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Molecular and General Genetics MGG

, Volume 257, Issue 5, pp 542–553 | Cite as

Targeted gene inactivation for the elucidation of deoxysugar biosynthesis in the erythromycin producer Saccharopolyspora erythraea

  • K. Salah-Bey
  • M. Doumith
  • J.-M. Michel
  • S. Haydock
  • J. Cortés
  • P. F. Leadlay
  • M.-C. Raynal
ORIGINAL PAPER

Abstract

The production of erythromycin A by Saccharopolysporaerythraea requires the synthesis of dTDP-D-desosamine and dTDP-L-mycarose, which serve as substrates for the transfer of the two sugar residues onto the macrolactone ring. The enzymatic activities involved in this process are largely encoded within the ery gene cluster, by two sets of genes flanking the eryA locus that encodes the polyketide synthase. We report here the nucleotide sequence of three such ORFs located immediately downstream of eryA, ORFs 7, 8 and 9. Chromosomal mutants carrying a deletion either in ORF7 or in one of the previously sequenced ORFs 13 and 14 have been constructed and shown to accumulate erythronolide B, as expected for eryB mutants. Similarly, chromosomal mutants carrying a deletion in either ORF8, ORF9, or one of the previously sequenced ORFs 17 and 18 have been constructed and shown to accumulate 3-α-mycarosyl erythronolide B, as expected for eryC mutants. The ORF13 (eryBIV ), ORF17 (eryCIV ) and ORF7 (eryBII ) mutants also synthesised small amounts of macrolide shunt metabolites, as shown by mass spectrometry. These results considerably strengthen previous tentative proposals for the pathways for the biosynthesis of dTDP-D-desosamine and dTDP-L-mycarose in Sac. erythraea and reveal that at least some of these enzymes can accommodate alternative substrates.

Key wordsSaccharopolyspora erythraea Erythromycin biosynthetic genes Desosamine Mycarose Glycosyltransferase 

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Copyright information

© Springer-Verlag Berlin Heidelberg 1998

Authors and Affiliations

  • K. Salah-Bey
    • 1
  • M. Doumith
    • 1
  • J.-M. Michel
    • 1
  • S. Haydock
    • 2
  • J. Cortés
    • 2
  • P. F. Leadlay
    • 2
  • M.-C. Raynal
    • 1
  1. 1.Infectious Disease Group, Hoechst Marion Roussel, 102 Route de Noisy, 93235 Romainville Cedex, France Fax: +1-33-1-49-91-52-57; e-mail: marie-cecile.raynal@hmrag.comFR
  2. 2.University of Cambridge, Department of Biochemistry, Tennis Court Road, Cambridge, CB2 1QW, UKGB

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