The reasons for the well-known significantly different behaviour of the anaerobic, gram-negative, ethanologenic bacterium Zymomonas mobilis during growth on fructose (i.e. decreased growth and ethanol yields, increased by-product formation) as compared to that on its second natural substrate, glucose, have remained unexplained. A xylose-fermenting recombinant strain of Z. mobilis that was recently constructed in our laboratory also unexpectedly displayed an increased formation of by-products and a strongly reduced growth rate as compared to the parent strain. Therefore, a comprehensive study employing recently developed NMR-based methods for the in vivo analysis of intracellular phosphorylated pool sizes and metabolic fluxes was undertaken to enable a global characterization of the intracellular metabolic state of Z. mobilis during growth on 13C-labelled glucose, fructose and xylose in defined continuous cultures. The 13C-NMR flux analysis indicated that ribose 5-phosphate is synthesized via the nonoxidative pentose phosphate pathway in Z. mobilis, and it identified a metabolic bottleneck in the recombinant xylose-fermenting Z. mobilis strain at the level of heterologous xylulokinase. The 31P-NMR analyses revealed a global alteration of the levels of intracellular phosphorylated metabolites during growth on fructose as compared to that on glucose. The results suggest that this is primarily caused by an elevated concentration of intracellular fructose 6-phosphate.
A new bioreactor system has been developed for in vivo NMR spectroscopy of microorganisms under defined physiological conditions. This cyclone reactor with an integrated NMR flow cell is continuously operated in the magnet of a 400‐MHz wide‐bore NMR spectrometer system. The residence times of medium and cells are decoupled by a circulation‐integrated cross‐flow microfiltration module to achieve higher cell densities as compared to continuous fermentations without cell retention (increase in cell density up to a factor of 10 in steady state). Volumetric mass transfer coefficients kLa of more than 1.0 s−1 are possible in the membrane cyclone reactor, ensuring adequate oxygen supply [oxygen transfer rate >15,000 mg O2 ·(L h)−1] of high cell densities. With the aid of the membrane cyclone reactor we were able to show, using continuous in vivo 31P NMR spectroscopy of anaerobic glucose fermentation by Zymomonas mobilis, that the NMR signal intensity was directly proportional to the cell concentration in the reactor. The concentration profiles of intracellular inorganic phosphate, NAD(H), NDP, NTP, UDP‐sugar, a cyclic pyrophosphate, two sugar phosphate pools, and extracellular inorganic phosphate were recorded after a shift from one steady state to another. The intracellular cyclic pyrophosphate had not been detected before in in vitro measurements of Zymomonas mobilis extracts due to the high instability of this compound. Using continuous in vivo 13C NMR spectroscopy of aerobic glucose utilization by Corynebacterium glutamicum at a density of 25 gcell dry weight · L−1, the membrane cyclone reactor served to measure the different dynamics of labeling in the carbon atoms of L‐lactate, L‐glutamate, succinate, and L‐lysine with a time resolution of 10 min after impressing a [1‐13C]‐glucose pulse.
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