Basfia succiniciproducens has been recently isolated as novel producer for succinate, an important platform chemical. In batch culture, the wild type exhibited a high natural yield of 0.75mol succinate (molglucose)(-1). Systems-wide C-13 metabolic flux analysis identified undesired fluxes through pyruvate-formate lyase (PflD) and lactate dehydrogenase (LdhA). The double deletion strain B. succiniciproducens ldhA pflD revealed a 45% improved product yield of 1.08molmol(-1). In addition, metabolic flux analysis unraveled the parallel in vivo activity of the oxidative and reductive branch of the TCA cycle in B. succiniciproducens, whereby the oxidative part mainly served for anabolism. The wild type re-directed surplus NADH via a cycle involving malic enzyme or via transhydrogenase, respectively, to supply NADPH for anabolism, because the fluxes through the oxidative PPP and isocitrate dehydrogenase, that also provide this cofactor, were not sufficient. This was not observed for the deletion mutants, B. succiniciproducens pflD and ldhA pflD, where PPP and isocitrate dehydrogenase flux alone matched with the reduced anabolic NADPH demand. The integration of the production performance into the theoretical flux space, computed by elementary flux mode analysis, revealed that B. succiniciproducens ldhA pflD reached 62% of the theoretical maximum yield. Biotechnol. Bioeng. 2013;110: 3013-3023. (c) 2013 Wiley Periodicals, Inc.