Allosteric regulation of phosphoenolpyruvate carboxylase (PEPC) controls the metabolic flux distribution of anaplerotic pathways. In this study, the feedback inhibition of Corynebacterium glutamicum PEPC 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 the ppc gene, encoding PEPC of the lysine-producing strain C. glutamicum LC298, resulted in ϳ37% improved lysine production. In vitro enzyme assays and 13 C-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.

Engineering of enzymes and pathways is generally required for the development of efficient strains for bioproduction processes. To this end, quantitative and reliable data of intracellular metabolites are highly desired, but often not available, especially for conditions more close to industrial applications, i.e. at high cell density and product concentration. Here, we investigated the intracellular metabolite profiles of an engineered l‐lysine‐producing Corynebacterium glutamicum strain and the corresponding wild‐type strain to assess the impacts of deregulation of product inhibition of the key enzymes aspartate kinase and phosphoenolpyruvate carboxylase and to identify potentials for their further improvement. A bioreactor system with automated fast‐sampling, filtration and on‐filter quenching of the metabolism was used for a more reliable determination of intracellular metabolites in batch cultures with optical cell density (OD660) up to 40. The l‐lysine‐producing strain showed substantially different metabolite profiles in the amino acid metabolism, including increased intracellular pool sizes in the l‐lysine‐, l‐homoserine‐ and l‐threonine pathways and decreased intracellular pool sizes for all other determined amino acids. By comparing data of in vitro inhibition of the engineered enzymes and determined intracellular concentrations of the inhibitors it was found that the inferred in vivo activities of these enzymes are still significantly below their in vitro maximums. This work demonstrates the usefulness of metabolic analysis for assessing the impact of engineered enzymes and identifying targets for further strain development.