<jats:title>ABSTRACT</jats:title> <jats:p> <jats:named-content content-type="genus-species">Streptomyces leeuwenhoekii</jats:named-content> , isolated from the hyperarid Atacama Desert, produces the new ansamycin-like compounds chaxamycins A to D, which possess potent antibacterial activity and moderate antiproliferative activity. We report the development of genetic tools to manipulate <jats:named-content content-type="genus-species">S. leeuwenhoekii</jats:named-content> and the identification and partial characterization of the 80.2-kb chaxamycin biosynthesis gene cluster, which was achieved by both mutational analysis in the natural producer and heterologous expression in <jats:named-content content-type="genus-species">Streptomyces coelicolor</jats:named-content> A3(2) strain M1152. Restoration of chaxamycin production in a nonproducing Δ <jats:italic>cxmK</jats:italic> mutant ( <jats:italic>cxmK</jats:italic> encodes 3-amino-5-hydroxybenzoic acid [AHBA] synthase) was achieved by supplementing the growth medium with AHBA, suggesting that mutasynthesis may be a viable approach for the generation of novel chaxamycin derivatives.