RESUMEN
Monoterpene indole alkaloids (MIAs) are a diverse family of complex plant secondary metabolites with many medicinal properties, including the essential anti-cancer therapeutics vinblastine and vincristine1. As MIAs are difficult to chemically synthesize, the world's supply chain for vinblastine relies on low-yielding extraction and purification of the precursors vindoline and catharanthine from the plant Catharanthus roseus, which is then followed by simple in vitro chemical coupling and reduction to form vinblastine at an industrial scale2,3. Here, we demonstrate the de novo microbial biosynthesis of vindoline and catharanthine using a highly engineered yeast, and in vitro chemical coupling to vinblastine. The study showcases a very long biosynthetic pathway refactored into a microbial cell factory, including 30 enzymatic steps beyond the yeast native metabolites geranyl pyrophosphate and tryptophan to catharanthine and vindoline. In total, 56 genetic edits were performed, including expression of 34 heterologous genes from plants, as well as deletions, knock-downs and overexpression of ten yeast genes to improve precursor supplies towards de novo production of catharanthine and vindoline, from which semisynthesis to vinblastine occurs. As the vinblastine pathway is one of the longest MIA biosynthetic pathways, this study positions yeast as a scalable platform to produce more than 3,000 natural MIAs and a virtually infinite number of new-to-nature analogues.
Asunto(s)
Antineoplásicos , Reactores Biológicos , Vías Biosintéticas , Ingeniería Metabólica , Saccharomyces cerevisiae , Vinblastina , Alcaloides de la Vinca , Antineoplásicos/química , Antineoplásicos/metabolismo , Antineoplásicos/provisión & distribución , Catharanthus/química , Genes Fúngicos , Genes de Plantas , Ingeniería Metabólica/métodos , Fosfatos de Poliisoprenilo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Triptófano , Vinblastina/biosíntesis , Vinblastina/química , Vinblastina/provisión & distribución , Alcaloides de la Vinca/biosíntesis , Alcaloides de la Vinca/química , Alcaloides de la Vinca/provisión & distribuciónRESUMEN
Secondary metabolome mining efforts in the myxobacterial multiproducer of natural products, Chondromyces crocatus Cm c5, resulted in the isolation and structure elucidation of crocagins, which are novel polycyclic peptides containing a tetrahydropyrrolo[2,3-b]indole core. The gene cluster was identified through an approach combining genome analysis, targeted gene inactivation in the producer, and inâ vitro experiments. Based on our findings, we developed a biosynthetic scheme for crocagin biosynthesis. These natural products are formed from the three C-terminal amino acids of a precursor peptide and thus belong to a novel class of ribosomally synthesized and post-translationally modified peptides (RiPPs). We demonstrate that crocaginâ A binds to the carbon storage regulator protein CsrA, thereby inhibiting the ability of CsrA to bind to its cognate RNA target.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Myxococcales/metabolismo , Péptidos Cíclicos/biosíntesis , Péptidos Cíclicos/química , Procesamiento Proteico-Postraduccional , Proteínas Ribosómicas/biosíntesis , Proteínas Ribosómicas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Productos Biológicos/química , Productos Biológicos/metabolismo , Espectroscopía de Resonancia Magnética con Carbono-13 , Espectrometría de Masas/métodos , Metaboloma , Mutagénesis , Péptidos Cíclicos/genética , Péptidos Cíclicos/metabolismo , Unión Proteica , Conformación Proteica , Espectroscopía de Protones por Resonancia Magnética , Proteínas Ribosómicas/genética , Proteínas Ribosómicas/metabolismoRESUMEN
Two macrolide glycosides with a unique scaffold were isolated from cultures of the myxobacterium Pyxidicoccus fallax. Their structures, including absolute configurations, were elucidated by a combination of NMR, MS, degradation, and molecular modeling techniques. Analysis of the proposed biosynthetic gene cluster led to insights into the biosynthesis of the polyketide and confirmed the structure assignment. The more active compound, disciformycinâ B, potently inhibits methicillin- and vancomycin-resistant Staphylococcus aureus.
