RESUMEN
Triacsins are an intriguing class of specialized metabolites possessing a conserved N-hydroxytriazene moiety not found in any other known natural products. Triacsins are notable as potent acyl-CoA synthetase inhibitors in lipid metabolism, yet their biosynthesis has remained elusive. Through extensive mutagenesis and biochemical studies, we here report all enzymes required to construct and install the N-hydroxytriazene pharmacophore of triacsins. Two distinct ATP-dependent enzymes were revealed to catalyze the two consecutive N-N bond formation reactions, including a glycine-utilizing, hydrazine-forming enzyme (Tri28) and a nitrite-utilizing, N-nitrosating enzyme (Tri17). This study paves the way for future mechanistic interrogation and biocatalytic application of enzymes for N-N bond formation.
Asunto(s)
Coenzima A Ligasas/metabolismo , Streptomyces aureofaciens/enzimología , Streptomyces aureofaciens/genética , Triazenos/metabolismo , Biocatálisis , Escherichia coli/genética , Glicina/química , Hidrazinas/química , Metabolismo de los Lípidos , Lípidos/química , Nitritos/química , Triazenos/químicaRESUMEN
Microbial biosynthetic gene clusters are a valuable source of bioactive molecules. However, because they typically represent a small fraction of genomic material in most metagenomic samples, it remains challenging to deeply sequence them. We present an approach to isolate and sequence gene clusters in metagenomic samples using microfluidic automated plasmid library enrichment. Our approach provides deep coverage of the target gene cluster, facilitating reassembly. We demonstrate the approach by isolating and sequencing type I polyketide synthase gene clusters from an Antarctic soil metagenome. Our method promotes the discovery of functional-related genes and biosynthetic pathways.
Asunto(s)
Vías Biosintéticas/genética , Metagenómica/métodos , Técnicas Analíticas Microfluídicas , Biblioteca Genómica , Dispositivos Laboratorio en un Chip , Plásmidos/genética , Sintasas Poliquetidas/genética , Microbiología del Suelo , Flujo de TrabajoRESUMEN
A facile method for the quick discovery and quantification of isonitrile compounds from microbial cultures was established based on the isonitrile-tetrazine click reaction. This method was successfully applied to the rediscovery of diisonitrile antibotic SF2768 from an unknown strain Streptomyces tsukubensis. Finally, an in situ reduction further enabled bioorthogonal ligation of primary and secondary isonitriles for the first time.
Asunto(s)
Productos Biológicos/análisis , Nitrilos/análisis , Streptomyces/química , Tetrazoles/química , Química Clic , Estructura MolecularRESUMEN
Despite intensive study for 50 years, the biochemical and genetic links between lysine metabolism and central metabolism in Pseudomonas putida remain unresolved. To establish these biochemical links, we leveraged random barcode transposon sequencing (RB-TnSeq), a genome-wide assay measuring the fitness of thousands of genes in parallel, to identify multiple novel enzymes in both l- and d-lysine metabolism. We first describe three pathway enzymes that catabolize l-2-aminoadipate (l-2AA) to 2-ketoglutarate (2KG), connecting d-lysine to the TCA cycle. One of these enzymes, P. putida 5260 (PP_5260), contains a DUF1338 domain, representing a family with no previously described biological function. Our work also identified the recently described coenzyme A (CoA)-independent route of l-lysine degradation that results in metabolization to succinate. We expanded on previous findings by demonstrating that glutarate hydroxylase CsiD is promiscuous in its 2-oxoacid selectivity. Proteomics of selected pathway enzymes revealed that expression of catabolic genes is highly sensitive to the presence of particular pathway metabolites, implying intensive local and global regulation. This work demonstrated the utility of RB-TnSeq for discovering novel metabolic pathways in even well-studied bacteria, as well as its utility a powerful tool for validating previous research.IMPORTANCEP. putida lysine metabolism can produce multiple commodity chemicals, conferring great biotechnological value. Despite much research, the connection of lysine catabolism to central metabolism in P. putida remained undefined. Here, we used random barcode transposon sequencing to fill the gaps of lysine metabolism in P. putida We describe a route of 2-oxoadipate (2OA) catabolism, which utilizes DUF1338-containing protein P. putida 5260 (PP_5260) in bacteria. Despite its prevalence in many domains of life, DUF1338-containing proteins have had no known biochemical function. We demonstrate that PP_5260 is a metalloenzyme which catalyzes an unusual route of decarboxylation of 2OA to d-2-hydroxyglutarate (d-2HG). Our screen also identified a recently described novel glutarate metabolic pathway. We validate previous results and expand the understanding of glutarate hydroxylase CsiD by showing that can it use either 2OA or 2KG as a cosubstrate. Our work demonstrated that biological novelty can be rapidly identified using unbiased experimental genetics and that RB-TnSeq can be used to rapidly validate previous results.
