Metabolic consequences of altered phosphoenolpyruvate carboxykinase activity in Corynebacterium glutamicum reveal anaplerotic regulation mechanisms in vivo

Corynebacterium glutamicum possesses high in viva activity of the gluconeogenic phosphoenolpyruvate carboxykinase (PEPCk) during growth on glucose, resulting together with anaplerotic carboxylation reactions in a PEP/pyruvate/oxaloacetate substrate cycle. The present study investigated the changes in intracellular fluxes and metabolite concentrations that are caused by altered PEPCk activity in L-lysine-producing C. glutamicum MH20-22B, applying a recently developed C-13 labeling-based strategy for anaplerotic flux resolution and quantification. Abolition of PEPCk activity by deletion of the respective pck gene resulted in increased intracellular concentrations of oxaloacetate, L-aspartate, a-ketoglutarate, pyruvate, and L-lysine and in a 60% enhanced flux toward L-lysine biosynthesis, whereas increasing the PEPCk activity by pck overexpression had opposite effects. The results of the combined measurements of enzyme activities, in viva fluxes, and metabolite concentrations were exploited to elucidate the in viva regulation of anaplerotic reactions in C. glutamicum, and implications for the metabolic engineering of amino-acid-producing strains are discussed. (C) 2001 Academic Press.