<jats:title>ABSTRACT</jats:title> <jats:p> Caprazamycins are antimycobacterials produced by <jats:italic>Streptomyces</jats:italic> sp. MK730-62F2. Previously, cosmid cpzLK09 was shown to direct the biosynthesis of caprazamycin aglycones, but not of intact caprazamycins. Sequence analysis of cpzLK09 identified 23 genes involved in the formation of the caprazamycin aglycones and the transfer and methylation of the sugar moiety, together with genes for resistance, transport, and regulation. In this study, coexpression of cpzLK09 in <jats:italic>Streptomyces coelicolor</jats:italic> M512 with pRHAM, containing all the required genes for dTDP- <jats:sc>l</jats:sc> -rhamnose biosynthesis, led to the production of intact caprazamycins. <jats:italic>In vitro</jats:italic> studies showed that Cpz31 is responsible for the attachment of the <jats:sc>l</jats:sc> -rhamnose to the caprazamycin aglycones, generating a rare acylated deoxyhexose. An <jats:sc>l</jats:sc> -rhamnose gene cluster was identified elsewhere on the <jats:italic>Streptomyces</jats:italic> sp. MK730-62F2 genome, and its involvement in caprazamycin formation was demonstrated by insertional inactivation of <jats:italic>cpzDIII</jats:italic> . The <jats:sc>l</jats:sc> -rhamnose subcluster was assembled with cpzLK09 using Red/ET-mediated recombination. Heterologous expression of the resulting cosmid, cpzEW07, led to the production of caprazamycins, demonstrating that both sets of genes are required for caprazamycin biosynthesis. Knockouts of <jats:italic>cpzDI</jats:italic> and <jats:italic>cpzDV</jats:italic> in the <jats:sc>l</jats:sc> -rhamnose subcluster confirmed that four genes, <jats:italic>cpzDII</jats:italic> , <jats:italic>cpzDIII</jats:italic> , <jats:italic>cpzDIV</jats:italic> , and <jats:italic>cpzDVI</jats:italic> , are sufficient for the biosynthesis of the deoxysugar moiety. The presented recombineering strategy may provide a useful tool for the assembly of biosynthetic building blocks for heterologous production of microbial compounds.