Asunto(s)
Antibacterianos/química , Antibacterianos/farmacología , Macrólidos/química , Macrólidos/farmacología , Myxococcales/química , Infecciones Estafilocócicas/tratamiento farmacológico , Staphylococcus aureus/efectos de los fármacos , Antibacterianos/aislamiento & purificación , Humanos , Macrólidos/aislamiento & purificación , Staphylococcus aureus Resistente a Meticilina/efectos de los fármacos , Resistencia a la VancomicinaRESUMEN
Actinobacteria, the bacterial phylum most renowned for natural product discovery, has been established as a valuable source for drug discovery and biotechnology but is underrepresented within accessible genome and strain collections. Herein, we introduce the Natural Products Discovery Center (NPDC), featuring 122,449 strains assembled over eight decades, the genomes of the first 8490 NPDC strains (7142 Actinobacteria), and the online NPDC Portal making both strains and genomes publicly available. A comparative survey of RefSeq and NPDC Actinobacteria highlights the taxonomic and biosynthetic diversity within the NPDC collection, including three new genera, hundreds of new species, and ~7000 new gene cluster families. Selected examples demonstrate how the NPDC Portal's strain metadata, genomes, and biosynthetic gene clusters can be leveraged using genome mining approaches. Our findings underscore the ongoing significance of Actinobacteria in natural product discovery, and the NPDC serves as an unparalleled resource for both Actinobacteria strains and genomes.
RESUMEN
The crocapeptins are described here as cyclic depsipeptides, isolated from cultures of the myxobacterium Chondromyces crocatus . Structure elucidation of the compounds revealed a cyanopeptolin-like skeleton, containing the characteristic amino-hydroxy-piperidone (Ahp)-heterocycle. Like the cyanopeptolins, the myxobacterial crocapeptins proved to be serine protease inhibitors. The nonribosomal origin of the peptide was confirmed by mutagenesis experiments, and the biosynthesis gene cluster was sequenced. It could be shown that the Ahp-heterocycle originates from a proline residue in the precursor molecule precrocapeptin, which is converted to crocapeptin by the tailoring enzymes CpnE and CpnF. Conversion of precrocapeptin isolated from a cpnF mutant into crocapeptin was achieved using recombinant CpnF, a cytochrome P450 enzyme responsible for hydroxylation of the proline residue in precrocapeptin. Addition of protein CpnE resulted in strongly increased conversion rates toward Ahp containing product. A mutant with 10-fold increased production of crocapeptin A was created through insertion of the Pnpt-promotor in front of the NRPS gene.
Asunto(s)
Sistema Enzimático del Citocromo P-450/metabolismo , Depsipéptidos/metabolismo , Myxococcales/enzimología , Piperidonas/metabolismo , Inhibidores de Serina Proteinasa/metabolismo , Depsipéptidos/química , Depsipéptidos/genética , Depsipéptidos/aislamiento & purificación , Hidroxilación , Familia de Multigenes , Mutación , Myxococcales/química , Myxococcales/genética , Myxococcales/metabolismo , Piperidonas/química , Piperidonas/aislamiento & purificación , Inhibidores de Serina Proteinasa/química , Inhibidores de Serina Proteinasa/genética , Inhibidores de Serina Proteinasa/aislamiento & purificaciónRESUMEN
Analysis of the genome sequence of the myxobacterium Chondromyces crocatus Cm c5 revealed the presence of numerous cryptic megasynthetase gene clusters, one of which we here assign to two previously unknown chlorinated metabolites by a comparative gene inactivation and secondary metabolomics approach. Structure elucidation of these compounds revealed a unique cyclic depsipeptide skeleton featuring ß- and δ-amide bonds of aspartic acid and 3-methyl ornithine moieties, respectively. Insights into their biosynthesis were obtained by targeted gene inactivation and feeding experiments employing isotope-labeled precursors. The compounds were produced ubiquitously by the species Chondromyces crocatus and were found to inhibit the carbon storage regulator-RNA interaction.
Asunto(s)
Depsipéptidos/metabolismo , Myxococcales/genética , Ácido Aspártico/química , Depsipéptidos/química , Silenciador del Gen , Genoma Bacteriano , Genómica , Espectroscopía de Resonancia Magnética , Metabolómica , Estructura Molecular , Familia de Multigenes , Myxococcales/metabolismoRESUMEN
Two new cyclic peptides, termed jahnellamides A and B, were isolated from the myxobacterium Jahnella sp. Their structures were solved by NMR, ESIMS, and chemical derivatizations. Jahnellamides are a new class of α-ketoamide-containing peptides comprised of nonproteinogenic amino acids, including α-keto-ß-methionine and 4-hydroxyglutamic acid. Moreover, in silico analysis of the genome sequence along with feeding experiments allowed us to identify and annotate a candidate nonribosomal peptide synthetase biosynthetic gene cluster containing a polyketide synthase module involved in the formation of the α-ketoamide moiety.