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Eukaryotic Cell, June 2003, p. 599-608, Vol. 2, No. 3
1535-9778/03/$08.00+0     DOI: 10.1128/EC.2.3.599-608.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Identification of In Vivo Enzyme Activities in the Cometabolism of Glucose and Acetate by Saccharomyces cerevisiae by Using 13C-Labeled Substrates

Margarida Moreira dos Santos, Andreas Karoly Gombert,{dagger} Bjarke Christensen, Lisbeth Olsson, and Jens Nielsen*

Center for Process Biotechnology, BioCentrum-DTU, Technical University of Denmark, DK-2800, Lyngby, Denmark

Received 4 December 2002/ Accepted 26 March 2003

A detailed characterization of the central metabolic network of Saccharomyces cerevisiae CEN.PK 113-7D was carried out during cometabolism of different mixtures of glucose and acetate, using aerobic C-limited chemostats in which one of these two substrates was labeled with 13C. To confirm the role of malic enzyme, an isogenic strain with the corresponding gene deleted was grown under the same conditions. The labeling patterns of proteinogenic amino acids were analyzed and used to estimate metabolic fluxes and/or make inferences about the in vivo activities of enzymes of the central carbon metabolism and amino acid biosynthesis. Malic enzyme flux increased linearly with increasing acetate fraction. During growth on a very-high-acetate fraction, the activity of malic enzyme satisfied the biosynthetic needs of pyruvate in the mitochondria, while in the cytosol pyruvate was supplied via pyruvate kinase. In several cases enzyme activities were unexpectedly detected, e.g., the glyoxylate shunt for a very-low-acetate fraction, phosphoenolpyruvate carboxykinase for an acetate fraction of 0.46 C-mol of acetate/C-mol of substrate, and glucose catabolism to CO2 via the tricarboxylic acid cycle for a very-high-acetate fraction. Cytoplasmic alanine aminotransferase activity was detected, and evidence was found that {alpha}-isopropylmalate synthase has two active forms in vivo, one mitochondrial and the other a short cytoplasmic form.

* Corresponding author. Mailing address: Center for Process Biotechnology, BioCentrum-DTU, Building 223, Technical University of Denmark, DK-2800 Lyngby, Denmark. Phone: 45 45 25 2696. Fax: 45 45 88 4148. E-mail: jn{at}biocentrum.dtu.dk.

{dagger} Present address: Department of Chemical Engineering, University of São Paulo, 05424-970 São Paulo SP, Brazil.

Eukaryotic Cell, June 2003, p. 599-608, Vol. 2, No. 3
1535-9778/03/$08.00+0     DOI: 10.1128/EC.2.3.599-608.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

  • Smedsgaard, J., Nielsen, J. (2005). Metabolite profiling of fungi and yeast: from phenotype to metabolome by MS and informatics. J Exp Bot 56: 273-286 [Abstract] [Full Text]  
  • Blank, L. M., Sauer, U. (2004). TCA cycle activity in Saccharomyces cerevisiae is a function of the environmentally determined specific growth and glucose uptake rates. Microbiology 150: 1085-1093 [Abstract] [Full Text]  
  • Nielsen, J. (2003). It Is All about Metabolic Fluxes. J. Bacteriol. 185: 7031-7035 [Full Text]  


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