Conserved cis and trans communication domains mediate module interaction in glycopeptide antibiotic NRPS assembly lines

Non-ribosomal peptide synthetases (NRPSs) assemble structurally complex and clinically important natural products, yet the mechanisms coordinating communication between multiple enzymes remain incompletely understood. Here, we dissected the NRPS system underlying biosynthesis of the glycopeptide antibiotic (GPA) balhimycin in Amycolatopsis balhimycina to elucidate principles of inter-enzyme communication. Genetic perturbation of short terminal structural elements markedly reduced balhimycin production, demonstrating their critical role in biosynthesis. AlphaFold3 predictions identified these elements as trans-COM domains mediating specific NRPS interactions, primarily through hydrophobic contacts. Quantitative binding studies using microscale thermophoresis confirmed the importance of the trans-COM domains for multiprotein interaction, extending current models of NRPS communication. Comparative structural analysis further uncovered a conserved class of cis-COM domains within condensation domains across GPA NRPS assembly lines. Together, our findings establish a unified model in which trans- and cis-interfaces cooperatively maintain assembly line fidelity, redefining NRPS architecture and enabling rational engineering strategies.