CARBON FLUX DISTRIBUTIONS AT THE PYRUVATE BRANCH POINT IN CORYNEBACTERIUM-GLUTAMICUM DURING LYSINE OVERPRODUCTION

In order to achieve the theoretical maximum yield of lysine from gluco se (75% molar), flux partitioning at principal branch points (glucose 6-phosphate, phosphoenolpyruvate, and pyruvate) in the primary metabol ic 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 dist ributions from extracellular measurements, in conjunction with two met abolic perturbations of the pyruvate dehydrogenase complex (PDC) to ex amine the possibility that flux partitioning at the pyruvate (Pyr) bra nch point (i.e., pyruvate availability) limits lysine yield. The two p erturbation 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 alteratio n in flux partition was observed at any of the three principal nodes i n the PDC-attenuated strain. However, the PDC mutation did cause a 70% uniform decrease in carbon flow throughout the network, which indicat es that the lysine yield is not limited solely by a weakly rigid Pyr b ranch point. The addition of FP did not affect the lysine synthesis ra te, but did temporarily redirect carbon flow away from the TCA cycle t oward pyruvate excretion, which further confirms that the lysine yield is not pyruvate-limited and that the Pyr branch point is not weakly r igid.