Targeted gene inactivation for the elucidation of deoxysugar biosynthesis in the erythromycin producer Saccharopolyspora erythraea.
The production of erythromycin A by Saccharopolyspora erythraea 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-alpha-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.
Salah-Bey
K
K
Infectious Disease Group, Hoechst Marion Roussel, Romainville, France.
Doumith
M
M
Michel
J M
JM
Haydock
S
S
Cortés
J
J
Leadlay
P F
PF
Raynal
M C
MC
D000595:Amino Acid Sequence; D000606:Amino Sugars / Q000378:metabolism; D001426:Bacterial Proteins; D003001:Cloning, Molecular; D004917:Erythromycin; D016695:Glycosyltransferases / Q000235:genetics*; D006601:Hexoses / Q000378:metabolism; D019746:Intramolecular Oxidoreductases / Q000235:genetics*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D005810:Multigene Family; D016296:Mutagenesis; D016366:Open Reading Frames; D010088:Oxidoreductases / Q000235:genetics*; D016930:Saccharopolyspora / Q000235:genetics*; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid
D000606:Amino Sugars; D001426:Bacterial Proteins; D006601:Hexoses; D009097:Multienzyme Complexes; C045835:mycarose; C110125:desosamine; D004917:Erythromycin; D010088:Oxidoreductases; C111820:eryBII protein, Saccharopolyspora erythraea; D016695:Glycosyltransferases; C111822:EryBV protein, Saccharopolyspora erythraea; C111821:EryCIII protein, Saccharopolyspora erythraea; D019746:Intramolecular Oxidoreductases; C111819:dTDP-4-keto-6-deoxyhexose 3,4-isomerase