The lipopeptides ramoplanin from sp. ATCC 33076 and enduracidin produced by 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 -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 infections. In this study we have determined the function of the putative mannosyltransferase encoded by 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 gene was cloned into the tetracycline inducible plasmid pMS17 and integrated into the genome of the enduracidin producer . Induction of expression in 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 Hpg 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.


Article metrics loading...

Loading full text...

Full text loading...



  1. Amir-Heidari B., Thirlway J., Micklefield J. (2008). Auxotrophic-precursor directed biosynthesis of nonribosomal lipopeptides with modified tryptophan residuesOrg Biomol Chem 6975978 [View Article][PubMed]. [Google Scholar]
  2. Berg S., Starbuck J., Torrelles J.B., Vissa V.D., Crick D.C., Chatterjee D., Brennan P.J. (2005). Roles of conserved proline and glycosyltransferase motifs of EmbC in biosynthesis of lipoarabinomannanJ Biol Chem 28056515663 [View Article][PubMed]. [Google Scholar]
  3. Berg S., Kaur D., Jackson M., Brennan P.J. (2007). The glycosyltransferases of Mycobacterium tuberculosis – roles in the synthesis of arabinogalactan, lipoarabinomannan, and other glycoconjugatesGlycobiology 1735R56R [View Article][PubMed]. [Google Scholar]
  4. Bernsel A., Viklund H., Falk J., Lindahl E., von Heijne G., Elofsson A. (2008). Prediction of membrane-protein topology from first principlesProc Natl Acad Sci U S A 10571777181 [View Article][PubMed]. [Google Scholar]
  5. Bibb M.J., Janssen G.R., Ward J.M. (1985). Cloning and analysis of the promoter region of the erythromycin resistance gene (ermE) of Streptomyces erythraeus Gene 38215226 [View Article][PubMed]. [Google Scholar]
  6. Birch H.L., Alderwick L.J., Rittmann D., Krumbach K., Etterich H., Grzegorzewicz A., McNeil M.R., Eggeling L., Besra G.S. (2009). Identification of a terminal rhamnopyranosyltransferase (RptA) involved in Corynebacterium glutamicum cell wall biosynthesisJ Bacteriol 19148794887 [View Article][PubMed]. [Google Scholar]
  7. Borghi A., Ferrari P., Gallo G.G., Zanol M., Zerilli L.F., Lancini G.C. (1991). Microbial de-mannosylation and mannosylation of teicoplanin derivativesJ Antibiot (Tokyo) 4414441451 [View Article][PubMed]. [Google Scholar]
  8. Castiglione F., Marazzi A., Meli M., Colombo G. (2005). Structure elucidation and 3D solution conformation of the antibiotic enduracidin determined by NMR spectroscopy and molecular dynamicsMagn Reson Chem 43603610 [View Article][PubMed]. [Google Scholar]
  9. Chen J.-S., Wang Y.-X., Shao L., Pan H.-X., Li J.-A., Lin H.-M., Dong X.-J., Chen D.-J. (2013). Functional identification of the gene encoding the enzyme involved in mannosylation in ramoplanin biosynthesis in Actinoplanes spBiotechnol Lett 3515011508 [View Article][PubMed]. [Google Scholar]
  10. Cudic P., Behenna D.C., Kranz J.K., Kruger R.G., Wand A.J., Veklich Y.I., Weisel J.W., McCafferty D.G. (2002). Functional analysis of the lipoglycodepsipeptide antibiotic ramoplaninChem Biol 9897906 [View Article][PubMed]. [Google Scholar]
  11. Di Palo S., Gandolfi R., Jovetic S., Marinelli F., Romano D., Molinari F. (2007). A new bacterial mannosidase for the selective modification of ramoplanin and its derivativesEnzyme Microb Technol 41806811 [View Article]. [Google Scholar]
  12. Fang X., Tiyanont K., Zhang Y., Wanner J., Boger D., Walker S. (2006). The mechanism of action of ramoplanin and enduracidinMol Biosyst 26976 [View Article][PubMed]. [Google Scholar]
