title: Alternative reactions at the interface of glycolysis and citric acid cycle in Saccharomyces cerevisiae.
abstract: Pyruvate and acetyl-coenzyme A, located at the interface between glycolysis and TCA cycle, are important intermediates in yeast metabolism and key precursors for industrially relevant products. Rational engineering of their supply requires knowledge of compensatory reactions that replace predominant pathways when these are inactivated. This study investigates effects of individual and combined mutations that inactivate the mitochondrial pyruvate-dehydrogenase (PDH) complex, extramitochondrial citrate synthase (Cit2) and mitochondrial CoA-transferase (Ach1) in Saccharomyces cerevisiae. Additionally, strains with a constitutively expressed carnitine shuttle were constructed and analyzed. A predominant role of the PDH complex in linking glycolysis and TCA cycle in glucose-grown batch cultures could be functionally replaced by the combined activity of the cytosolic PDH bypass and Cit2. Strongly impaired growth and a high incidence of respiratory deficiency in pda1Δ ach1Δ strains showed that synthesis of intramitochondrial acetyl-CoA as a metabolic precursor requires activity of either the PDH complex or Ach1. Constitutive overexpression of AGP2, HNM1, YAT2, YAT1, CRC1 and CAT2 enabled the carnitine shuttle to efficiently link glycolysis and TCA cycle in l-carnitine-supplemented, glucose-grown batch cultures. Strains in which all known reactions at the glycolysis-TCA cycle interface were inactivated still grew slowly on glucose, indicating additional flexibility at this key metabolic junction.
authors.affiliation: Department of Biotechnology, Delft University of Technology, Julianalaan 67, NL-2628 BC Delft, The Netherlands.
authors.forename: Harmen M
authors.initials: HM
authors.lastname: van Rossum
authors.affiliation: Department of Biotechnology, Delft University of Technology, Julianalaan 67, NL-2628 BC Delft, The Netherlands.
authors.forename: Barbara U
authors.initials: BU
authors.lastname: Kozak
authors.affiliation: Department of Biotechnology, Delft University of Technology, Julianalaan 67, NL-2628 BC Delft, The Netherlands.
authors.forename: Matthijs S
authors.initials: MS
authors.lastname: Niemeijer
authors.affiliation: Department of Biotechnology, Delft University of Technology, Julianalaan 67, NL-2628 BC Delft, The Netherlands.
authors.forename: Hendrik J
authors.initials: HJ
authors.lastname: Duine
authors.affiliation: Department of Biotechnology, Delft University of Technology, Julianalaan 67, NL-2628 BC Delft, The Netherlands.
authors.forename: Marijke A H
authors.initials: MA
authors.lastname: Luttik
authors.affiliation: DSM Biotechnology Center, Alexander Fleminglaan 1, NL-2613 AX Delft, The Netherlands.
authors.forename: Viktor M
authors.initials: VM
authors.lastname: Boer
authors.affiliation: Institute for Molecular Bio Sciences, Goethe University, Max-von-Laue-Straße 9, D-60438 Frankfurt, Germany.
authors.forename: Peter
authors.initials: P
authors.lastname: Kötter
authors.affiliation: Department of Biotechnology, Delft University of Technology, Julianalaan 67, NL-2628 BC Delft, The Netherlands.
authors.forename: Jean-Marc G
authors.initials: JM
authors.lastname: Daran
authors.affiliation: Department of Biotechnology, Delft University of Technology, Julianalaan 67, NL-2628 BC Delft, The Netherlands.
authors.forename: Antonius J A
authors.initials: AJ
authors.lastname: van Maris
authors.affiliation: Department of Biotechnology, Delft University of Technology, Julianalaan 67, NL-2628 BC Delft, The Netherlands j.t.pronk@tudelft.nl.
authors.forename: Jack T
authors.initials: JT
authors.lastname: Pronk
mesh_terms: D000105:Acetyl Coenzyme A / Q000378:metabolism; D002950:Citrate (si)-Synthase / Q000235:genetics; D002952:Citric Acid Cycle; D015256:Coenzyme A-Transferases / Q000235:genetics; D017353:Gene Deletion; D015870:Gene Expression; D006019:Glycolysis; D060847:Metabolic Engineering; D064688:Metabolic Flux Analysis; D053858:Metabolic Networks and Pathways / Q000235:genetics; D011768:Pyruvate Dehydrogenase Complex / Q000235:genetics; D019289:Pyruvic Acid / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics
chemical_list: D011768:Pyruvate Dehydrogenase Complex; D029701:Saccharomyces cerevisiae Proteins; D000105:Acetyl Coenzyme A; D019289:Pyruvic Acid; D002950:Citrate (si)-Synthase; C000603661:Ach1 protein, S cerevisiae; D015256:Coenzyme A-Transferases
keywords: Ach1; Cit2; PDH complex; Saccharomyces cerevisiae; carnitine shuttle; glycolysis-TCA cycle interface