1887

Abstract

Spiramycin, a 16-membered macrolide antibiotic used in human medicine, is produced by ; it comprises a polyketide lactone, platenolide, to which three deoxyhexose sugars are attached. In order to characterize the gene cluster governing the biosynthesis of spiramycin, several overlapping cosmids were isolated from an gene library, by hybridization with various probes (spiramycin resistance or biosynthetic genes, tylosin biosynthetic genes), and the sequences of their inserts were determined. Sequence analysis showed that the spiramycin biosynthetic gene cluster spanned a region of over 85 kb of contiguous DNA. In addition to the five previously described genes that encode the type I polyketide synthase involved in platenolide biosynthesis, 45 other genes have been identified. It was possible to propose a function for most of the inferred proteins in spiramycin biosynthesis, in its regulation, in resistance to the produced antibiotic or in the provision of extender units for the polyketide synthase. Two of these genes, predicted to be involved in deoxysugar biosynthesis, were inactivated by gene replacement, and the resulting mutants were unable to produce spiramycin, thus confirming their involvement in spiramycin biosynthesis. This work reveals the main features of spiramycin biosynthesis and constitutes a first step towards a detailed molecular analysis of the production of this medically important antibiotic.

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2007-12-01
2019-10-24
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References

  1. Altschul, S. F., Madden, T. L., Schaffer, A. A., Zhang, J., Zhang, Z., Miller, W. & Lipman, D. J. ( 1997; ). Gapped blast and psi-blast: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402.[CrossRef]
    [Google Scholar]
  2. Arisawa, A., Kawamura, N., Tsunekawa, H., Okamura, K., Tone, H. & Okamoto, R. ( 1993; ). Cloning and nucleotide sequences of two genes involved in the 4″-O-acylation of macrolide antibiotics from Streptomyces thermotolerans. Biosci Biotechnol Biochem 57, 2020–2025.[CrossRef]
    [Google Scholar]
  3. Bate, N., Butler, A. R., Gandecha, A. R. & Cundliffe, E. ( 1999; ). Multiple regulatory genes in the tylosin biosynthetic cluster of Streptomyces fradiae. Chem Biol 6, 617–624.[CrossRef]
    [Google Scholar]
  4. Bate, N., Butler, A. R., Smith, I. P. & Cundliffe, E. ( 2000; ). The mycarose-biosynthetic genes of Streptomyces fradiae, producer of tylosin. Microbiology 146, 139–146.
    [Google Scholar]
  5. Bibb, M. J. ( 2005; ). Regulation of secondary metabolism in streptomycetes. Curr Opin Microbiol 8, 208–215.[CrossRef]
    [Google Scholar]
  6. Bignell, D. R., Bate, N. & Cundliffe, E. ( 2007; ). Regulation of tylosin production: role of a TylP-interactive ligand. Mol Microbiol 63, 838–847.
    [Google Scholar]
  7. Borisova, S. A., Zhao, L., Melançon, C. E., III, Kao, C. L. & Liu, H. W. ( 2004; ). Characterization of the glycosyltransferase activity of DesVII: analysis of and implications for the biosynthesis of macrolide antibiotics. J Am Chem Soc 126, 6534–6535.[CrossRef]
    [Google Scholar]
  8. Brown, E. D. ( 2005; ). Conserved P-loop GTPases of unknown function in bacteria: an emerging and vital ensemble in bacterial physiology. Biochem Cell Biol 83, 738–746.[CrossRef]
    [Google Scholar]
  9. Burgett, S. G., Kuhstoss, S. A., Rao, R. N., Richardson, M. A. & Rosteck, P. R., Jr ( 1999; ). Platenolide synthase gene. United States Patent 5,945,320.
