Derivatives of Escherichia coli C were previously described for succinate production by combining the deletion of genes that disrupt fermentation pathways for alternative products (ldhA::FRT, adhE::FRT, ackA::FRT, focA-pflB::FRT, mgsA, poxB) with growth-based selection for increased ATP production. The resulting strain, KJ073, produced 1.2 mol of succinate per mol glucose in mineral salts medium with acetate, malate, and pyruvate as significant co-products. KJ073 has been further improved by removing residual recombinase sites (FRT sites) from the chromosomal regions of gene deletion to create a strain devoid of foreign DNA, strain KJ091(ΔldhA ΔadhE ΔackA ΔfocA-pflB ΔmgsA ΔpoxB). KJ091 was further engineered for improvements in succinate production. Deletion of the threonine decarboxylase (tdcD; acetate kinase homologue) and 2-ketobutyrate formate-lyase (tdcE; pyruvate formate-lyase homologue) reduced the acetate level by 50% and increased succinate yield (1.3 mol mol−1 glucose) by almost 10% as compared to KJ091 and KJ073. Deletion of two genes involved in oxaloacetate metabolism, aspartate aminotransferase (aspC) and the NAD+-linked malic enzyme (sfcA) (KJ122) significantly increased succinate yield (1.5 mol mol−1 glucose), succinate titer (700 mM), and average volumetric productivity (0.9 g L−1 h−1). Residual pyruvate and acetate were substantially reduced by further deletion of pta encoding phosphotransacetylase to produce KJ134 (ΔldhA ΔadhE ΔfocA-pflB ΔmgsA ΔpoxB ΔtdcDE ΔcitF ΔaspC ΔsfcA Δpta-ackA). Strains KJ122 and KJ134 produced near theoretical yields of succinate during simple, anaerobic, batch fermentations using mineral salts medium. Both may be useful as biocatalysts for the commercial production of succinate. Biotechnol. Bioeng. © 2008 Wiley Periodicals, Inc.