Carbohydrate moieties are important components of natural products, which are often imperative for the solubility and biological activity of the compounds. The aromatic polyketide alnumycin A contains an extraordinary sugar-like 4′-hydroxy-5′-hydroxymethyl-2′,7′-dioxane moiety attached via a carbon-carbon bond to the aglycone. Here we have extensively investigated the biosynthesis of the dioxane unit through 13 C labeling studies, gene inactivation experiments and enzymatic synthesis. We show that AlnA and AlnB, members of the pseudouridine glycosidase and haloacid dehalogenase enzyme families, respectively, catalyze C-ribosylation conceivably through Michael-type addition of D-ribose-5-phosphate and dephosphorylation. The ribose moiety may be attached both in furanose (alnumycin C) and pyranose (alnumycin D) forms. The C 1 0 -C 2 0 bond of alnumycin C is subsequently cleaved and the ribose unit is rearranged into an unprecedented dioxolane (cisbicyclo[3.3.0]-2′,4′,6′-trioxaoctan-3′β-ol) structure present in alnumycin B. The reaction is catalyzed by Aln6, which belongs to a previously uncharacterized enzyme family. The conversion was accompanied with consumption of O 2 and formation of H 2 O 2 , which allowed us to propose that the reaction may proceed via hydroxylation of C1′ followed by retro-aldol cleavage and acetal formation. Interestingly, no cofactors could be detected and the reaction was also conducted in the presence of metal chelating agents. The last step is the conversion of alnumycin B into the final end-product alnumycin A catalyzed by Aln4, an NADPH-dependent aldo-keto reductase. This characterization of the dioxane biosynthetic pathway sets the basis for the utilization of C-C bound ribose, dioxolane and dioxane moieties in the generation of improved biologically active compounds.ribose-5-phosphate | natural product | biosynthesis | Streptomyces