  10. Butler, A. R., Bate, N. & Cundliffe, E. ( 1999; ). Impact of thioesterase activity on tylosin biosynthesis in Streptomyces fradiae. Chem Biol 6, 287–292.[CrossRef]
    [Google Scholar]
  11. Butler, A. R., Bate, N., Kiehl, D. E., Kirst, H. A. & Cundliffe, E. ( 2002; ). Genetic engineering of aminodeoxyhexose biosynthesis in Streptomyces fradiae. Nat Biotechnol 20, 713–716.[CrossRef]
    [Google Scholar]
  12. Champney, W. S. & Tober, C. L. ( 2000; ). Specific inhibition of 50S ribosomal subunit formation in Staphylococcus aureus cells by 16-membered macrolide, lincosamide, and streptogramin B antibiotics. Curr Microbiol 41, 126–135.[CrossRef]
    [Google Scholar]
  13. Chaveroche, M. K., Ghigo, J. M. & d'Enfert, C. ( 2000; ). A rapid method for efficient gene replacement in the filamentous fungus Aspergillus nidulans. Nucleic Acids Res 28, E97 [CrossRef]
    [Google Scholar]
  14. Choulet, F., Aigle, B., Gallois, A., Mangenot, S., Gerbaud, C., Truong, C., Francou, F.-X., Fourrier, C., Guérineau, M. & other authors ( 2006; ). Evolution of the terminal regions of the Streptomyces linear chromosome. Mol Biol Evol 23, 2361–2369.[CrossRef]
    [Google Scholar]
  15. Cong, L. & Piepersberg, W. ( 2007; ). Cloning and characterization of genes encoded in dTDP-d-mycaminose biosynthetic pathway from a midecamycin-producing strain, Streptomyces mycarofaciens. Acta Biochim Biophys Sin (Shanghai) 39, 187–193.[CrossRef]
    [Google Scholar]
  16. Cundliffe, E., Bate, N., Butler, A., Fish, S., Gandecha, A. & Merson-Davies, L. ( 2001; ). The tylosin-biosynthetic genes of Streptomyces fradiae. Antonie van Leeuwenhoek 79, 229–234.[CrossRef]
    [Google Scholar]
  17. Doumith, M., Weingarten, P., Wehmeier, U. F., Salah-Bey, K., Benhamou, B., Capdevila, C., Michel, J. M., Piepersberg, W. & Raynal, M. C. ( 2000; ). Analysis of genes involved in 6-deoxyhexose biosynthesis and transfer in Saccharopolyspora erythraea. Mol Gen Genet 264, 477–485.[CrossRef]
    [Google Scholar]
  18. Gaisser, S., Bohm, G. A., Cortes, J. & Leadlay, P. F. ( 1997; ). Analysis of seven genes from the eryAI-eryK region of the erythromycin biosynthetic gene cluster in Saccharopolyspora erythraea. Mol Gen Genet 256, 239–251.[CrossRef]
    [Google Scholar]
  19. Gaisser, S., Bohm, G. A., Doumith, M., Raynal, M. C., Dhillon, N., Cortes, J. & Leadlay, P. F. ( 1998; ). Analysis of eryBI, eryBIII and eryBVII from the erythromycin biosynthetic gene cluster in Saccharopolyspora erythraea. Mol Gen Genet 258, 78–88.[CrossRef]
    [Google Scholar]
  20. Gale, E. F., Cundliffe, E., Reynolds, P. E., Richmond, M. H. & Waring, J. M. ( 1981; ). The Molecular Basis of Antibiotic Action. New York: Wiley.
  21. Geistlich, M., Losick, R., Turner, J. R. & Rao, R. N. ( 1992; ). Characterization of a novel regulatory gene governing the expression of a polyketide synthase gene in Streptomyces ambofaciens. Mol Microbiol 6, 2019–2029.[CrossRef]
    [Google Scholar]
  22. Gourmelen, A., Blondelet-Rouault, M.-H. & Pernodet, J.-L. ( 1998; ). Characterization of a glycosyl transferase inactivating macrolides, encoded by gimA from Streptomyces ambofaciens. Antimicrob Agents Chemother 42, 2612–2619.
    [Google Scholar]
  23. Gust, B., Challis, G. L., Fowler, K., Kieser, T. & Chater, K. F. ( 2003; ). PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc Natl Acad Sci U S A 100, 1541–1546.[CrossRef]
    [Google Scholar]
  24. Hansen, J. L., Ippolito, J. A., Ban, N., Nissen, P., Moore, P. B. & Steitz, T. A. ( 2002; ). The structures of four macrolide antibiotics bound to the large ribosomal subunit. Mol Cell 10, 117–128.[CrossRef]
    [Google Scholar]
  25. Hara, O. & Hutchinson, C. R. ( 1992; ). A macrolide 3-O-acyltransferase gene from the midecamycin-producing species Streptomyces mycarofaciens. J Bacteriol 174, 5141–5144.
