Ravidomycin (RM) is one of the most active benzo[d]naptho[1,2-b]pyran-6-one anticancer antibiotics. A C-glycosidically linked aminosugar (4’-O-acetyl-D-ravidosamine) is a unique feature of this antibiotic. With an aim to generate RM analogues through combinatorial biosynthesis, the genomic library of S. ravidus was constructed in the pOJ446 cosmid vector. Screening of the cosmid library with an internal sequence of NDP-glucose 4,6-dehydratase and β-ketoacyl synthase probes yielded 3 different cosmids. Sequencing of one of the cosmids namely cosRav32 revealed a cluster of 30 open reading frames (ORFs): 2 regulatory genes, 23 RM biosynthetic genes and 5 other genes of unknown function. Heterologous expression of cosRav32 in Streptomyces lividans TK24 resulted in the production of 4’-O-deacetyl ravidomycin E, prejadomycin, homo-2,3-dehydroUWM6 and pregilvocarcin-o-quinone. The results clearly indicated that the cosRav32 covers most of the RM gene cluster. However, the absence of RM could be due to the lack of RM resistance enzymes in S. lividans TK24. In addition, TDP-D-ravidosamine was synthesized enzymatically in vitro through the activities of RavD (TDP-glucose-synthase), RavE (TDP-glucose 4,6-dehydratase), RavIM (TDP-4-keto-6-deoxy-D-glucose-3,4-ketoisomerase), RavAMT (TDP-3-keto-6-deoxy-D-galactose-3-aminotransferase) and RavNMT (TDP-3-amino-3,6-dideoxy-D-galactose-N,N-dimethyltransferase). Isolation and characterization of the RM gene cluster paves the way for the generation of its analogues through combinatorial biosynthesis.