Uncovering Microbial Nitrogenous Chemical Diversity through Isotope-Guided Molecular Networking

Abstract Nitrogen-containing natural products are pivotal sources of bioactive molecules. However, their comprehensive investigation by metabolomics remains challenging due to background interference from primary metabolites and their high structural diversity including peptides, alkaloids, nucleosides, and others. Here, we present NitroVISION, an integrated pipeline that combines stable isotope labeling with targeted molecular networking to selectively detect nitrogen-containing metabolites from a complex mixture. Applied to five well-characterized Streptomyces strains, NitroVISION identified 42 nitrogen-containing natural products across 23 distinct compound classes, from which we characterized menaxinnamide, a new subclass of nonribosomal peptides incorporating a methylnaphthoquinone unit, and octapiperamide, a novel ribosomal peptide featuring a unique piperazine ring. Our work reveals the hidden chemical space of nitrogenous natural products and paves the way for the systematic exploration of microbial metabolic dark matter.