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Applied and Environmental Microbiology, February 2007, p. 718-729, Vol. 73, No. 3
0099-2240/07/$08.00+0     doi:10.1128/AEM.01532-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Shewanella oneidensis MR-1 Fluxome under Various Oxygen Conditions ^,

Yinjie J. Tang,^1,3, Judy S. Hwang,^1,2, David E. Wemmer,^1,2,4 and Jay D. Keasling^1,3,5*

Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720,¹ Biophysics Graduate Groupand,² Departments of Chemical Engineering,³ Chemistry,⁴ Bioengineering, University of California, Berkeley, California 94720⁵

Received 5 July 2006/ Accepted 1 November 2006

The central metabolic fluxes of Shewanella oneidensis MR-1 were examined under carbon-limited (aerobic) and oxygen-limited (microaerobic) chemostat conditions, using ¹³C-labeled lactate as the sole carbon source. The carbon labeling patterns of key amino acids in biomass were probed using both gas chromatography-mass spectrometry (GC-MS) and ¹³C nuclear magnetic resonance (NMR). Based on the genome annotation, a metabolic pathway model was constructed to quantify the central metabolic flux distributions. The model showed that the tricarboxylic acid (TCA) cycle is the major carbon metabolism route under both conditions. The Entner-Doudoroff and pentose phosphate pathways were utilized primarily for biomass synthesis (with a flux below 5% of the lactate uptake rate). The anaplerotic reactions (pyruvate to malate and oxaloacetate to phosphoenolpyruvate) and the glyoxylate shunt were active. Under carbon-limited conditions, a substantial amount (9% of the lactate uptake rate) of carbon entered the highly reversible serine metabolic pathway. Under microaerobic conditions, fluxes through the TCA cycle decreased and acetate production increased compared to what was found for carbon-limited conditions, and the flux from glyoxylate to glycine (serine-glyoxylate aminotransferase) became measurable. Although the flux distributions under aerobic, microaerobic, and shake flask culture conditions were different, the relative flux ratios for some central metabolic reactions did not differ significantly (in particular, between the shake flask and aerobic-chemostat groups). Hence, the central metabolism of S. oneidensis appears to be robust to environmental changes. Our study also demonstrates the merit of coupling GC-MS with ¹³C NMR for metabolic flux analysis to reduce the use of ¹³C-labeled substrates and to obtain more-accurate flux values.

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* Corresponding author. Mailing address: Berkeley Center for Synthetic Biology, University of California, Berkeley, CA 94720. Phone: (510) 642-4862. Fax: (510) 495-2630. E-mail: Keasling{at}berkeley.edu.

Published ahead of print on 10 November 2006.

Supplemental material for this article may be found at http://aem.asm.org/.

These authors contributed equally to the work.

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Applied and Environmental Microbiology, February 2007, p. 718-729, Vol. 73, No. 3
0099-2240/07/$08.00+0     doi:10.1128/AEM.01532-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

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