<jats:title>ABSTRACT</jats:title><jats:p>Doxorubicin, one of the most widely used anticancer drugs, is composed of a tetracyclic polyketide aglycone and<jats:sc>l</jats:sc>-daunosamine as a deoxysugar moiety, which acts as an important determinant of its biological activity. This is exemplified by the fewer side effects of semisynthetic epirubicin (4′-<jats:italic>epi</jats:italic>-doxorubicin). An efficient combinatorial biosynthetic system that can convert the exogenous aglycone ε-rhodomycinone into diverse glycosylated derivatives of doxorubicin or its biosynthetic intermediates, rhodomycin D and daunorubicin, was developed through the use of<jats:named-content content-type="genus-species">Streptomyces venezuelae</jats:named-content>mutants carrying plasmids that direct the biosynthesis of different nucleotide deoxysugars and their transfer onto aglycone, as well as the postglycosylation modifications. This system improved epirubicin production from ε-rhodomycinone by selecting a substrate flexible glycosyltransferase, AknS, which was able to transfer the unnatural sugar donors and a TDP-4-ketohexose reductase, AvrE, which efficiently supported the biosynthesis of TDP-4-<jats:italic>epi</jats:italic>-<jats:sc>l</jats:sc>-daunosamine. Furthermore, a range of doxorubicin analogs containing diverse deoxysugar moieties, seven of which are novel rhodomycin D derivatives, were generated. This provides new insights into the functions of deoxysugar biosynthetic enzymes and demonstrates the potential of the<jats:named-content content-type="genus-species">S. venezuelae-</jats:named-content>based combinatorial biosynthetic system as a simple biological tool for modifying structurally complex sugar moieties attached to anthracyclines as an alternative to chemical syntheses for improving anticancer agents.