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Acetate scavenging activity in Escherichia coli: interplay of acetyl–CoA synthetase and the PEP–glyoxylate cycle in chemostat cultures

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Abstract

Impairment of acetate production in Escherichia coli is crucial for the performance of many biotechnological processes. Aerobic production of acetate (or acetate overflow) results from changes in the expression of central metabolism genes. Acetyl−CoA synthetase scavenges extracellular acetate in glucose-limited cultures. Once converted to acetyl−CoA, it can be catabolized by the tricarboxylic acid cycle or the glyoxylate pathway. In this work, we assessed the significance of these pathways on acetate overflow during glucose excess and limitation. Gene expression, enzyme activities, and metabolic fluxes were studied in E. coli knock-out mutants related to the glyoxylate pathway operon and its regulators. The relevance of post-translational regulation by AceK-mediated phosphorylation of isocitrate dehydrogenase for pathway functionality was underlined. In chemostat cultures performed at increasing dilution rates, acetate overflow occurs when growing over a threshold glucose uptake rate. This threshold was not affected in a glyoxylate-pathway-deficient strain (lacking isocitrate lyase, the first enzyme of the pathway), indicating that it is not relevant for acetate overflow. In carbon-limited chemostat cultures, gluconeogenesis (maeB, sfcA, and pck), the glyoxylate operon and, especially, acetyl−CoA synthetase are upregulated. A mutant in acs (encoding acetyl−CoA synthetase) produced acetate at all dilution rates. This work demonstrates that, in E. coli, acetate production occurs at all dilution rates and that overflow is the result of unbalanced synthesis and scavenging activities. The over-expression of acetyl−CoA synthetase by cAMP−CRP-dependent induction limits this phenomenon in cultures consuming glucose at low rate, ensuring the recycling of the acetyl−CoA and acetyl−phosphate pools, although establishing an energy-dissipating substrate cycle.

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Acknowledgments

This work was supported by MCYT project BIO2008-04500-C02-01, and Fundación Séneca-CARM project 08660/PI/08. S. Renilla and J. M. Pastor are recipients of FPU fellowships from MCINN; S. Castaño-Cerezo is a recipient of FPI fellowship from Fundación Séneca-CARM. V. Bernal holds a post-doctoral contract with the University of Murcia. We thank the National Institute of Genetics of Japan for kind gift of Keio Collection strains.

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  1. Department of Biochemistry and Molecular Biology B and Immunology, Faculty of Chemistry, University of Murcia, Murcia, Spain

    Sergio Renilla, Vicente Bernal, Sara Castaño-Cerezo, José M. Pastor, José L. Iborra & Manuel Cánovas

  2. Institute of Molecular Systems Biology, ETH Zürich, Zürich, CH-8093, Switzerland

    Tobias Fuhrer & Uwe Sauer

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  1. Sergio Renilla
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  2. Vicente Bernal
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  3. Tobias Fuhrer
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  4. Sara Castaño-Cerezo
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  5. José M. Pastor
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Correspondence to Vicente Bernal or Manuel Cánovas.

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Sergio Renilla and Vicente Bernal equally contributed to this work.

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Renilla, S., Bernal, V., Fuhrer, T. et al. Acetate scavenging activity in Escherichia coli: interplay of acetyl–CoA synthetase and the PEP–glyoxylate cycle in chemostat cultures. Appl Microbiol Biotechnol 93, 2109–2124 (2012). https://doi.org/10.1007/s00253-011-3536-4

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