Dihydrochalcomycin from Streptomyces sp. KCTC 0041BP is a 16-membered macrolide antibiotic containing two deoxysugars (d-chalcose and d-mycinose) that are O-glycosylated at the C-5 and C-20 positions, respectively. The desosamine sugar cassette was constructed from pikromycin-deoxysugar biosynthetic genes and transformed into Streptomyces sp. GerSM1, which was engineered for deletion of the genes related to TDP-d-chalcose biosynthesis (gerB, gerN and gerMI). Novel 16-membered macrolides (5-O-desosaminyl derivatives of dihydrochalcomycin) were detected by ESI-MS, LC/MS, and MS/MS thereby demonstrating combinatorial biosynthesis of the deoxysugar in 16-membered macrolide antibiotics.