Corynebacterium glutamicum was engineered for the production of l -valine from glucose by deletion of the aceE gene encoding the E1p enzyme of the pyruvate dehydrogenase complex and additional overexpression of the ilvBNCE genes encoding the l -valine biosynthetic enzymes acetohydroxyacid synthase, isomeroreductase, and transaminase B. In the absence of cellular growth, C. glutamicum Δ aceE showed a relatively high intracellular concentration of pyruvate (25.9 mM) and produced significant amounts of pyruvate, l -alanine, and l -valine from glucose as the sole carbon source. Lactate or acetate was not formed. Plasmid-bound overexpression of ilvBNCE in C. glutamicum Δ aceE resulted in an approximately 10-fold-lower intracellular pyruvate concentration (2.3 mM) and a shift of the extracellular product pattern from pyruvate and l -alanine towards l -valine. In fed-batch fermentations at high cell densities and an excess of glucose, C. glutamicum Δ aceE (pJC4ilvBNCE) produced up to 210 mM l -valine with a volumetric productivity of 10.0 mM h ⁻¹ (1.17 g l ⁻¹ h ⁻¹ ) and a maximum yield of about 0.6 mol per mol (0.4 g per g) of glucose.

Metabolic cofactors such as NADH and ATP play important roles in a large number of cellular reactions, and it is of great interest to dissect the role of these cofactors in different aspects of metabolism. Toward this goal, we overexpressed NADH oxidase and the soluble F1-ATPase in Escherichia coli to lower the level of NADH and ATP, respectively. We used a global interaction network, comprising of protein interactions, transcriptional regulation, and metabolic networks, to integrate data from transcription profiles, metabolic fluxes, and the metabolite levels. We identified high-scoring networks for the two strains. The results revealed a smaller, but denser network for perturbations of ATP level, compared with that of NADH level. The action of many global transcription factors such as ArcA, Fnr, CRP, and IHF commonly involved both NADH and ATP, whereas others responded to either ATP or NADH. Overexpressing NADH oxidase invokes response in widespread aspects of metabolism involving the redox cofactors (NADH and NADPH), whereas ATPase has a more focused response to restore ATP level by enhancing proton translocation mechanisms and repressing biosynthesis. Interestingly, NADPH played a key role in restoring redox homeostasis through the concerted activity of isocitrate dehydrogenase and UdhA transhydrogenase. We present a reconciled network of regulation that illustrates the overlapping and distinct aspects of metabolism controlled by NADH and ATP. Our study contributes to the general understanding of redox and energy metabolism and should help in developing metabolic engineering strategies in E. coli.