Targeted pathway mining reveals novel modified hexapeptides from the demiguisin family

Following the discovery of the piperazic acid (Piz)-containing hexapeptide demiguisin A from a toxic bird-associated Amycolatopsis strain, we aimed to investigate the structural diversity and bioactive potential of this natural product family. Here, we used the dmg biosynthetic gene cluster from Amycolatopsis sp. PS_44_ISF1 as a pathway mining probe to search for alternative demiguisin A producers. A global search identified only one other soil-derived Amycolatopsis sp. NBC_01488, encoding a highly syntenic pathway. Under feather-mimicking conditions, PS_44_ISF1 demonstrated enhanced growth and metabolite production relative to NBC_01488. Despite phylogenomic distance, both strains produced shared metabolites, while NBC_01488 yielded more abundant demiguisins, enabling the isolation of five novel derivatives characterized by two Piz-moieties with different hydroxylation patterns and glycine truncation. Two congeners displayed potent activity against the avian pathogen Mycobacterium intracellulare and moderate activity against Mycobacterium tuberculosis. These findings show that targeted pathway mining guided the expansion of the chemical diversity of Piz-modifications and demiguisin congeners with promising anti-Mycobacterium activity.