  13. Hamburger J.B., Hoertz A.J., Lee A., Senturia R.J., McCafferty D.G., Loll P.J. (2009). A crystal structure of a dimer of the antibiotic ramoplanin illustrates membrane positioning and a potential Lipid II docking interfaceProc Natl Acad Sci U S A 1061375913764 [View Article][PubMed]. [Google Scholar]
  14. He H. (2005). Mannopeptimycins, a novel class of glycopeptide antibiotics active against Gram-positive bacteriaAppl Microbiol Biotechnol 67444452 [View Article][PubMed]. [Google Scholar]
  15. Hobbs G., Frazer C.M., Gardner D.C.J., Cullum J.A., Oliver S.G. (1989). Dispersed growth of Streptomyces in liquid cultureAppl Microbiol Biotechnol 31272277. [Google Scholar]
  16. Hofmann K., Stoffel W. (1993). tmbase – A database of membrane spanning protein segmentsBiol Chem Hoppe Seyler 374166. [Google Scholar]
  17. Kieser T., Bibb M.J., Buttner M.J., Chater K.F., Hopwood D.A. (2000). Practical Streptomyces Genetics., NorwichThe John Innes Foundation. [Google Scholar]
  18. Lewis R.A., Nunns L., Thirlway J., Carroll K., Smith C.P., Micklefield J. (2011). Active site modification of the β-ketoacyl-ACP synthase FabF3 of Streptomyces coelicolor affects the fatty acid chain length of the CDA lipopeptidesChem Commun (Camb) 4718601862 [View Article][PubMed]. [Google Scholar]
  19. Liu J., Mushegian A. (2003). Three monophyletic superfamilies account for the majority of the known glycosyltransferasesProtein Sci 1214181431 [View Article][PubMed]. [Google Scholar]
  20. MacNeil D.J., Gewain K.M., Ruby C.L., Dezeny G., Gibbons P.H., MacNeil T. (1992). Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vectorGene 1116168 [View Article][PubMed]. [Google Scholar]
  21. Magarvey N.A., Haltli B., He M., Greenstein M., Hucul J.A. (2006). Biosynthetic pathway for mannopeptimycins, lipoglycopeptide antibiotics active against drug-resistant Gram-positive pathogensAntimicrob Agents Chemother 5021672177 [View Article][PubMed]. [Google Scholar]
  22. Mahlert C., Kopp F., Thirlway J., Micklefield J., Marahiel M.A. (2007). Stereospecific enzymatic transformation of α-ketoglutarate to (2S,3R)-3-methyl glutamate during acidic lipopeptide biosynthesisJ Am Chem Soc 1291201112018 [View Article][PubMed]. [Google Scholar]
  23. McCafferty D.G., Cudic P., Frankel B.A., Barkallah S., Kruger R.G., Li W. (2002). Chemistry and biology of the ramoplanin family of peptide antibioticsBiopolymers 66261284 [View Article][PubMed]. [Google Scholar]
  24. Milne C., Powell A., Jim J., Al Nakeeb M., Smith C.P., Micklefield J. (2006). Biosynthesis of the (2S,3R)-3-methyl glutamate residue of nonribosomal lipopeptidesJ Am Chem Soc 1281125011259 [View Article][PubMed]. [Google Scholar]
  25. Möller S., Croning M.D., Apweiler R. (2001). Evaluation of methods for the prediction of membrane spanning regionsBioinformatics 17646653 [View Article][PubMed]. [Google Scholar]
  26. Neary J.M., Powell A., Gordon L., Milne C., Flett F., Wilkinson B., Smith C.P., Micklefield J. (2007). An asparagine oxygenase (AsnO) and a 3-hydroxyasparaginyl phosphotransferase (HasP) are involved in the biosynthesis of calcium-dependent lipopeptide antibioticsMicrobiology 153768776 [View Article][PubMed]. [Google Scholar]
  27. Nogami I., Shirafuji H., Matsumura S. (1984). Production of enduracidin and micro-organisms therefor US Patent 4465771.
  28. Powell A., Al Nakeeb M., Wilkinson B., Micklefield J. (2007a). Precursor-directed biosynthesis of nonribosomal lipopeptides with modified glutamate residuesChem Commun (Camb)(26), 26832685 [View Article][PubMed]. [Google Scholar]
  29. Powell A., Borg M., Amir-Heidari B., Neary J.M., Thirlway J., Wilkinson B., Smith C.P., Micklefield J. (2007b). Engineered biosynthesis of nonribosomal lipopeptides with modified fatty acid side chainsJ Am Chem Soc 1291518215191 [View Article][PubMed]. [Google Scholar]
