AEM Accepts, published online ahead of print on 10 November 2006

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Appl. Environ. Microbiol. doi:10.1128/AEM.01532-06
Copyright (c) 2006, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

The Shewanella oneidensis MR-1 fluxome under various oxygen conditions

Yinjie J. Tang, Judy S. Hwang, David E. Wemmer, and Jay D. Keasling^*

Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA 94720, Biophysics Graduate Group, Departments of Chemical Engineering, Chemistry, and Bioengineering, University of California, Berkeley, CA 94720

^* To whom correspondence should be addressed. Email: Keasling{at}berkeley.edu.

The central metabolic fluxes of Shewanella oneidensis MR-1 were examined under carbon-limited (aerobic) and oxygen-limited (micro-aerobic) 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 GC-MS and ¹³C-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 (flux below 5% of the lactate uptake rate). The anapleurotic 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 micro-aerobic conditions fluxes through the TCA cycle decreased and acetate production increased compared to carbon-limited conditions, and flux from glyoxlate to glycine (serine-glyoxylate aminotransferase) became measurable. Although flux distributions under aerobic, micro-aerobic, and shake-flask culture conditions were different, the relative flux ratios of some central metabolic reactions did not vary significantly (in particular, between shake flask and aerobic chemostat). Hence, S. oneidensis central metabolism 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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