Characterization of the Postaglycone Modifications in Ristomycin Biosynthesis

Amycolatopsis 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 A. sp. TNS106 mutants and characterized accumulated derivatives to delineate the postaglycone tailoring steps for ristomycin maturation. In vitro 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 Enterococcus. 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.