Deregulation of Feedback Inhibition of Phosphoenolpyruvate Carboxylase for Improved Lysine Production in Corynebacterium glutamicum

Abstract

ABSTRACTAllosteric regulation of phosphoenolpyruvate carboxylase (PEPC) controls the metabolic flux distribution of anaplerotic pathways. In this study, the feedback inhibition ofCorynebacterium glutamicumPEPC was rationally deregulated, and its effect on metabolic flux redistribution was evaluated. Based on rational protein design, six PEPC mutants were designed, and all of them showed significantly reduced sensitivity toward aspartate and malate inhibition. Introducing one of the point mutations (N917G) into theppcgene, encoding PEPC of the lysine-producing strainC. glutamicumLC298, resulted in ∼37% improved lysine production.In vitroenzyme assays and13C-based metabolic flux analysis showed ca. 20 and 30% increases in the PEPC activity and corresponding flux, respectively, in the mutant strain. Higher demand for NADPH in the mutant strain increased the flux toward pentose phosphate pathway, which increased the supply of NADPH for enhanced lysine production. The present study highlights the importance of allosteric regulation on the flux control of central metabolism. The strategy described here can also be implemented to improve other oxaloacetate-derived products.