    [Google Scholar]
  26. Haydock, S. F., Appleyard, A. N., Mironenko, T., Lester, J., Scott, N. & Leadlay, P. F. ( 2005; ). Organization of the biosynthetic gene cluster for the macrolide concanamycin A in Streptomyces neyagawaensis ATCC 27449. Microbiology 151, 3161–3169.[CrossRef]
    [Google Scholar]
  27. Heathcote, M. L., Staunton, J. & Leadlay, P. F. ( 2001; ). Role of type II thioesterases: evidence for removal of short acyl chains produced by aberrant decarboxylation of chain extender units. Chem Biol 8, 207–220.[CrossRef]
    [Google Scholar]
  28. Hong, J. S., Park, S. J., Parajuli, N., Park, S. R., Koh, H. S., Jung, W. S., Choi, C. Y. & Yoon, Y. J. ( 2007; ). Functional analysis of DesVIII homologues involved in glycosylation of macrolide antibiotics by interspecies complementation. Gene 386, 123–130.[CrossRef]
    [Google Scholar]
  29. Hutchinson, C. R. & McDaniel, R. ( 2001; ). Combinatorial biosynthesis in microorganisms as a route to new antimicrobial, antitumor and neuroregenerative drugs. Curr Opin Investig Drugs 2, 1681–1690.
    [Google Scholar]
  30. Ikeda, H., Nonomiya, T., Usami, M., Ohta, T. & Omura, S. ( 1999; ). Organization of the biosynthetic gene cluster for the polyketide anthelmintic macrolide avermectin in Streptomyces avermitilis. Proc Natl Acad Sci U S A 96, 9509–9514.[CrossRef]
    [Google Scholar]
  31. Ishikawa, J. & Hotta, K. ( 1999; ). FramePlot: a new implementation of the frame analysis for predicting protein-coding regions in bacterial DNA with a high G+C content. FEMS Microbiol Lett 174, 251–253.[CrossRef]
    [Google Scholar]
  32. Kamra, P., Gokhale, R. S. & Mohanty, D. ( 2005; ). SEARCHGTr: a program for analysis of glycosyltransferases involved in glycosylation of secondary metabolites. Nucleic Acids Res 33, W220–W225
    [Google Scholar]
  33. Katz, L. ( 1997; ). Manipulation of modular polyketide synthases. Chem Rev 97, 2557–2576.[CrossRef]
    [Google Scholar]
  34. Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F. & Hopwood, D. A. ( 2000; ). Practical Streptomyces Genetics. Norwich: John Innes Foundation.
  35. Kuhstoss, S., Huber, M., Turner, J. R., Paschal, J. W. & Rao, R. N. ( 1996; ). Production of a novel polyketide through the construction of a hybrid polyketide synthase. Gene 183, 231–236.[CrossRef]
    [Google Scholar]
  36. Long, P. F., Wilkinson, C. J., Bisang, C. P., Cortes, J., Dunster, N., Oliynyk, M., McCormick, E., McArthur, H., Mendez, C. & other authors ( 2002; ). Engineering specificity of starter unit selection by the erythromycin-producing polyketide synthase. Mol Microbiol 43, 1215–1225.[CrossRef]
    [Google Scholar]
  37. Marchler-Bauer, A. & Bryant, S. H. ( 2004; ). CD-Search: protein domain annotations on the fly. Nucleic Acids Res 32, W327–W331
    [Google Scholar]
  38. McCabe, R. E. & Oster, S. ( 1989; ). Current recommendations and future prospects in the treatment of toxoplasmosis. Drugs 38, 973–987.[CrossRef]
    [Google Scholar]
  39. McDaniel, R., Welch, M. & Hutchinson, C. R. ( 2005; ). Genetic approaches to polyketide antibiotics. 1. Chem Rev 105, 543–558.[CrossRef]
    [Google Scholar]
  40. Melançon, C. E., III & Liu, H. W. ( 2007; ). Engineered biosynthesis of macrolide derivatives bearing the non-natural deoxysugars 4-epi-d-mycaminose and 3-N-monomethylamino-3-deoxy-d-fucose. J Am Chem Soc 129, 4896–4897.[CrossRef]
    [Google Scholar]
  41. Melançon, C. E., III, Takahashi, H. & Liu, H. W. ( 2004; ). Characterization of tylM3/tylM2 and mydC/mycB pairs required for efficient glycosyltransfer in macrolide antibiotic biosynthesis. J Am Chem Soc 126, 16726–16727.[CrossRef]
    [Google Scholar]
  42. Melançon, C. E., III, Yu, W. L. & Liu, H. W. ( 2005; ). TDP-mycaminose biosynthetic pathway revised and conversion of desosamine pathway to mycaminose pathway with one gene. J Am Chem Soc 127, 12240–12241.[CrossRef]
    [Google Scholar]
  43. Melançon, C. E., III, Hong, L., White, J. A., Liu, Y. N. & Liu, H. W. ( 2007; ). Characterization of TDP-4-keto-6-deoxy-d-glucose-3,4-ketoisomerase from the d-mycaminose biosynthetic pathway of Streptomyces fradiae: in vitro activity and substrate specificity studies. Biochemistry 46, 577–590.[CrossRef]
    [Google Scholar]
  44. Merson-Davies, L. A. & Cundliffe, E. ( 1994; ). Analysis of five tylosin biosynthetic genes from the tyllBA region of the Streptomyces fradiae genome. Mol Microbiol 13, 349–355.[CrossRef]
    [Google Scholar]
  45. Miller, V. L. & Mekalanos, J. J. ( 1988; ). A novel suicide vector and its use in construction of insertion mutations: osmoregulation of outer membrane proteins and virulence determinants in Vibrio cholerae requires toxR. J Bacteriol 170, 2575–2583.
