RESUMEN
Most biosynthetic gene clusters (BGCs) are functionally inaccessible by using fermentation methods. Bioinformatic-coupled total synthesis provides an alternative approach for accessing BGC-encoded bioactivities. To date, synthetic bioinformatic natural product (synBNP) methods have focused on lipopeptides containing simple lipids. Here we increase the bioinformatic and synthetic complexity of the synBNP approach by targeting BGCs that encode N-cinnamoyl lipids. This led to our synthesis of cinnamosyn, a 10-mer N-cinnamoyl-containing peptide that is cytotoxic to human cells.
Asunto(s)
Productos Biológicos , Productos Biológicos/química , Productos Biológicos/farmacología , Productos Biológicos/síntesis química , Humanos , Estructura Molecular , Biología Computacional , Familia de Multigenes , Lipopéptidos/química , Lipopéptidos/farmacología , Lipopéptidos/síntesis química , Cinamatos/química , Cinamatos/farmacología , Cinamatos/síntesis química , Antineoplásicos/farmacología , Antineoplásicos/química , Antineoplásicos/síntesis químicaRESUMEN
Bacterial genomes contain large reservoirs of biosynthetic gene clusters (BGCs) that are predicted to encode unexplored natural products. Heterologous expression of previously unstudied BGCs should facilitate the discovery of additional therapeutically relevant bioactive molecules from bacterial culture collections, but the large-scale manipulation of BGCs remains cumbersome. Here, we describe a method to parallelize the identification, mobilization and heterologous expression of BGCs. Our solution simultaneously captures large numbers of BGCs by cloning the genomes of a strain collection in a large-insert library and uses the CONKAT-seq (co-occurrence network analysis of targeted sequences) sequencing pipeline to efficiently localize clones carrying intact BGCs which represent candidates for heterologous expression. Our discovery of several natural products, including an antibiotic that is active against multi-drug resistant Staphylococcus aureus, demonstrates the potential of leveraging economies of scale with this approach to systematically interrogate cryptic BGCs contained in strain collections.
Asunto(s)
Productos Biológicos , Staphylococcus aureus Resistente a Meticilina , Antibacterianos , Productos Biológicos/metabolismo , Vías Biosintéticas/genética , Genoma Bacteriano/genética , Staphylococcus aureus Resistente a Meticilina/genética , Familia de MultigenesRESUMEN
The emergence of multidrug-resistant bacteria poses a threat to global health and necessitates the development of additional in vivo active antibiotics with diverse modes of action. Directly targeting menaquinone (MK), which plays an important role in bacterial electron transport, is an appealing, yet underexplored, mode of action due to a dearth of MK-binding molecules. Here we combine sequence-based metagenomic mining with a motif search of bioinformatically predicted natural product structures to identify six biosynthetic gene clusters that we predicted encode MK-binding antibiotics (MBAs). Their predicted products (MBA1-6) were rapidly accessed using a synthetic bioinformatic natural product approach, which relies on bioinformatic structure prediction followed by chemical synthesis. Among these six structurally diverse MBAs, four make up two new MBA structural families. The most potent member of each new family (MBA3, MBA6) proved effective at treating methicillin-resistant Staphylococcus aureus infection in a murine peritonitis-sepsis model. The only conserved feature present in all MBAs is the sequence 'GXLXXXW', which we propose represents a minimum MK-binding motif. Notably, we found that a subset of MBAs were active against Mycobacterium tuberculosis both in vitro and in macrophages. Our findings suggest that naturally occurring MBAs are a structurally diverse and untapped class of mechanistically interesting, in vivo active antibiotics.
Asunto(s)
Antibacterianos/metabolismo , Antibacterianos/uso terapéutico , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Vitamina K 2/metabolismo , Animales , Farmacorresistencia Bacteriana Múltiple , Femenino , Humanos , Macrófagos/efectos de los fármacos , Macrófagos/microbiología , Metagenómica/métodos , Staphylococcus aureus Resistente a Meticilina/patogenicidad , Ratones , Pruebas de Sensibilidad Microbiana , Mycobacterium tuberculosis/efectos de los fármacos , Peritonitis/tratamiento farmacológico , Peritonitis/microbiología , Infecciones Estafilocócicas/tratamiento farmacológico , Vitamina K 2/aislamiento & purificaciónRESUMEN
Tuberculosis (TB) remains one of the deadliest infectious diseases. Unfortunately, the development of antibiotic resistance threatens our current therapeutic arsenal, which has necessitated the discovery and development of novel antibiotics against drug-resistant Mycobacterium tuberculosis (Mtb). Cyclomarin A and rufomycin I are structurally related cyclic heptapeptides assembled by nonribosomal peptide synthetases (NRPSs), which show potent anti-Mtb activity with a new cellular target, the caseinolytic protein ClpC1. An NRPS adenylation domain survey using DNA extracted from â¼2000 ecologically diverse soils found low cyclomarin/rufomycin biosynthetic diversity. In this survey, a family of cyclomarin/rufomycin-like biosynthetic gene clusters (BGC) that encode metamarin, an uncommon cyclomarin congener with potent activity against both Mtb H37Rv and multidrug-resistant Mtb clinical isolates was identified. Metamarin effectively inhibits Mtb growth in murine macrophages and increases the activities of ClpC1 ATPase and the associated ClpC1/P1/P2 protease complex, thus causing cell death by uncontrolled protein degradation.
