Previous pathway engineering work demonstrated that the desosamine biosynthetic pathway in Streptomyces venezuelae could be converted to an efficient mycaminose biosynthesizing pathway by replacement of DesI with the hexose 3,4-ketoisomerase Tyl1a. In this work, FdtA, a ketoisomerase homologous to Tyl1a which catalyzes conversion of the Tyl1a substrate to the C-4 epimer of the Tyl1a product, was used to replace DesI. The ability of desosamine pathway enzymes DesV, DesVI, DesVII, and DesVIII to accept substrates with inverted C-4 stereochemistry in the mutant expressing FdtA resulted in formation of macrolide derivatives bearing 4-epi-D-mycaminose, a sugar heretofore unobserved in Nature. Interestingly, minor glycosylated macrolides bearing another non-natural sugar, 3-N-monomethylamino-3-deoxy-D-fucose, were also produced by this mutant. An explanation for the formation of these unexpected new compounds is presented, and the implications of this work for combinatorial biosynthesis of new antibiotics are discussed.