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Abstract

In order to achieve the theoretical maximum yield of lysine from glucose (75% molar), flux partitioning at principal branch points (glucose 6-phosphate, phosphoenolpyruvate, and pyruvate) in the primary metabolic network of Corynebacterium glutamicum must be significantly altered from that observed during balanced growth, so that lysine precursors are synthesized in optimal stoichiometric ratios. In this article, we employ flux analysis, which provides a snapshot of metabolic flux distributions from extracellular measurements, in conjunction with two metabolic perturbations of the pyruvate dehydrogenase complex (PDC) to examine the possibility that flux partitioning at the pyruvate (Pyr) branch point (i. e., pyruvate availability) limits lysine yield. The two perturbation experiments involved (1) isolation and flux analysis of a PDC-attenuated mutant of C. glutamicum and (2) flux analysis following the addition of fluoropyruvate (FP) to a standard fermentation during the initial period of lysine overproduction. No significant alteration in flux partition was observed at any of the three principal nodes in the PDC-attenuated strain. However, the PDC mutation did cause a 70% uniform decrease in carbon flow throughout the network, which indicates that the lysine yield is not limited solely by a weakly rigid Pyr branch point. The addition of FP did not affect the lysine synthesis rate, but did temporarily redirect carbon flow away from the TCA cycle toward pyruvate excretion, which further confirms that the lysine yield is not pyruvate-limited and that the Pyr branch point is not weakly rigid.