A mutant strain of Streptomyces venezuelae was engineered by deletion of the entire gene cluster related to biosynthesis of the endogenous deoxysugar (TDP-D-desosamine) and replacement with genes required for biosynthesis of an intermediate sugar (TDP-4-keto-6-deoxy-D-glucose) or an exogenous sugar (TDP-D-olivose), from the oleandomycin and urdamycin deoxysugar pathways. The 'sugar-flexible' glycosyltransferase (DesVII) was able to attach the intermediate sugar and the new sugar to both 12- and 14-membered macrolactones thus producing quinovose or olivose glycosylated 10-deoxymethynolide and narbonolide, respectively. In addition, hydroxylated analogs of the new metabolites were detected. These results demonstrate a successful attempt of engineering the deoxysugar pathway for generation of novel hybrid macrolide antibiotics.