TARGETED GENE INACTIVATION FOR THE ELUCIDATION OF DEOXYSUGAR BIOSYNTHESIS IN THE ERYTHROMYCIN PRODUCER SACCHAROPOLYSPORA-ERYTHRAEA

The production of erythromycin A by Saccharopolyspora erythraea requir es the synthesis of dTDP-D-desosamine and dTDP-L-mycarose, which serve as substrates for the transfer of the two sugar residues onto the mac rolactone ring. The enzymatic activities involved in this process are largely encoded within the ery gene cluster, by two sets of genes flan king the eryA locus that encodes the polyketide synthase. We report he re the nucleotide sequence of three such ORFs located immediately down stream of eryA, ORFs 7, 8 and 9. Chromosomal mutants carrying a deleti on either in ORF7 or in one of the previously sequenced ORFs 13 and 14 have been constructed and shown to accumulate erythronolide B, as exp ected for eryB mutants. Similarly, chromosomal mutants carrying a dele tion 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 13, as expected for eryC mutants. The ORF13 (eryBIV), O RF17 (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 p athways for the biosynthesis of dTDP-D-desosamine and dTDP-L-mycarose in Sac. erythraea and reveal that at least some of these enzymes can a ccommodate alternative substrates.