Untersuchungen zur $\textit{in vivo}$ anaplerotischer Stoffwechselwege in $\textit{Corynebacterium glutamicum}$ mittels $^{13}$C-Markierungstechnik

$\textit{Corynebacterium glutamicum}$ is an industrially important producer of the amino acid L-lysine. Metabolic precursors of lysine biosynthesis are oxaloacetate and pyruvate. Oxaloacetate itself is synthesized by the anaplerotic enzymes pyruvate carboxylase and phosphoenolpyruvate (PEP) carboxylase. In addition, $\textit{C. glutamicum}$ possesses the enzymes PEP carboxykinase, oxaloacetate decarboxylase and malic enzyme, which catalyze the decarboxylation of oxaloacetate or malate back to PEP or pyruvate. To quantify these individual fluxes within the anaplerosis $\textit{in vivo, C. glutamicum}$ was grown on a mixture of differently $^{13}$C-labeled glucoses and $^{13}$C-labeled lactate as a cosubstrate. Experimental $^{13}$C labeling patterns ofproteinogenic amino acids were used in a computer-based estimation procedure to yield intracellular metabolic fluxes. The results showed that pyruvate carboxylase is the principal anaplerotic enzyme in vivo carrying 70 - 90% of the anaplerotic flux. The results further showed that 40 - 70% of anaplerotically synthesized oxaloacetate was decarboxylated by PEP carboxykinase, constituting an enzymatic substrate cycle involving the metabolites PEP, pyruvate and oxaloacetate. Oxaloacetate decarboxylase and malic enzyme were inactive in vivo. The effect of PEP carboxykinase activity on central metabolism and amino acid production was investigated in mutant strains of L-lysine producing $\textit{C. glutamicum}$ MH20-22B with altered expression of the $\textit{pck}$ gene encoding this enzyme. $\textit{pck}$ deletion resulted in increased intracellular concentrations of oxaloacetate, L-aspartate, $\alpha$-ketoglutarate, pyruvate and L-lysine, and in a 60% enhanced flux towards L-lysine biosynthesis, whereas $\textit{pck}$ overexpression had opposite effects. Hence, PEP carboxykinase activity has a clearly adverse influence on lysine production. Intracellular flux and concentration data were combined with enzyme activity measurements to elucidate the $\textit{in vivo}$ regulation of anaplerotic reactions in $\textit{C. glutamicum}$, and an appropriate mathematical model was developed. By means of this model, oxaloacetate and $\alpha$-ketoglutarate could be identified as inhibitors of pyruvate carboxylase, whereas aspartate was ruled out as a significant inhibitor under $\textit{in vivo}$ conditions. To date, the effect of these metabolites on the pyruvate carboxylase of $\textit{C. glutamicum}$ could not be determined $\textit{in vitro}$ due to the instability of this enzyme. Implications for the engineering of amino acid-producing strains are discussed