Streptomyces coelicolor produces fatty acids for both primary metabolism and for production of the components of natural products such as undecylprodiginine. Primary metabolism makes the longer and predominantly branched-chain fatty acids, while undecylprodiginine utilizes shorter and almost exclusively straight chain fatty acids. The first step in fatty acid biosynthetic process is catalyzed by FabH (β-ketoacyl synthase III), which catalyzes a decarboxylative condensation of an acyl-CoA primer with malonyl-acyl carrier protein (ACP). The resulting 3-ketoacyl-ACP product is reduced by NADPH- Malonyl-FabC is not a substrate for RedP, indicating that ACP specificity is one of the factors that permit a separation between prodiginine and fatty acid biosynthetic processes. In contrast to RedP, FabH was active with all pairings but demonstrated the greatest catalytic efficiency with isobutyryl-CoA using malonylFabC. Lower catalytic efficiency was observed using an acetyl-CoA and malonyliii FabC pairing consistent with the observation that in streptomycetes, a broad mixture of fatty acids are biosynthesized, with those derived from branched chain acyl-CoA starter units predominating. Diminished but demonstrable FabH activity was also observed using malonyl-RedQ, with the same preference for isobutyrylCoA over acetyl-CoA, completing biochemical and genetic evidence that in the absence of RedP this enzyme can also play a role in prodiginine biosynthesis, producing branched alkyl chain prodiginines.The identification and characterization of both enzymes FabG and FabI was also carried out. A series of straight and branched-chain β-ketoacyl and enoyl substrates tethered to either NAC or ACP were synthesized and used to elucidate the functional role and substrate specificity of these enzymes. Kinetic analysis demonstrates that of the three S. coelicolor enzymes, SCO1815 and SCO1345 have NADPH-dependent β-ketoacyl-reductase activity, in contrast to SCO1346, which has NADH-dependent β-ketoacyl-reductase activity. Spectrophotometric assays revealed that all three FabGs are capable of utilizing both straight and branched-chain β-ketoacyl-NAC substrates. These results are consistent with FabGs role in fatty acid and undecylprodiginine biosynthesis, wherein it processes branched-chain for primary metabolism as well as straight-chain products for undecylprodiginine biosynthesis. LC/MS assays demonstrate that these FabG enzymes do not discriminate between primary metabolism ACP (FabC) and secondary metabolism ACP (RedQ) (except for SCO1345, which does not have any activity with RedQ). This relaxed substrate specificity allows these enzymes to process 3-ketoacyl-FabC substrates for fatty acid biosynthesis as well as 3-