Cosmid 16F4 contains 25 kb of the elloramycin biosynthetic pathway of Streptomyces olivaceus Tu2353. Transformation of this cosmid into a polyketide synthase (PKS)-deleted mutant of the urdamycin producer, Streptomyces fradiae Tu2717/Delta PKS and into the mithramycin producer Streptomyces argillaceus ATCC 12956 resulted in the production of several novel glycosylated tetracenomycins. Four of the structures of these elloramycin analogues (3, 5-7) were elucidated. They carry various deoxysugar moieties (D-olivose, L-rhodinose, D-mycarose, and a disaccharide consisting of two 1,3-linked D-olivoses) attached at C-8-O of the same aglycon, 8-demethyltetracenomycin C (4), The transfer of the sugars is not catalyzed by glycosyltransferases of the S. fradiae or S. argillaceus strains since the novel hybrid tetracenomycins are also produced by a S. argillaceus mutant carrying cosmid 16F4 but lacking all the known mithramycin glycosyltransferases. Furthermore, a Streptomyces lividans strain containing cosmid 16F4 produced the novel tetracenomycins only when a second plasmid containing the cloned mithramycin sugar biosynthetic genes bur lacking glycosyltransferase genes was also present. The glycosyl transfer therefore must be catalyzed by an elloramycin glycosyltransferase encoded by cosmid 16F4. Apparently, this glycosyltransferase is able to catalyze the glycosylation of 8-demethyltetracenomycin C (4, = 12a-demethylelloramycinone) using various D- and L-sugars including a disaccharide. Its future use for combinatorial biosynthetic approaches is discussed.