Enduracidin B

Enduracidins (1, 2) and ramoplanin (3) are structurally and functionally closely related lipodepsipeptide antibiotics. They are active against multi-drug-resistant Gram-positive pathogens, including MRSA. Each peptide contains one chlorinated non-proteinogenic amino acid residue, Cl(2)-Hpg or Cl-Hpg. To investigate the timing of halogenation and the importance of chlorination on bioactivity and bioavailability of enduracidin, and to probe the substrate specificity and portability of the ramoplanin halogenase, we constructed the mutant strain SfDelta30 in which the enduracidin halogenase gene orf30 had been deleted and complemented it with the ramoplanin counterpart orf20. We also expressed orf20 in the enduracidin wild-type producer. Metabolite analysis revealed SfDelta30 produced the novel analogues dideschloroenduracidins A (4) and B (5), while the recombinant strains SfDelta30R20 and SfR20 produced monodeschloroenduracidins A (6) and B (7) and a trichlorinated enduracidin (8), respectively. In addition, orf30 self-complementation yielded the strain SfDelta30E30, which is capable of producing six peptides including 6 and 7. MS/MS analysis positioned the single chlorine atom in 6 at Hpg(13) and localized the third chlorine atom in 8 to Hpg(11). Biological evaluation of these enduracidin analogues indicated that all retained activity against Staphylococcus aureus. Our findings lay the foundation for further utilization of enduracidin and ramoplanin halogenases in combinatorial biosynthesis.

A new multi-residue method for the determination of seven polypeptides, namely, polymixin B1, polymixin B2, polymixin E1 (colistin A), polymixin E2 (colistin B), enduracidin A (enramycin A), enduracidin B (enramycin B), and bacitracin A, in food of animal origin was developed and validated for chicken muscle tissue. Chicken muscle tissue was extracted with acidified methanol (1 % TFA). After homogenization, shaking, and centrifugation, the acidified methanol extract was decanted. A second extraction was performed with methanol (1 % TFA) and formic acid (1 %) 25:75, v/v. The pooled extract was cleaned up and concentrated on a solid-phase extraction cartridge. The retained analytes were eluted with methanol/acetonitrile. The extract was evaporated to dryness, reconstituted in mobile phase, filtered, and quantified by LC-MS/MS under ESI conditions. The method has a LOQ of 50.0 μg/kg for polymixin E2 (colistin B), 39.0 μg/kg for polymixin E1 (colistin A), 74.0 μg/kg for polymixin B1, 71.0 μg/kg for polymixin B2, 66.0 μg/kg for enduracidin A, 50.0 μg/kg for enduracidin B, and 30.0 μg/kg for bacitracin A in chicken muscle tissues. This is the first sensitive, suitable, multi-residue method reported for the seven polypeptide drug residues in chicken muscle tissue.

The lipopeptides ramoplanin from Actinoplanes sp. ATCC 33076 and enduracidin produced by Streptomyces fungicidicus are effective antibiotics against a number of drug-resistant Gram-positive pathogens. While these two antibiotics share a similar cyclic peptide structure, comprising 17 amino acids with an N-terminal fatty acid side chain, ramoplanin has a di-mannose moiety that enduracidin lacks. The mannosyl substituents of ramoplanin enhance aqueous solubility, which was important in the development of ramoplanin as a potential treatment for Clostridium difficile infections. In this study we have determined the function of the putative mannosyltransferase encoded by ram29 from the ramoplanin biosynthetic gene cluster. Bioinformatics revealed that Ram29 is an integral membrane protein with a putative DxD motif that is suggested to bind to, and activate, a polyprenyl phosphomannose donor and an extracytoplasmic C-terminal domain that is predicted to bind the ramoplanin aglycone acceptor. The ram29 gene was cloned into the tetracycline inducible plasmid pMS17 and integrated into the genome of the enduracidin producer S. fungicidicus. Induction of ram29 expression in S. fungicidicus resulted in the production of monomannosylated enduracidin derivatives, which are not present in the WT strain. Tandem MS analysis showed that mannosylation occurs on the Hpg11 residue of enduracidin. In addition to confirming the function of Ram29, these findings demonstrate how the less common, membrane-associated, polyprenyl phosphosugar-dependent glycosyltransferases can be used in natural product glycodiversification. Such a strategy may be valuable in future biosynthetic engineering approaches aimed at improving the physico-chemical and biological properties of bioactive secondary metabolites including antibiotics.