Engineered biosynthesis of enduracidin lipoglycopeptide antibiotics using the ramoplanin mannosyltransferase Ram29

Abstract

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.

Recent advances in engineering nonribosomal peptide assembly lines

“…This ultimately means that while ramoplanin has potentially found a role in the treatment of Clostridium difficile infections (undergoing phase 3 trials), the related enduracidin is relegated to use as an animal feed additive. 35,36 If enduracidin was mannosylated in a similar manner to ramoplanin then it would potentially make a much better drug candidate. The ramoplanin gene cluster contains a gene, ram29, which encodes for an integral membrane protein that is homologous to gene products found in several other mannosylated natural product gene clusters.…”

“…This extracytoplasmic domain is not present in the other mannosyltransferases and is suggested to be responsible for binding the ramoplanin aglycone. 36 Employing the assumption that the structural similarities between the two lipopeptide structures would allow the binding and subsequent mannosylation of enduracidin, ram29 was expressed in the enduracidin-producing Streptomyces fungicidicus. An expression cassette containing the ram29 gene along with its native Shine-Dalgarno sequence under the control of the tetracycline inducible promoter and integrated at the FC31 site on the Streptomyces chromosome failed to produce any evidence of mannosylated enduracidin.…”

New Found Hope for Antibiotic Discovery: Lipid II Inhibitors

“…Also, the macrocyclic peptides 10 , 11 and 13 (Figure ) possess lipophilic side chains that are vital for their antimicrobial activities, possibly through membrane insertion or hindering PBPs from reaching lipid II . A noticeable structural difference is the unique dimannosyl group that is present only in ramoplanin A2 ( 10 ), which is likely to provide enhanced hydrolytic stability and solubility …”

Enhancement of Enduracidin Accumulation from Sweet Sorghum Juice via Alleviation of Oxidative Damage in Streptomyces Fungicidicus M30

“…Enduracidin is a lipopeptide antibiotic being widely used as an excellent animal growth promoter due to its high safety, low toxicity, low residue, and powerful antibacterial effect [1][2][3][4]. With growth of the enduracidin industry, the global demand for enduracidin continues to increase.…”