Targeted gene inactivation for the elucidation of deoxysugar biosynthesis in the erythromycin producer Saccharopolyspora erythraea |
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Authors: | K Salah-Bey M Doumith J-M Michel S Haydock J Cortés P F Leadlay and M-C Raynal |
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Institution: | (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.com, FR;(2) University of Cambridge, Department of Biochemistry, Tennis Court Road, Cambridge, CB2 1QW, UK, GB |
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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.
Received: 29 July 1997 / Accepted: 16 October 1997 |
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Keywords: | Saccharopolyspora erythraea Erythromycin biosynthetic genes Desosamine Mycarose Glycosyltransferase |
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