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.