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This work concerns applications of NMR techniques in metabolic engineering studies. As demonstrated here, NMR is a valuable tool in analyzing intracellular metabolic and energetic states. When combined with growth and fermentation studies, it greatly enhances the understanding of cellular responses to particular genetic modifications.

A novel on-line NMR system has been developed in this work which extends the capacities of in vivo NMR to growing cell cultures. This new approach eliminates possible artifacts and time limitations associated with conventional NMR methods. Furthermore, experiments can be conducted under well-defined conditions. New types of physiological states and experimental regimes are accessible with this on-line system.

Steady-state levels of ATP and [...] have been measured using the on-line NMR system for strains with and without Vitreoscilla hemoglobin (VHb) (GRO21 and MG1655, respectively) during their growth under oxygen-limited conditions. GRO21 has similar levels of ATP and [...] while exhibiting a higher specific growth rate, indicating that the net ATP accumulation rate is enhanced. Studies on ATP synthase (ATPase) kinetics show that the presence of VHb accelerates ATP synthesis rate catalyzed by ATPase by 30%.

Switching from the phosphotransferase system (PTS) to the galactose-proton symport system for glucose uptake brings about many changes in cell energetics and metabolism. The extra energy cost in the non-PTS glucose uptake has a greater effect on the specific growth rates in anaerobic conditions than in aerobic conditions. Fermentation patterns are significantly different in the non-PTS strain, compared to the PTS strain. The non-PTS strain has a much lower NTP and [...] and higher NDP; NAD(H) is also considerably reduced. Different levels of glycolytic intermediates have also been observed in these two strains.

The non-PTS strain carrying a phenylalanine overproduction plasmid exhibits much lower energy level (NTP and ApH), lower sugar phosphate (S-P) total concentration, NAD(H) and PEP concentrations than its PTS counterpart. The compositions of S-P are significantly different between the two strains. Phenylalanine production is not increased in the non-PTS strain and a larger portion of carbon is oxidized to CO2.

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