    [Google Scholar]
  46. Nielsen, H., Engelbrecht, J., Brunak, S. & von Heijne, G. ( 1997; ). Identification of prokaryotic and eukaryotic signal peptides and prediction of their cleavage sites. Protein Eng 10, 1–6.[CrossRef]
    [Google Scholar]
  47. Omura, S., Kitao, C., Hamada, H. & Ikeda, H. ( 1979; ). Bioconversion and biosynthesis of 16-membered macrolide antibiotics. X. Final steps in the biosynthesis of spiramycin, using enzyme inhibitor: cerulenin. Chem Pharm Bull (Tokyo) 27, 176–182.[CrossRef]
    [Google Scholar]
  48. Omura, S., Sadakane, N., Tanaka, Y. & Matsubara, H. ( 1983; ). Chimeramycins: new macrolide antibiotics produced by hybrid biosynthesis. J Antibiot (Tokyo) 36, 927–930.[CrossRef]
    [Google Scholar]
  49. Pang, X., Aigle, B., Girardet, J. M., Mangenot, S., Pernodet, J.-L., Decaris, B. & Leblond, P. ( 2004; ). Functional angucycline-like antibiotic gene cluster in the terminal inverted repeats of the Streptomyces ambofaciens linear chromosome. Antimicrob Agents Chemother 48, 575–588.[CrossRef]
    [Google Scholar]
  50. Pernodet, J.-L., Alegre, M. T., Blondelet-Rouault, M.-H. & Guérineau, M. ( 1993; ). Resistance to spiramycin in Streptomyces ambofaciens, the producer organism, involves at least two different mechanisms. J Gen Microbiol 139, 1003–1011.[CrossRef]
    [Google Scholar]
  51. Pernodet, J.-L., Fish, S., Blondelet-Rouault, M.-H. & Cundliffe, E. ( 1996; ). The macrolide-lincosamide-streptogramin B resistance phenotypes characterized by using a specifically deleted, antibiotic-sensitive strain of Streptomyces lividans. Antimicrob Agents Chemother 40, 581–585.
    [Google Scholar]
  52. Pernodet, J.-L., Gourmelen, A., Blondelet-Rouault, M.-H. & Cundliffe, E. ( 1999; ). Dispensable ribosomal resistance to spiramycin conferred by srmA in the spiramycin producer Streptomyces ambofaciens. Microbiology 145, 2355–2364.
    [Google Scholar]
  53. Quiros, L. M., Aguirrezabalaga, I., Olano, C., Mendez, C. & Salas, J. A. ( 1998; ). Two glycosyltransferases and a glycosidase are involved in oleandomycin modification during its biosynthesis by Streptomyces antibioticus. Mol Microbiol 28, 1177–1185.[CrossRef]
    [Google Scholar]
  54. Rascher, A., Hu, Z., Viswanathan, N., Schirmer, A., Reid, R., Nierman, W. C., Lewis, M. & Hutchinson, C. R. ( 2003; ). Cloning and characterization of a gene cluster for geldanamycin production in Streptomyces hygroscopicus NRRL 3602. FEMS Microbiol Lett 218, 223–230.[CrossRef]
    [Google Scholar]
  55. Raynal, A., Karray, F., Tuphile, K., Darbon-Rongère, E. & Pernodet, J.-L. ( 2006; ). Excisable cassettes: new tools for functional analysis of Streptomyces genomes. Appl Environ Microbiol 72, 4839–4844.[CrossRef]
    [Google Scholar]
  56. Richardson, M. A., Kuhstoss, S., Solenberg, P., Schaus, N. A. & Rao, R. N. ( 1987; ). A new shuttle cosmid vector, pKC505, for streptomycetes: its use in the cloning of three different spiramycin-resistance genes from a Streptomyces ambofaciens library. Gene 61, 231–241.[CrossRef]
    [Google Scholar]
  57. Richardson, M. A., Kuhstoss, S., Huber, M. L., Ford, L., Godfrey, O., Turner, J. R. & Rao, R. N. ( 1990; ). Cloning of spiramycin biosynthetic genes and their use in constructing Streptomyces ambofaciens mutants defective in spiramycin biosynthesis. J Bacteriol 172, 3790–3798.
