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Applied and Environmental Microbiology, April 2002, p. 1760-1771, Vol. 68, No. 4
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.4.1760-1771.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Intracellular Carbon Fluxes in Riboflavin-Producing Bacillus subtilis during Growth on Two-Carbon Substrate Mixtures

Michael Dauner,¹ Marco Sonderegger,¹ Michel Hochuli,² Thomas Szyperski,^2, Kurt Wüthrich,² Hans-Peter Hohmann,³ Uwe Sauer,^1* and James E. Bailey¹

Institute of Biotechnology,¹ Institute of Molecular Biology and Biophysics, ETH Zürich, CH-8093 Zürich, Switzerland,² Roche Vitamins Inc., Nutley, New Jersey 07110-1199³

Received 24 July 2001/ Accepted 8 January 2002

Metabolic responses to cofeeding of different carbon substrates in carbon-limited chemostat cultures were investigated with riboflavin-producing Bacillus subtilis. Relative to the carbon content (or energy content) of the substrates, the biomass yield was lower in all cofeeding experiments than with glucose alone. The riboflavin yield, in contrast, was significantly increased in the acetoin- and gluconate-cofed cultures. In these two scenarios, unusually high intracellular ATP-to-ADP ratios correlated with improved riboflavin yields. Nuclear magnetic resonance spectra recorded with amino acids obtained from biosynthetically directed fractional ¹³C labeling experiments were used in an isotope isomer balancing framework to estimate intracellular carbon fluxes. The glycolysis-to-pentose phosphate (PP) pathway split ratio was almost invariant at about 80% in all experiments, a result that was particularly surprising for the cosubstrate gluconate, which feeds directly into the PP pathway. The in vivo activities of the tricarboxylic acid cycle, in contrast, varied more than twofold. The malic enzyme was active with acetate, gluconate, or acetoin cofeeding but not with citrate cofeeding or with glucose alone. The in vivo activity of the gluconeogenic phosphoenolpyruvate carboxykinase was found to be relatively high in all experiments, with the sole exception of the gluconate-cofed culture.

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* Corresponding author. Mailing address: Institute of Biotechnology, ETH Zürich, CH-8093 Zürich, Switzerland. Phone: 41-1-633 36 72. Fax: 41-1-633 10 51. E-mail: sauer{at}biotech.biol.ethz.ch.

Present address: Department of Chemistry, State University of New York at Buffalo, Buffalo, NY 14260.

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Applied and Environmental Microbiology, April 2002, p. 1760-1771, Vol. 68, No. 4
0099-2240/02/$04.00+0     DOI: 10.1128/AEM.68.4.1760-1771.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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