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Journal of Bacteriology, June 2000, p. 3088-3096, Vol. 182, No. 11
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.

Quantitative Determination of Metabolic Fluxes during Coutilization of Two Carbon Sources: Comparative Analyses with Corynebacterium glutamicum during Growth on Acetate and/or Glucosedagger

Volker F. Wendisch,* Albert A. de Graaf, Hermann Sahm, and Bernhard J. EikmannsDagger

Institute of Biotechnology 1, Research Center Jülich, D-52425 Jülich, Germany

Received 18 October 1999/Accepted 6 March 2000

Growth of Corynebacterium glutamicum on mixtures of the carbon sources glucose and acetate is shown to be distinct from growth on either substrate alone. The organism showed nondiauxic growth on media containing acetate-glucose mixtures and simultaneously metabolized these substrates. Compared to those for growth on acetate or glucose alone, the consumption rates of the individual substrates were reduced during acetate-glucose cometabolism, resulting in similar total carbon consumption rates for the three conditions. By 13C-labeling experiments with subsequent nuclear magnetic resonance analyses in combination with metabolite balancing, the in vivo activities for pathways or single enzymes in the central metabolism of C. glutamicum were quantified for growth on acetate, on glucose, and on both carbon sources. The activity of the citric acid cycle was high on acetate, intermediate on acetate plus glucose, and low on glucose, corresponding to in vivo activities of citrate synthase of 413, 219, and 111 nmol · (mg of protein)-1 · min-1, respectively. The citric acid cycle was replenished by carboxylation of phosphoenolpyruvate (PEP) and/or pyruvate (30 nmol · [mg of protein]-1 · min-1) during growth on glucose. Although levels of PEP carboxylase and pyruvate carboxylase during growth on acetate were similar to those for growth on glucose, anaplerosis occurred solely by the glyoxylate cycle (99 nmol · [mg of protein]-1 · min-1). Surprisingly, the anaplerotic function was fulfilled completely by the glyoxylate cycle (50 nmol · [mg of protein]-1 · min-1) on glucose plus acetate also. Consistent with the predictions deduced from the metabolic flux analyses, a glyoxylate cycle-deficient mutant of C. glutamicum, constructed by targeted deletion of the isocitrate lyase and malate synthase genes, exhibited impaired growth on acetate-glucose mixtures.

* Corresponding author. Mailing address: Institute of Biotechnology 1, Research Center Jülich, D-52428 Jülich, Germany, Phone: 49-2461-615169. Fax: 49-2461-612710. E-mail: v.wendisch{at}fz-juelich.de.

dagger Dedicated to Rudolf K. Thauer on the occasion of his 60th birthday.

Dagger Present address: Abt. Mikrobiologie und Biotechnologie, University of Ulm, D-89061 Ulm, Germany.

Journal of Bacteriology, June 2000, p. 3088-3096, Vol. 182, No. 11
0021-9193/00/$04.00+0
Copyright © 2000, American Society for Microbiology. All rights reserved.


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