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Purpose:To investigate strategies for the generation of analogues of the anticancer antibiotic gilvocarcin V based on knowledge of the biosynthetic pathway and its enzymes.Methods:Three pathways of gilvocarcin-type anticancer drugs were cloned and sequenced,using PKS-2 (KS) and deoxysugar pathway (4,6-dehydratase) gene probes.[1] Enzymes of the polyketide-derived moieties and the deoxysugar pathways were overexpressed as His6-tagged proteins and purified with Ni-affinity chromatography using standard E,coli overexpression vectors,such as pET2ga.RedirectR-PCR targeting based gene inactivation and heterologously expressed plasmids containing deoxy-or deoxyaminosugar pathways were used for analogue generation.