  30. Rodríguez-García A., Combes P., Pérez-Redondo R., Smith M.C.A., Smith M.C.M. (2005). Natural and synthetic tetracycline-inducible promoters for use in the antibiotic-producing bacteria Streptomyces Nucleic Acids Res 33e87 [View Article][PubMed]. [Google Scholar]
  31. Shirling E.B., Gottlieb D. (1966). Methods for characterization of Streptomyces speciesInt J Syst Bacteriol 16313340 [View Article]. [Google Scholar]
  32. Skovierová H., Larrouy-Maumus G., Pham H., Belanová M., Barilone N., Dasgupta A., Mikusová K., Gicquel B., Gilleron M., other authors. (2010). Biosynthetic origin of the galactosamine substituent of arabinogalactan in Mycobacterium tuberculosis J Biol Chem 2854134841355 [View Article][PubMed]. [Google Scholar]
  33. Song F., Guan Z., Raetz C.R.H. (2009). Biosynthesis of undecaprenyl phosphate-galactosamine and undecaprenyl phosphate-glucose in Francisella novicida Biochemistry 4811731182 [View Article][PubMed]. [Google Scholar]
  34. Sosio M., Stinchi S., Beltrametti F., Lazzarini A., Donadio S. (2003). The gene cluster for the biosynthesis of the glycopeptide antibiotic A40926 by Nonomuraea speciesChem Biol 10541549 [View Article][PubMed]. [Google Scholar]
  35. Sosio M., Kloosterman H., Bianchi A., de Vreugd P., Dijkhuizen L., Donadio S. (2004). Organization of the teicoplanin gene cluster in Actinoplanes teichomyceticus Microbiology 15095102 [View Article][PubMed]. [Google Scholar]
  36. Thirlway J., Lewis R., Nunns L., Al Nakeeb M., Styles M., Struck A.W., Smith C.P., Micklefield J. (2012). Introduction of a non-natural amino acid into a nonribosomal peptide antibiotic by modification of adenylation domain specificityAngew Chem Int Ed Engl 5171817184 [View Article][PubMed]. [Google Scholar]
  37. Tusnády G.E., Simon I. (2001). The hmmtop transmembrane topology prediction serverBioinformatics 17849850 [View Article][PubMed]. [Google Scholar]
  38. Uguru G.C., Milne C., Borg M., Flett F., Smith C.P., Micklefield J. (2004). Active-site modifications of adenylation domains lead to hydrolysis of upstream nonribosomal peptidyl thioester intermediatesJ Am Chem Soc 12650325033 [View Article][PubMed]. [Google Scholar]
  39. Viklund H., Elofsson A. (2004). Best α-helical transmembrane protein topology predictions are achieved using hidden Markov models and evolutionary informationProtein Sci 1319081917 [View Article][PubMed]. [Google Scholar]
  40. Viklund H., Elofsson A. (2008). octopus: improving topology prediction by two-track ANN-based preference scores and an extended topological grammarBioinformatics 2416621668 [View Article][PubMed]. [Google Scholar]
  41. Walker S., Chen L., Hu Y., Rew Y., Shin D., Boger D.L. (2005). Chemistry and biology of ramoplanin: a lipoglycodepsipeptide with potent antibiotic activityChem Rev 105449476 [View Article][PubMed]. [Google Scholar]
  42. Wehmeier S., Varghese A.S., Gurcha S.S., Tissot B., Panico M., Hitchen P., Morris H.R., Besra G.S., Dell A., Smith M.C.M. (2009). Glycosylation of the phosphate binding protein, PstS, in Streptomyces coelicolor by a pathway that resembles protein O-mannosylation in eukaryotesMol Microbiol 71421433 [View Article][PubMed]. [Google Scholar]
  43. Wilkinson B., Micklefield J. (2007). Mining and engineering natural-product biosynthetic pathwaysNat Chem Biol 3379386 [View Article][PubMed]. [Google Scholar]
  44. Wu M.C., Law B., Wilkinson B., Micklefield J. (2012). Bioengineering natural product biosynthetic pathways for therapeutic applicationsCurr Opin Biotechnol 23931940 [View Article][PubMed]. [Google Scholar]
  45. Yin X., Zabriskie T.M. (2006). The enduracidin biosynthetic gene cluster from Streptomyces fungicidicus Microbiology 15229692983 [View Article][PubMed]. [Google Scholar]

Data & Media loading...


Supplementary Data


Most cited this month Most Cited RSS feed

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error