    [Google Scholar]
  58. Salzberg, S. L., Delcher, A. L., Kasif, S. & White, O. ( 1998; ). Microbial gene identification using interpolated Markov models. Nucleic Acids Res 26, 544–548.[CrossRef]
    [Google Scholar]
  59. Sambrook, J. & Russell, D. W. ( 2001; ). Molecular Cloning: a Laboratory Manual, 3rd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory.
  60. Schoner, B., Geistlich, M., Rosteck, P., Jr, Rao, R. N., Seno, E., Reynolds, P., Cox, K., Burgett, S. & Hershberger, C. ( 1992; ). Sequence similarity between macrolide-resistance determinants and ATP-binding transport proteins. Gene 115, 93–96.[CrossRef]
    [Google Scholar]
  61. Schönfeld, W. & Kirst, H. A. (editors) ( 2002; ). Macrolide Antibiotics. Basel: Birkhaüser Verlag.
  62. Simon, R., Priefer, U. & Pühler, A. ( 1983; ). A broad host range mobilization system for in vivo genetic engineering: transposon mutagenesis in gram negative bacteria. Biotechnology (N Y) 1, 784–791.[CrossRef]
    [Google Scholar]
  63. Smith, C. R. ( 1988; ). The spiramycin paradox. J Antimicrob Chemother 22 (Suppl. B), 141–144.
    [Google Scholar]
  64. Stratigopoulos, G., Bate, N. & Cundliffe, E. ( 2004; ). Positive control of tylosin biosynthesis: pivotal role of TylR. Mol Microbiol 54, 1326–1334.[CrossRef]
    [Google Scholar]
  65. Summers, R. G., Donadio, S., Staver, M. J., Wendt-Pienkowski, E., Hutchinson, C. R. & Katz, L. ( 1997; ). Sequencing and mutagenesis of genes from the erythromycin biosynthetic gene cluster of Saccharopolyspora erythraea that are involved in l-mycarose and d-desosamine production. Microbiology 143, 3251–3262.[CrossRef]
    [Google Scholar]
  66. Takahashi, H., Liu, Y.-N., Chen, H. & Liu, H.-W. ( 2005; ). Biosynthesis of TDP-l-mycarose: the specificity of a single enzyme governs the outcome of the pathway. J Am Chem Soc 127, 9340–9341.[CrossRef]
    [Google Scholar]
  67. Waldron, C., Matsushima, P., Rosteck, P. R., Jr, Broughton, M. C., Turner, J., Madduri, K., Crawford, K. P., Merlo, D. J. & Baltz, R. H. ( 2001; ). Cloning and analysis of the spinosad biosynthetic gene cluster of Saccharopolyspora spinosa. Chem Biol 8, 487–499.[CrossRef]
    [Google Scholar]
  68. Wu, K., Chung, L., Revill, W. P., Katz, L. & Reeves, C. D. ( 2000; ). The FK520 gene cluster of Streptomyces hygroscopicus var. ascomyceticus (ATCC 14891) contains genes for biosynthesis of unusual polyketide extender units. Gene 251, 81–90.[CrossRef]
    [Google Scholar]
  69. Yu, D., Ellis, H. M., Lee, E. C., Jenkins, N. A., Copeland, N. G. & Court, D. L. ( 2000; ). An efficient recombination system for chromosome engineering in Escherichia coli. Proc Natl Acad Sci U S A 97, 5978–5983.[CrossRef]
    [Google Scholar]
  70. Zhao, Z., Hong, L. & Liu, H. W. ( 2005; ). Characterization of protein encoded by spnR from the spinosyn gene cluster of Saccharopolyspora spinosa: mechanistic implications for forosamine biosynthesis. J Am Chem Soc 127, 7692–7693.[CrossRef]
    [Google Scholar]
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