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

Abstract

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 intro 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.\n\nSignificance StatementMycobacterium tuberculosis is the leading causative agent of tuberculosis (TB), of which millions of deaths occur annually. A putative lipopeptide biosynthetic gene cluster has been shown to be essential for the survival of this pathogen in hosts, and homologous gene clusters have also been found in all pathogenic mycobacteria and other species of Actinobacteria. We have identified the function of these gene clusters in making a new family of isonitrile lipopeptides. The biosynthesis has several unique features, including an unprecedented mechanism for isonitrile synthesis. Our results have further suggested that these biosynthetic gene clusters play a role in metal transport, and thus have shed light on a new metal transport system that is crucial for virulence of pathogenic mycobacteria.