Biosynthesis of Isonitrile Lipopeptides by Conserved Non-ribosomal Peptide Synthetase Gene Clusters in Actinobacteria

A putative lipopeptide biosynthetic gene cluster is conserved in many species of Actinobacteria, including Mycobacterium tuberculosis and Mycobacterium 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 new 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 non-heme iron(II)-dependent oxidase homologue, and the acylation of both amino groups of Lys by the same isonitrile acyl chain facilitated by a single condensation domain of a non-ribosomal peptide synthetase (NRPS). 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.