Updated sequence of ramoplanin biosynthetic gene cluster from Actinoplanes ramoplaninifer ATCC 33,076.

Actinoplanes ramoplaninifer ATCC 33,076 is the only known producer of the glycolipodepsipeptide antibiotic ramoplanin. Ramoplanin (Rmp) interferes with peptidoglycan biosynthesis by binding to lipid II and consequently impeding the transglycosylation reactions. Rmp exhibits excellent antimicrobial properties against Gram-positive bacteria, including methicillin- and vancomycin-resistant strains of staphylococci and enterococci, as well as Clostridioides difficile, showing no cross-resistance with glycopeptides and β-lactams. Rmp is poorly absorbed in the gastrointestinal tract when administered orally, exhibits poor local tolerability after intravenous injections and scarce tissue penetrability upon intramuscular injection. Moreover, Rmp is reported to be rather instable in the bloodstream. Consequently, Rmp oral capsule has reached phase II and III clinical trials for the eradication of vancomycin-resistant Enterococcus faecium and Clostridioides difficile in the gastrointestinal tract, respectively. The modification of Rmp through combinatorial biosynthesis might open new perspectives for this antibiotic, enhancing and/or modifying its pharmacokinetics. To understand the genetics underlying Rmp biosynthesis, its biosynthetic gene cluster (BGC) was sequenced two decades ago, revealing a 16-modular non-ribosomal peptide synthetase (NRPS) machinery, which is unusual considering that ramoplanin is a 17 amino acid peptide. Bearing in mind that older sequencing approaches often produced errors when facing iterative DNA segments (as is the case with NRPS genes), we aimed to resequence the ramoplanin BGC using new sequencing approaches. Thus, in the current paper, we present a new sequence of the ramoplanin BGC (ramo), revealing a conventional 17-modular NRPS machinery. Additionally, we reannotated the ramo genes, providing new insights into Rmp biosynthesis.