Polyketides represent a significant fraction of all natural products. Many possess pharmacological activity which makes them attractive drug candidates. The production of the parent macrocyclic aglycones is catalyzed by multi-modular polyketide synthases utilizing short-chain acyl-CoA monomers. When producing polyketides through heterologous hosts, one must not only functionally express the synthase itself, but activate the machinery used to generate the required substrate acyl-CoA's. As a result, metabolic engineering of these pathways is necessary for high-level production of heterologous polyketides. In this study, we over-express three different pathways for provision of the two substrates (propionyl-CoA and (2S)-methylmalonyl-CoA) utilized for the biosynthesis of 6-deoxyerythronolide B (6-dEB; the macrolactone precursor of erythromycin): (1) a propionate -> propionyl-CoA -> (2S)-methylmalonyl-CoA pathway, (2) a methylmalonate -> methylmalonyl-CoA -> propionyl-CoA pathway, and (3) a succinate -> succinyl-CoA -> (2R)-methylmalonyl-CoA -> (2S)-methylmalonyl-CoA -> propionyl-CoA pathway. The current study revealed that propionate is a necessary component for greater than 5mg L-1 titers. Deletion of the propionyl-CoA:succinate CoA transferase (ygfH) or over-expression of the transcriptional activator of short chain fatty acid uptake improved titer to over 100 mg L-1, while the combination of the two improved titer to over 130 mg L-1. The addition of exogenous methylmalonate could also improve titer to over 100 mg L-1. Expression of a Streptomyces coelicolor A3(2) methylmalonyl-CoA epimerase, in conjunction with over-expression of Escherichia coli's native methylmalonyl-CoA mutase, allowed for the incorporation of exogenously fed succinate into the 6-dEB core. Biotechnol. Bioeng. 2011; 108: 1360-1371. (C) 2011 Wiley Periodicals, Inc.