Characterization of the Postaglycone Modifications in Ristomycin Biosynthesis

<i>Amycolatopsis</i> sp. TNS106 produces ristomycin (ristocetin), a type III glycopeptide antibiotic (GPA) featuring extensive glycosyl modifications and potent antimicrobial activity against Gram-positive pathogens. Unlike the well-documented nonribosomal peptide synthetase-assembled peptide scaffold, the timing and specificity of its postassembly tailoring steps remain poorly understood. In this study, we generated a series of <i>A.</i> sp. TNS106 mutants and characterized accumulated derivatives to delineate the postaglycone tailoring steps for ristomycin maturation. <i>In vitro</i> biochemical reactions confirmed the function and timing of the RgtfB and RgtfC glycosyltransferases and MtfA carboxyl methyltransferase. By integrating our findings with prior studies, we propose a comprehensive model for the sequential glycosylation and C-terminal methylation processes governing ristomycin biosynthesis. Notably, we identified multiple ristomycin glycoforms that demonstrate improved antimicrobial activity compared to the parent molecule. Strikingly, removal of the ristosamine on β-hydroxytyrosine 6 (βht6) or the presence of rhamnose on 4-hydroxyphenylglycine 4 (Hpg4) markedly impaired its antibacterial activity, particularly against vancomycin-resistant <i>Enterococcus</i>. The engineered strains constructed here provide a versatile platform for generating novel ristomycin analogs through combinatorial biosynthesis or chemical synthesis, advancing the development of new-to-nature GPAs with improved potency.