Identification of in vivo enzyme activities in the cometabolism of glucose and acetate by Saccharomyces cerevisiae by using 13C-labeled substrates.
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 (13)C. 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 CO(2) 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.
dos Santos
Margarida Moreira
MM
Center for Process Biotechnology, BioCentrum-DTU, Technical University of Denmark, DK-2800, Lyngby, Denmark.
Gombert
Andreas Karoly
AK
Christensen
Bjarke
B
Olsson
Lisbeth
L
Nielsen
Jens
J
D000085:Acetates / Q000378:metabolism*; D000596:Amino Acids / Q000378:metabolism; D018533:Biomass; D002244:Carbon / Q000378:metabolism*; D002952:Citric Acid Cycle; D003600:Cytosol / Q000378:metabolism; D017353:Gene Deletion; D005800:Genes, Fungal; D005947:Glucose / Q000378:metabolism*; D008291:Malate Dehydrogenase / Q000235:genetics / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D009154:Mutation; D011770:Pyruvate Kinase / Q000378:metabolism; D019289:Pyruvic Acid / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
D000085:Acetates; D000596:Amino Acids; D002244:Carbon; D019289:Pyruvic Acid; D008291:Malate Dehydrogenase; D011770:Pyruvate Kinase; D005947:Glucose