Dihydrochalcomycin (GERI-155), produced by Streptomyces sp. KCTC-0041BP isolated from Korean soil, is a 16-membered macrolide antibiotic consisting of two deoxysugar moieties at C-5 and C-20 positions of a branched lactone ring. The cloning and sequencing of a gene cluster for dihydrochalcomycin biosynthesis revealed a 63-kb nucleotide region containing 25 open reading frames (ORFs). The products of all of these 25 ORFs playa role in dihydrochalcomycin biosynthesis and self-resistance against the compounds synthesized. At the core of this cluster lies a 39.6-kb polyketide synthase (PKS) region encoding eight modules in five giant multifunctional protein-coding genes (gerSI-SV). The genes responsible for the biosynthesis of deoxysugar moieties, D-chalcose and D-mycinose, and their modification and attachment were found on either side of this PKS region. The involvement of this gene cluster in dihydrochalcomycin biosynthesis was confirmed by disruption of the dehydratase (DH) domain in module 3 of the PKS gene and by metabolite analysis.