The intracellular carbon flux distribution in wild-type and pyruvate kinase-deficient Escherichia coli was estimated using biosynthetically directed fractional 13 C labeling experiments with [U-13 C 6 ]glucose in glucoseor ammonia-limited chemostats, two-dimensional nuclear magnetic resonance (NMR) spectroscopy of cellular amino acids, and a comprehensive isotopomer model. The general response to disruption of both pyruvate kinase isoenzymes in E. coli was a local flux rerouting via the combined reactions of phosphoenolpyruvate (PEP) carboxylase and malic enzyme. Responses in the pentose phosphate pathway and the tricarboxylic acid cycle were strongly dependent on the environmental conditions. In addition, high futile cycling activity via the gluconeogenic PEP carboxykinase was identified at a low dilution rate in glucose-limited chemostat culture of pyruvate kinase-deficient E. coli, with a turnover that is comparable to the specific glucose uptake rate. Furthermore, flux analysis in mutant cultures indicates that glucose uptake in E. coli is not catalyzed exclusively by the phosphotransferase system in glucose-limited cultures at a low dilution rate. Reliability of the flux estimates thus obtained was verified by statistical error analysis and by comparison to intracellular carbon flux ratios that were independently calculated from the same NMR data by metabolic flux ratio analysis.The central carbon pathways fulfill anabolic and catabolic functions by providing cofactors and building blocks for macromolecular synthesis as well as energy. While some singlegene knockout mutations in central metabolism preclude growth on glucose, a majority of such variations can potentially be compensated for either by isoenzymes (39) or by a rerouting of carbon fluxes through alternative pathways (7,15,20,42). Usually, the phenotypes of knockout mutants is characterized by quantitative physiological analysis, and conclusions on intracellular metabolism are then based on qualitative interpretation of these results. To reveal cause and effect relationships, however, it is important to gain deeper insight into the complex metabolic responses at the level of intracellular metabolite concentrations and fluxes. These intracellular carbon fluxes, or in vivo reaction rates, are per se nonmeasurable quantities that cannot usually be inferred directly from in vitro enzyme activities because not all in vivo effector concentrations are known. Hence, the intracellular reaction rates are commonly estimated by methods of metabolic flux analysis, which provide a holistic perspective on metabolism (52, 54).The most common approach is based on flux balancing analysis within a stoichiometric model of cellular metabolism (26,41, 50, 52). In this approach, uptake and secretion rates and biosynthetic requirements are balanced in a stoichiometric model, assuming quasi-steady-state material balances on the intermediates. In many cases, however, limited extracellular data and stoichiometric constraints lead to underdetermined systems, so that addition...

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Many attempts to engineer cellular metabolism have failed due to the complexity of cellular functions. Mathematical and computational methods are needed that can organize the available experimental information, and provide insight and guidance for successful metabolic engineering. Two such methods are reviewed here. Both methods employ a (log)linear kinetic model of metabolism that is constructed based on enzyme kinetics characteristics. The first method allows the description of the dynamic responses of metabolic systems subject to spatiotemporal variations in their parameters. The second method considers the product‐oriented, constrained optimization of metabolic reaction networks using mixed‐integer linear programming methods. The optimization framework is used in order to identify the combinations of the metabolic characteristics of the glycolytic enzymes from yeast and bacteria that will maximize ethanol production. The methods are also applied to the design of microbial ethanol production metabolism. The results of the calculations are in qualitative agreement with experimental data presented here. Experiments and calculations suggest that, in resting Escherichia coli cells, ethanol production and glucose uptake rates can be increased by 30% and 20%, respectively, by overexpression of a deregulated pyruvate kinase, while increase in phosphofructokinase expression levels has no effect on ethanol production and glucose uptake rates. © 1998 John Wiley & Sons, Inc. Biotechnol Bioeng 58:154–161, 1998.

Glycolytic fluxes in resting Escherichia coli were enhanced by overexpression of heterologous pyruvate kinases (Pyk) from Bacillus stearothermophilus and Zymomonas mobilis, but not homologous Pyk. Compared to the control, an increase of 10% in specific glucose consumption and of 15% in specific ethanol production rates was found in anaerobic resting cells, expressing the heterologous Pyks, that were harvested from exponentially growing aerobic cultures. A further increase in glycolytic flux was achieved by simultaneous overexpression of E. coli phosphofructokinase (Pfk) and Pyk with specific glucose consumption and ethanol production rates of 25% and 35% greater, respectively, than the control. Fluxes to lactate were not significantly affected, contrary to previous observations with resting cells harvested from anaerobically growing cultures. To correlate the physiology of resting cells with the physiology of cells prior to harvest, we determined the relevant growth parameters from aerobic and anaerobic precultures. We conclude that glycolytic fluxes in E. coli with submaximal (aerobic) metabolic activity can be increased by overexpression of pyruvate kinases which do not require allosteric activation or co‐overexpression with Pfk. However, such improvements require more extensive engineering in E. coli with near maximal (anaerobic) metabolic activity. © 2000 John Wiley & Sons, Inc. Biotechnol Bioeng 67: 623–627, 2000.