Metabolic state of Zymomonas mobilis in glucose-, fructose-, and xylose-fed continuous cultures as analysed by 13 C- and 31 P-NMR spectroscopy

Abstract

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.

Optimization of a synthetic medium for ethanol production by xylose-fermenting Zymomonas mobilis using response surface methodology

“…The process ethanol yield in the final optimum medium reached 73.2% of the theoretical yield, which is 36.3% higher than the ethanol yield obtained in a synthetic xylose-fed (70 g/L) continuous culture broth [20]. Interestingly, the two media have very similar compositions, except for 11 extra minor components in the continuous culture broth [20]. Thus, the enhanced ethanol production obtained in the final synthetic medium may be the result of the removal of components which have negative effects on ethanol production, and of the addition of the optimal concentration of each nutrient.…”

“…The process ethanol yield in the final optimum medium reached 73.2% of the theoretical yield, which is 36.3% higher than the ethanol yield obtained in a synthetic xylose-fed (70 g/L) continuous culture broth [20]. Interestingly, the two media have very similar compositions, except for 11 extra minor components in the continuous culture broth [20].…”

“…This indicates that calcium pantothenate is a necessary growth factor for recombinant Z. mobilis. Thus, calcium pantothenate was added to the synthetic medium at a concentration of 2.74 mg/L, according to De Graaf et al [20]. The significance of each variable was determined by the method of Wu et al [12] on the basis of ethanol production (Table S1).…”

Respiratory behaviour of a Zymomonas mobilis adhB::kanr mutant supports the hypothesis of two alcohol dehydrogenase isoenzymes catalysing opposite reactions

“…It must be emphasized that the measured bulk reactant ratios for the ADH reaction in both strains were closely similar, generally exceeding K eq by 3 orders of magnitude, and hence could not explain the extremely different responses of the strains to the ethanol pulse. Taking the measured values for ethanol, acetaldehyde and intracellular NADH concentrations (Table 2), 6.5 for intracellular pH [18], 3.3 ll per mg of dry weight for the intracellular volume [19], and (for the lowest estimate of the reactant ratios) assuming that NAD + makes up most of the 5 mM intracellular NAD(P)(H) pool, detected in Z. mobilis by NMR [20], we come to reactant ratios of 2.6 · 10 À9 M for ZM6 and 12.6 · 10 À9 M for the ADH II-deficient strain. These are indeed the lowest estimates of the reactant ratios, because: (i) by assuming the intracellular acetaldehyde/ethanol ratio equal to that of the outer medium, we are neglecting the fact that acetaldehyde is a more volatile compound than ethanol, and hence, the true intracellular acetaldehyde/ethanol ratio might be higher; (ii) a lower value for the intracellular volume of Z. mobilis has been reported by Osman et al [21], which would result in a higher measured intracellular NADH concentrations.…”

Rewiring carbon catabolite repression for microbial cell factory

“…Several lines of evidence indicate that the slow growth rate of Z. mobilis in xylose medium might be responsible for this CCR. It has been established that the energy content of xylose-grown cells were less than that in glucose grown cells (79). Further, xylitol, sugar alcoholic byproduct of xylose metabolism, is a potent inhibitor of cell growth.…”