Using an acyl-acyl carrier protein (ACP) as a starter unit, type Ⅱ polyketide synthases (PKSs) generate a wide range of polyketide products by successive decarboxylative condensation with the two-carkon donor malonyl (ACP). In vitro experimetts have demonstrated that polketide biosynthesis in reconstituted PKS systems requires the fatty acid synthase (FAS) enzyme malonyl CoA: ACP acyltransferase (FabD) from streptomycets. It has also been shown that holo-ACPs from a typeII PKS can catalyze self -malonylation in the presence of malonyl CoA and negate this FabD requirement. The relative roles of FabD an ACP self -malonylatin in PKS biosynthesis in vivo are still not known. Results: We have examined the ACO specificity of the Streptomyces glaucescens FabD and shown that it reacts specifically with monomeric forms of ACP, with comparable kcat IKM values for ACPs from both type II PJS and FAS systems. Incubations of tetracenomycin ACP(TcmM) withthe Escherichia coli FAS ACP (Acp P) unexpectedly revaled that, in addition to the selfmalonylation process, TcmM can catalyze the malonylation of AcpP. The ACP is two value for the TcmM-catalyzed malonylation of S. glaucescens FAS ACP is two orders of magnitude smaller than that observed for the Fabd-CAtalyzed process. Conclusion: The abilityof APKS ACP to catalyze malonylation of a FAS ACP is a surprising finding and demonstrates for the first time that PKS ACP and FabD can catalyze the same reation. The differences in the acatalytic efficiency of these two proteins rationalizes in vitro observations that FabD-independent polyketide biosynthesis proceeds only at high concentrations of a PKS ACP.