The glucose-6-phosphate (Glc6P) and 6-phosphogluconate (6PG) dehydrogenases of the amino-acid-producing bacterium Corynebacterium glutamicum were purified to homogeneity and kinetically characterized. The Glc6P dehydrogenase was a heteromultimeric complex, which consists of Zwf and OpcA subunits. The product inhibition pattern of the Glc6P dehydrogenase was consistent with an ordered bi-bi mechanism. The 6PG dehydrogenase was found to operate according to a Theorell±Chance ordered bi-ter mechanism. Both enzymes were inhibited by NADPH and the 6PG dehydrogenase additionally by ATP, fructose 1,6-bisphosphate (Fru1,6P 2 ), d-glyceraldehyde 3-phosphate (Gra3P), erythrose 4-phosphate and ribulose 5-phosphate (Rib5P). The inhibition by NADPH was considered to be most important, with inhibition constants of around 25 mm for both enzymes.Intracellular metabolite concentrations were determined in two isogenic strains of C. glutamicum with plasmid-encoded NAD-and NADP-dependent glutamate dehydrogenases. NADP 1 and NADPH levels were between 130 mm and 290 mm, which is very much higher than the respective K m and K i values. The Glc6P concentration was around 500 mm in both strains.The in vivo fluxes through the oxidative part of the pentose phosphate pathway calculated on the basis of intracellular metabolite concentrations and the kinetic constants of the purified enzymes determined in vitro were in agreement with the same fluxes determined by NMR after 13 C-labelling. From the derived kinetic model thus validated, it is concluded that the oxidative pentose phosphate pathway in C. glutamicum is mainly regulated by the ratio of NADPH and NADP 1 concentrations and the specific enzyme activities of both dehydrogenases.

To evaluate the importance of reactions within the central metabolism under different flux burdens the fluxes within the pentose phosphate pathway (PPP), as well as the other reactions of the central metabolism, were intensively analyzed and quantitated. For this purpose, Corynebacterium glutamicum was grown with [1‐13C]glucose to metabolic and isotopic steady state and the fractional enrichments in precursor metabolites (e.g., pentose 5‐phosphate) were quantified. Matrix calculus was used to express these data together with metabolite mass data. The detailed analysis of the dependence of 13C enrichments on exchange fluxes enabled the transketolase‐catalyzed exchange rate (2 pentose 5‐phosphate ↔ sedoheptulose 7‐phosphate + glyceraldehyde 3‐phosphate) to be quantified as 74.3% (molar metabolite flux) at a net flux of 10.3% and the exchange rate (pentose 5‐phosphate + erythrose 4‐phosphate ↔ fructose 6‐phosphate + glyceraldehyde 3‐phosphate) to be quantified as 5.6% at a net flux of 8.1%. The flux entering the tricarboxylic acid cycle was 93.3%. The same comprehensive flux analysis as performed for the nonexcreting condition was done with the identical strain that had been forced to excrete L‐glutamate. Because we had already quantified the fluxes for L‐lysine excretion with an isogenic strain, three directly comparable flux situations are thus available. Consequently, this comparison permits a direct cause‐and‐effect relationship to be specified. In response to the different flux burdens of the cell, the PPP flux decreased from a maximum of 67% to 26%, with the glycolytic flux increasing accordingly. The carbon flux through isocitrate dehydrogenase increased from 20% to 36%. The bidirectional carbon flux between pyruvate and oxaloacetate decreased from 36% to 9%. Since the cause of the three different flux states was the allelic exchange in the final L‐lysine assembling pathway or the glutamate export activity, respectively, the flexible response is the effect. This shows conclusively the enormous flexibility within the central metabolism of C. glutamicum to supply precursors upon their withdrawal for the synthesis of amino acids. © 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 56: 168–180, 1997.

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.

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