in human commensal bacterial species have been identified that produce molecules with antibiotic activity, suggesting another source of interesting NRPS pathways may be the commensal and pathogenic bacteria that live on the human body. The ESKAPE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) have been identified as a significant cause of human bacterial infections that are frequently multidrug resistant. The emerging resistance profile of these organisms has prompted calls from multiple international agencies to identify novel antibacterial targets and develop new approaches to treat infections from ESKAPE pathogens. Each of these species contains several NRPS biosynthetic gene clusters. While some have been well characterized and produce known natural products with important biological roles in microbial physiology, others have yet to be investigated. This review catalogs the NRPS pathways of ESKAPE pathogens. The exploration of novel NRPS products may lead to a better understanding of the chemical communication used by human pathogens and potentially to the discovery of novel therapeutic approaches.

Gene sequence diversity of the nonribosomal peptide and polyketide natural products in Changbaishan soil correlates with changes in landscape belts

Peptidic natural products (PNPs) and non ribosomal peptides (NRPs) are chemical compounds produced by living organisms. These peptides often exhibit interesting bioactivities. In this thesis I present a collection of innovative bioinformatic platforms dedicated to PNPs with the aim of facilitating the structural analysis of these compounds, especially for MS experiments. First, I introduce rBAN, a retro-biosynthesis tool mainly dedicated to the monomeric annotation of NRP chemical structures. Secondly, I present the KFP application, a straightforward program for NRP chemical formula detection and MS peak annotation, which implements a prediction method based on the Kendrick mass defect. Lastly, the thesis culminates with NRPro, a new platform for the MS/MS analysis of PNPs through automatic annotation and dereplication. These three tools were integrated in the Norine database, the only resource that is exclusively dedicated to NRPs.

To delineate the time of onset and the mechanism of delayed toxic effects produced by streptothricin antibiotics in rats, the tissue antibiotic distribution, histopathologic features of the kidney, serum biochemical changes and antibiotic metabolites recovered in the urine after administration of a radiolabeled compound were investigated. Rats showed a pattern of tissue antibiotic distribution similar to that observed in the mouse. Microscopic examination of the kidney revealed lesions which became evident at 48 hr after the injection. Serum biochemical tests showed a rapid elevation of blood urea nitrogen and creatinine nitrogen levels from 48 hr post injection, with abnormalities in the cellulose acetate membrane electrophoresis pattern of serum proteins. Thus, nephrotoxic effects evoked by the antibiotic became evident about 48 hr after the injection, indicating that the time of onset of delayed toxicity of the compound is approximately 48 hr after dosing. As for the mechanism of the delayed toxic effects, evidence has been obtained for the formation of a toxic metabolite with an opened lactam ring, or an"acid compound"excreted in the rat urine. These findings support our previous inference, based on studies in mice.

In order to investigate the mode of the delayed toxicity of streptothricin antibiotics, 14C-glycyl-racemomycin-A and racemomycin-D were each administered intravenously to mice and rats in a seuviving dose. As to the distribution of 14C-glycyl-racemomycin-A in the organs of mice, studies confirmed us that it was distributed in kidney in a high concentration in comparison with the distribution in the other organs. Therefore, pathohistological reviews were carried out on the kidney which was whitened macroscopically. As a result, severe toxicity wide-spread in the cortex renis was observed in the kidney. From these results, it was recogenized that streptothricin antibiotics produce nephrotoxicity. As to the developmental mechanism of the delayed toxicity, the authors presented the assumption that after the administered antibiotic us, in vivo, transformed into acid, lactam ring of which is broken from examination of radiochromatogram of metabolite in urine, and that the acute toxicity of that acid is responsible for the delayed toxicity.