The macrolide antibiotic concanamycin A has been identified as an exceptionally potent inhibitor of the vacuolar (V-type) ATPase. Such compounds have been mooted as the basis of a potential drug treatment for osteoporosis, since the V-ATPase is involved in the osteoclast-mediated bone resorption that underlies this common condition. To enable combinatorial engineering of altered concanamycins, the biosynthetic gene cluster governing the biosynthesis of concanamycin A has been cloned from Streptomyces neyagawaensis and shown to span a region of over 100 kbp of contiguous DNA. An efficient transformation system has been developed for S. neyagawaensis and used to demonstrate the role of the cloned locus in the formation of concanamycin A. Sequence analysis of the 28 ORFs in the region has revealed key features of the biosynthetic pathway, in particular the biosynthetic origin of portions of the backbone, which arise from the unusual polyketide building blocks ethylmalonyl-CoA and methoxymalonyl-ACP, and the origin of the pendant deoxysugar moiety 49-O-carbamoyl-29-deoxyrhamnose, as well as the presence of a modular polyketide synthase (PKS) encoded by six giant ORFs. Examination of the methoxymalonyl-specific acyltransferase (AT) domains has led to recognition of an amino acid sequence motif which can be used to distinguish methylmalonyl-CoA-from methoxymalonyl-ACP-specific AT domains in natural PKSs. INTRODUCTIONPolyketide macrolides constitute a large family of bioactive natural products, including many compounds with important clinical applications in both human and veterinary medicine. Concanamycin A (folimycin) is an unusual macrolide, first isolated from Streptomyces diastatochromogenes S-45 (Kinashi et al., 1984), whose absolute configuration was established as shown in Fig. 1 (Westley et al., 1984). Concanamycin A and closely related compounds (concanamycins B-F) are characterized by an 18-membered tetraenic macrolide ring incorporating a methyl enol ether, and by a b-hydroxyhemiacetal side chain. They are closely related to the 16-membered macrolides called bafilomycins (Goetz et al., 1985) and also share structural features with the antifungal macrodiolide, elaiophylin A (Arcamone et al., 1959) whose biosynthetic pathway we recently elucidated (Haydock et al., 2004). These unusual macrolides have been dubbed the 'plecomacrolides' (from the Greek word plectos, describing their unusually folded side chain) (Bindseil & Zeeck, 1994).The concanamycins exhibit a wide range of important biological activities: antiviral (Omura et al., 1983), antiprotozoal (Omura et al., 1983 and antineoplastic (SekiAsano et al., 1994). These activities are thought to be due, at least in part, to their potent inhibition of the vacuolar (Vtype) H + -ATPase (Muroi et al., 1993) mediated by their binding to the membrane-spanning V 0 component of the ATPase (Drose et al., 2001). Interest in these compounds has increased recently with the recognition of the importance of the V-type ATPase in osteoclast-mediated resorption of bone (Lait...