Asunto(s)
Metagenoma , Mycobacterium tuberculosis/efectos de los fármacos , Oligopéptidos/farmacología , Microbiología del Suelo , Animales , Antituberculosos , Proteínas Bacterianas , Línea Celular , Proteínas de Choque Térmico , Macrófagos , Ratones , Pruebas de Sensibilidad Microbiana , Estructura MolecularRESUMEN
The ichip (isolation chip) was employed for the first time in a marine sponge (Xestospongia muta), and a putatively new bacterial species, Alteromonas sp. RKMC-009, was isolated. Strain RKMC-009 produces a novel N-acyltyrosine (1) that is appended with a rare α-methyl substituent within the aminoacyl moiety and also exhibits Gram-positive antibacterial activity. We determined through an SAR experiment that the α-methyl is necessary for Staphylococcus activity of 1 and that it enhances Enterococcus activity.
Asunto(s)
Alteromonas/química , Antibacterianos/farmacología , Proteínas Bacterianas/farmacología , Enterococcus/efectos de los fármacos , Staphylococcus/efectos de los fármacos , Animales , Antibacterianos/química , Antibacterianos/aislamiento & purificación , Proteínas Bacterianas/química , Proteínas Bacterianas/aislamiento & purificación , Pruebas de Sensibilidad Microbiana , Conformación Molecular , PoríferosRESUMEN
We report the draft whole-genome sequence of Alteromonas sp. strain RKMC-009, which was isolated in situ from the sponge Xestospongia muta in San Salvador, The Bahamas, using an isolation chip (ichip). Automated biosynthetic gene cluster analysis using antiSMASH 4.0 predicted the presence of 22 biosynthetic gene clusters.
RESUMEN
A new cyclic lipodepsipeptide, fusaristatin C (1), was obtained from the fungus Pithomyces sp. RKDO 1698, which was isolated from the Caribbean octocoral Eunicea fusca. The 2D structure of fusaristatin C was elucidated using NMR spectroscopy and mass spectrometry, while the absolute configuration of the sole chiral amino acid residue (l-serine) was determined using Marfey's method. 3-Hydroxy-2,11-dimethyltetradecanoic acid (HDMT) was cleaved from 1, and the absolute configuration at the C-3 position was determined using Mosher's ester analysis. Subsequent J-based configuration analysis of 1 allowed for assignment of the C-2 configuration. Fusaristatin C exhibited no antimicrobial activity or cytotoxicity.
Asunto(s)
Ascomicetos/química , Animales , Línea Celular Tumoral , Chlorocebus aethiops , Ensayos de Selección de Medicamentos Antitumorales , Células HeLa , Humanos , Espectroscopía de Resonancia Magnética , Espectrometría de Masas , Pruebas de Sensibilidad Microbiana , Estructura Molecular , Células VeroRESUMEN
The amphiphilic siderophore imaqobactin was isolated from the Arctic bacterium Variovorax sp. RKJM285, a strain isolated from marine sediment collected from an inlet near Clyde River, Nunavut, Canada. The 2D structure of imaqobactin was determined by a combination of LC-HRMS, MS/MS, and NMR spectroscopic methods. The absolute configuration of the depsipeptide core was determined by Marfey's analysis, and the relative configuration of the 4,7-diamino-3-hydroxy-2-methylheptanoic acid moiety was determined by NOESY and selective NOE experiments. The photoreductive properties of imaqobactin were tested and are discussed. Initial tests for antimicrobial and cytotoxic activity of imaqobactin were also performed, identifying moderate antimicrobial activity.
Asunto(s)
Organismos Acuáticos/química , Bacterias/química , Sideróforos/química , Sideróforos/farmacología , Antiinfecciosos/química , Antiinfecciosos/farmacología , Canadá , Línea Celular Tumoral , Citotoxinas/química , Citotoxinas/farmacología , Humanos , Células MCF-7 , Biología Marina/métodos , Resonancia Magnética Nuclear Biomolecular/métodos , Péptidos Cíclicos/química , Péptidos Cíclicos/farmacología , Espectrometría de Masas en Tándem/métodosRESUMEN
Polyketide synthase (PKS) derived natural products are biosynthesized by head-to-tail addition of acetate and malonate extender units resulting in linear extended-polyketide chains. Despite the well-documented structural diversity associated with PKS-derived natural products, C-C chain branching deviating from the usual linear pattern is relatively rare. Herein, type-II PKS angucyclic natural products containing a hemiaminal functionality were identified and proposed as the parent of a series of C-C-branched analogues. These C-C linked acetate or pyruvate branching units were located at the α-positions on the extended polyketide chains of jadomycins incorporating 3- and 4-aminomethylbenzoic acids. Labeling studies utilizing [1-13C]-d-glucose provided mechanistic evidence that the C-C bond formation occurred as a result of a previously unidentified post-PKS processing, additional to the enzymes encoded within the biosynthetic gene cluster. Selected compounds were evaluated in cytotoxic or antimicrobial assays.