Biosynthesis of triacsin featuring an N-hydroxytriazene pharmacophore.

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.

Microfluidic automated plasmid library enrichment for biosynthetic gene cluster discovery.

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.

Facile Discovery and Quantification of Isonitrile Natural Products via Tetrazine-Based Click Reactions.

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 . Finally, an reduction further enabled bioorthogonal ligation of primary and secondary isonitriles for the first time.

Massively Parallel Fitness Profiling Reveals Multiple Novel Enzymes in Lysine Metabolism.

Despite intensive study for 50 years, the biochemical and genetic links between lysine metabolism and central metabolism in remain unresolved. To establish these biochemical links, we leveraged andom arcode rasposon uencing (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.

Identifying the Biosynthetic Gene Cluster for Triacsins with an N-Hydroxytriazene Moiety.

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.

Biosynthesis of isonitrile lipopeptides by conserved nonribosomal peptide synthetase gene clusters in Actinobacteria.

A putative lipopeptide biosynthetic gene cluster is conserved in many species of Actinobacteria, including and , 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.

Influence of Cross-Linking on the Physical Properties and Cytotoxicity of Polyhydroxyalkanoate (PHA) Scaffolds for Tissue Engineering.

In this study, an unsaturated copolyester, poly[()-3-hydroxybutyrate--()-3-hydroxy-10-undecenoate] (PHBU), was produced by an engineered strain of , 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.