Abstract:
Glycopeptide antibiotics (GPAs) are natural products from actinomycetes, classified into five types based on their structural diversity. Clinically relevant GPAs such as vancomycin and teicoplanin are crucial for treating infection caused by multi-resistant Gram-positive bacteria. The biosynthesis of balhimycin, a vancomycin-type GPA produced by Amycolatopsis balhimycina, serves as a model system of GPA biosynthesis. While individual catalytic steps have been characterized, the coordination between biosynthetic enzymes and export mechanisms remains poorly understood. The present work focuses on an in-depth examination of GPA biosynthesis in Amycolatopsis balhimycina, emphasizing the integration of biosynthesis and export during balhimycin production. The first part of the work investigated Tba's substrate specificity, its functional role in transport processes, and its impact on the organization of the balhimycin biosynthesis complex. Deletion of tba showed a decrease in balhimycin production, which could only be restored when heterologous transporter genes of the same GPA type were integrated. Molecular dynamics simulations and mutational analysis confirmed that modifications like glycosylation and chlorination were shown to have no impact on export efficiency, whereas glycosylation was crucial for antibacterial action. Proximity-dependent labeling provided evidence for a putative interaction between Tba and the balhimycin biosynthetic machinery. This indicates that Tba may be involved not only in the export of balhimycin but also in having a functional role within a biosynthetic microcompartment. The second part of this work investigated the protein-protein interactions involved in balhimycin biosynthesis. In vivo and in vitro experiments demonstrated that distinct docking domains (DDs) are crucial for interactions between the NRPS modules. Furthermore, MbtH-like proteins (MLPs), which serve as cofactors for the adenylation domains (A-domains) of NRPSs, have been shown to interact with NRPS modules. Gene deletion indicated and bacterial two-hybrid assays confirmed that, several genome-encoded MLP genes could compensate for each other. AlphaFold analysis predicted the binding interface between MLPs and A-domains, and mutational analysis confirmed important residues involved in the binding. Furthermore, the cytochrome P450 monooxygenases OxyA and OxyC, which facilitate oxidative crosslinking of amino acids, interacted with the X-domain of the NRPS, identified as a recruitment domain for these oxygenases. No related interaction could be found for OxyB. Native PAGE analysis and proximity-dependent labeling experiments corroborated the intimate spatial arrangement of the NRPSs with the halogenase BhaA, which catalyzes the chlorination of β-hydroxytyrosine during peptide synthesis on the NRPS. The present work demonstrates that balhimycin biosynthesis is a strictly regulated and spatially organized process in which export, peptide assembly, and modifications are interlinked via a network of unique protein-protein interactions.
Actinomycetes