Interspecies complementation in Saccharopolyspora erythraea: elucidation of the function of oleP1, oleG1 and oleG2 from the oleandomycin biosyntheticgene cluster of Streptomyces antibioticus and generation of new erythromycin derivatives

Two glycosyltransferase genes, oleG1 and oleG2, and a putative isomerase ge ne, oleP1, have previously been identified in the oleandomycin biosynthetic gene cluster of Streptomyces antibioticus. In order to identify which of t hese two glycosyltransferases encodes the desosaminyltransferase and which the oleandrosyltransferase, interspecies complementation has been carried o ut, using two mutant strains of Saccharopolyspora erythraea, one strain car rying an internal deletion in the eryCIII (desosaminyltransferase) gene and the other an internal deletion in the eryBV (mycarosyltransferase) gene. E xpression of the oleG1 gene in the eryCIII deletion mutant restored the pro duction of erythromycin A (although at a low level), demonstrating that ole G1 encodes the desosaminyltransferase required for the biosynthesis of olea ndomycin and indicating that, as in erythromycin biosynthesis, the neutral sugar is transferred before the aminosugar onto the macrocyclic ring. Signi ficantly, when an intact oleG2 gene (presumed to encode the oleandrosyltran sferase) was expressed in the eryBV deletion mutant, antibiotic activity wa s also restored and, in addition to erythromycin A, new bioactive compounds were produced with a good yield. The neutral sugar residue present in thes e compounds was identified as L-rhamnose attached at position C-3 of an ery thronolide B or a 6-deoxyerythronolide B lactone ring, thus indicating a re laxed specificity of the oleandrosyltransferase, OleG2, for both the activa ted sugar and the macrolactone substrate. The oleP1 gene located immediatel y upstream of oleG1 was likewise introduced into an eryCII deletion mutant of Sac. erythraea, and production of erythromycin A was again restored, dem onstrating that the function of OleP1 is identical to that of EryCII in the biosynthesis of dTDP-D-desosamine, which we have previously proposed to be a dTDP-4-keto-6-deoxy-D-glucose 3,4-isomerase.