Asunto(s)
Aptitud Genética , Lisina/metabolismo , Pseudomonas putida/enzimología , Pseudomonas putida/genética , Redes y Vías MetabólicasRESUMEN
Triacsins are a family of natural products having in common an N-hydroxytriazene moiety not found in any other known secondary metabolites. Though many studies have examined the biological activity of triacsins in lipid metabolism, their biosynthesis has remained unknown. Here we report the identification of the triacsin biosynthetic gene cluster in Streptomyces aureofaciens ATCC 31442. Bioinformatic analysis of the gene cluster led to the discovery of the tacrolimus producer Streptomyces tsukubaensis NRRL 18488 as a new triacsin producer. In addition to targeted gene disruption to identify necessary genes for triacsin production, stable isotope feeding was performed in vivo to advance the understanding of N-hydroxytriazene biosynthesis.
Asunto(s)
Familia de Multigenes , Triazenos/metabolismo , Biología Computacional , Inhibidores Enzimáticos/metabolismo , Enzimas/genética , Genes Bacterianos , Mutación , Streptomyces/genética , Streptomyces aureofaciens/genéticaRESUMEN
A putative lipopeptide biosynthetic gene cluster is conserved in many species of Actinobacteria, including Mycobacterium tuberculosis and M. marinum, but the specific function of the encoding proteins has been elusive. Using both in vivo heterologous reconstitution and in vitro biochemical analyses, we have revealed that the five encoding biosynthetic enzymes are capable of synthesizing a family of isonitrile lipopeptides (INLPs) through a thio-template mechanism. The biosynthesis features the generation of isonitrile from a single precursor Gly promoted by a thioesterase and a nonheme iron(II)-dependent oxidase homolog and the acylation of both amino groups of Lys by the same isonitrile acyl chain facilitated by a single condensation domain of a nonribosomal peptide synthetase. In addition, the deletion of INLP biosynthetic genes in M. marinum has decreased the intracellular metal concentration, suggesting the role of this biosynthetic gene cluster in metal transport.
Asunto(s)
Actinobacteria/enzimología , Lipopéptidos/biosíntesis , Familia de Multigenes , Mycobacterium tuberculosis/enzimología , Péptido Sintasas/metabolismo , Actinobacteria/genética , Transporte Biológico , Catálisis , Cromatografía , Cromatografía por Intercambio Iónico , Escherichia coli/enzimología , Escherichia coli/genética , Ácidos Grasos/química , Eliminación de Gen , Lisina/química , Metales , Mutación , Mycobacterium marinum/enzimología , Mycobacterium marinum/genética , Mycobacterium tuberculosis/genética , Péptido Sintasas/genética , Dominios Proteicos , Ribosomas/químicaRESUMEN
In this study, an unsaturated copolyester, poly[(R)-3-hydroxybutyrate-co-(R)-3-hydroxy-10-undecenoate] (PHBU), was produced by an engineered strain of Escherichia coli, cross-linked via thiol-ene click chemistry, and analyzed for improved physical properties and biocompatibility with human mesenchymal stem cells. By cross-linking the PHBU polymer, an increase in tensile strength of greater than 200% to 26.2 MPa was observed, resulting in a material with physical properties closer to those relevant for soft tissue replacement. Results showed that this chemically cross-linked polyester did not exhibit significant cytotoxicity toward human cells after chemical modification. The chemically modifiable copolyester described here could potentially be used as a replacement for an assortment of tissues currently without viable material alternatives in the field of tissue-engineering.