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Applied and Environmental Microbiology, June 2007, p. 3859-3864, Vol. 73, No. 12
0099-2240/07/$08.00+0     doi:10.1128/AEM.02986-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Flux Analysis of Central Metabolic Pathways in Geobacter metallireducens during Reduction of Soluble Fe(III)-Nitrilotriacetic Acid{triangledown} ,§

Yinjie J. Tang,1,2,7,{dagger} Romy Chakraborty,3,7,{dagger} Héctor García Martín,4 Jeannie Chu,1,2 Terry C. Hazen,3,7 and Jay D. Keasling1,2,5,6,7*

Synthetic Biology Department, Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720,1 Department of Chemical Engineering, University of California at Berkeley, Berkeley, California 94720,2 Center for Environmental Biotechnology, Lawrence Berkeley National Laboratory, Berkeley, California 94720,3 DOE Joint Genome Institute, 2800 Mitchell Drive, Walnut Creek, California 94598,4 Department of Bioengineering, University of California at Berkeley, Berkeley, California 94720,5 California Institute for Quantitative Biomedical Research (QB3), University of California at Berkeley, Berkeley, California 94720,6 Virtual Institute for Microbial Stress and Survival,{ddagger} ,{ddagger}

Received 22 December 2006/ Accepted 18 April 2007

We analyzed the carbon fluxes in the central metabolism of Geobacter metallireducens strain GS-15 using 13C isotopomer modeling. Acetate labeled in the first or second position was the sole carbon source, and Fe-nitrilotriacetic acid was the sole terminal electron acceptor. The measured labeled acetate uptake rate was 21 mmol/g (dry weight)/h in the exponential growth phase. The resulting isotope labeling pattern of amino acids allowed an accurate determination of the in vivo global metabolic reaction rates (fluxes) through the central metabolic pathways using a computational isotopomer model. The tracer experiments showed that G. metallireducens contained complete biosynthesis pathways for essential metabolism, and this strain might also have an unusual isoleucine biosynthesis route (using acetyl coenzyme A and pyruvate as the precursors). The model indicated that over 90% of the acetate was completely oxidized to CO2 via a complete tricarboxylic acid cycle while reducing iron. Pyruvate carboxylase and phosphoenolpyruvate (PEP) carboxykinase were present under these conditions, but enzymes in the glyoxylate shunt and malic enzyme were absent. Gluconeogenesis and the pentose phosphate pathway were mainly employed for biosynthesis and accounted for less than 3% of total carbon consumption. The model also indicated surprisingly high reversibility in the reaction between oxoglutarate and succinate. This step operates close to the thermodynamic equilibrium, possibly because succinate is synthesized via a transferase reaction, and the conversion of oxoglutarate to succinate is a rate-limiting step for carbon metabolism. These findings enable a better understanding of the relationship between genome annotation and extant metabolic pathways in G. metallireducens.

* Corresponding author. Mailing address: Berkeley Center for Synthetic Biology, 717 Potter Street, Building 977, Mail code 3224, University of California, Berkeley, CA 94720-3224. Phone: (510) 495-2620. Fax: (510) 495-2630. E-mail: keasling{at}berkeley.edu

{triangledown} Published ahead of print on 27 April 2007.

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

{dagger} These authors made equal contributions to the study.

http://vimss.lbl.gov.

Applied and Environmental Microbiology, June 2007, p. 3859-3864, Vol. 73, No. 12
0099-2240/07/$08.00+0     doi:10.1128/AEM.02986-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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