Using our recently developed sensor reactor approach, lysine-producing, nongrowing Corynebacterium glutamicum MH20-22B cells were subjected to serial C-13-labeling experiments for flux analysis during the leucine-limited fed-batch production phase in a 300-L bioreactor. Based on two-dimensional (2D) nuclear magnetic resonance (NMR) measurements of C-13-labeling patterns of cytoplasmic free metabolites, metabolic flux distributions in the central metabolism were successfully determined. Focusing on the highly concentrated metabolite L-glutamate, the working hypothesis was validated that the equilibration of labeling patterns in intracellular pools was much faster (up to 9.45 min) than the labeling period (3 h) used in the experiments. Analysis of anaplerotic reactions revealed that highly selective lysine production was accompanied by a significant reduction of decarboxylating reactions from 10 mol% to only 2 mol%, whereas PEP/pyruvate-carboxylating fluxes remained constant at about 40 mol% of consumed glucose. These results support the conclusion that an optimized C. glutamicum L-lysine producer should possess increased PEP carboxylase and/or pyruvate carboxylase activity combined with downregulated, decarboxylating fluxes consuming oxaloacetate/malate. The findings also illustrate the usefulness of the sensor reactor approach in the study of industrial fermentations. (C) 2004 Wiley Periodicals, Inc.