A plasmid (pLN2) was generated in which genes involved in the biosynthesis of L-oleandrose in the oleandomycin producer Streptomyces antibioticus ATCC11891 were cloned. pLN2 was used to direct the biosynthesis of different deoxysugars by exchanging and/or adding genes from other antibiotic biosynthetic clusters. Transfer of the synthesized deoxysugars to the tetracenomycin C aglycon, 8-demethyl-tetracenomycin C, through the use of the "sugar flexible" glycosyltransferase ElmGT, validated the system. Several pLN2 derivatives were constructed by replacement of the oleU 4-ketoreductase gene by different 4-ketoreductase genes. Some of them, such as EryBIV and UrdR, reduced the keto group of the 4-keto intermediates, generating L-olivosyl and D-olivosyl derivatives, respectively. The system was also used to generate an L-rhamnosyl derivative (through a two-gene deletion) and an L-rhodinosyl derivative (through a combination of a gene replacement and a gene addition).

A 9.8-kb DNA region from the oleandomycin gene cluster in Streptomyces antibioticus was cloned. Sequence analysis revealed the presence of 8 open reading frames encoding different enzyme activities involved in the biosynthesis of one of the two 2,6-deoxysugars attached to the oleandomycin aglycone: L-oleandrose (the oleW, oleV, oleL, and oleU genes) and D-desosamine (the oleNI and oleT genes), or of both (the oleS and oleE genes). A Streptomyces albus strain harboring the oleG2 glycosyltransferase gene integrated into the chromosome was constructed. This strain was transformed with two different plasmid constructs (pOLV and pOLE) containing a set of genes proposed to be required for the biosynthesis of dTDP-L-olivose and dTDP-L-oleandrose, respectively. Incubation of these recombinant strains with the erythromycin aglycon (erythronolide B) gave rise to two new glycosylated compounds, identified as L-3-O-olivosyl-and L-3-O-oleandrosyl-erythronolide B, indicating that pOLV and pOLE encode all enzyme activities required for the biosynthesis of these two 2,6-dideoxysugars. A pathway is proposed for the biosynthesis of these two deoxysugars in S. antibioticus.