New erythromycin derivatives from Saccharopolyspora erythraea using sugar O-methyltransferases from the spinosyn biosynthetic gene cluster

Using a previously developed expression system based on the erythromycin-pr oducing strain of Saccharopolyspora erythraea, O-methyltransferases from th e spinosyn biosynthetic gene cluster of Saccharopolyspora spinosa have been shown to modify a rhamnosyl sugar attached to a 14-membered polyketide mac rolactone. The spnI, spnK and spnH methyltransferase genes were expressed i ndividually in the S. erythraea mutant SGT2, which is blocked both in endog enous macrolide biosynthesis and in ery glycosyltransferases eryBV and eryC III. Exogenous 3-O-rhamnosyl-erythronolide B was efficiently converted into 3-O-(2'-O-methylrhamnosyl)-erythronolide B by the S. erythraea SGT2 (spnI) strain only. When 3-O-(2'-O-methylrhamnosyl)-erythronolide IS was, in turn , fed to a culture of S. erythraea SGT2 (spnK), 3-O-(2',3'-bis-O-methylrham nosyl)-erythronolide B was identified in the culture supernatant, whereas S . erythraea SGT2 (spnH) was without effect. These results confirm the ident ity of the 2'- and 3'-O-methyltransferases, and the specific sequence in wh ich they act, and they demonstrate that these methyltransferases may be use d to methylate rhamnose units in other polyketide natural products with the same specificity as in the spinosyn pathway. In contrast, 3-O-(2',3'-bis-O -methylrhamnosyl)-erythronolide IS was found not to be a substrate for the 4'-O-methyltransferase SpnH. Although rhamnosyl-erythromycins did not serve directly as substrates for the spinosyn methyltransferases, methylrhamnosy l-erythromycins were obtained by subsequent conversion of the corresponding methylrhamnosyl-erythronolide precursors using the S. erythraea strain SGT 2 housing EryCIII, the desosaminyltransferase of the erythromycin pathway. 3-O-(2'-O-methylrhamnosyl)-erythromycin D was tested and found to be signif icantly active against a strain of erythromycin-sensitive Bacillus subtilis .