title: Replacement of the Saccharomyces cerevisiae acetyl-CoA synthetases by alternative pathways for cytosolic acetyl-CoA synthesis.
abstract: Cytosolic acetyl-coenzyme A is a precursor for many biotechnologically relevant compounds produced by Saccharomyces cerevisiae. In this yeast, cytosolic acetyl-CoA synthesis and growth strictly depend on expression of either the Acs1 or Acs2 isoenzyme of acetyl-CoA synthetase (ACS). Since hydrolysis of ATP to AMP and pyrophosphate in the ACS reaction constrains maximum yields of acetyl-CoA-derived products, this study explores replacement of ACS by two ATP-independent pathways for acetyl-CoA synthesis. After evaluating expression of different bacterial genes encoding acetylating acetaldehyde dehydrogenase (A-ALD) and pyruvate-formate lyase (PFL), acs1Δ acs2Δ S. cerevisiae strains were constructed in which A-ALD or PFL successfully replaced ACS. In A-ALD-dependent strains, aerobic growth rates of up to 0.27 h(-1) were observed, while anaerobic growth rates of PFL-dependent S. cerevisiae (0.20 h(-1)) were stoichiometrically coupled to formate production. In glucose-limited chemostat cultures, intracellular metabolite analysis did not reveal major differences between A-ALD-dependent and reference strains. However, biomass yields on glucose of A-ALD- and PFL-dependent strains were lower than those of the reference strain. Transcriptome analysis suggested that reduced biomass yields were caused by acetaldehyde and formate in A-ALD- and PFL-dependent strains, respectively. Transcript profiles also indicated that a previously proposed role of Acs2 in histone acetylation is probably linked to cytosolic acetyl-CoA levels rather than to direct involvement of Acs2 in histone acetylation. While demonstrating that yeast ACS can be fully replaced, this study demonstrates that further modifications are needed to achieve optimal in vivo performance of the alternative reactions for supply of cytosolic acetyl-CoA as a product precursor.
authors.affiliation: Department of Biotechnology, Delft University of Technology, Kluyver Centre for Genomics of Industrial Fermentation, Julianalaan 67, 2628 BC Delft, The Netherlands.
authors.forename: Barbara U
authors.initials: BU
authors.lastname: Kozak
authors.affiliation: Department of Biotechnology, Delft University of Technology, Kluyver Centre for Genomics of Industrial Fermentation, Julianalaan 67, 2628 BC Delft, The Netherlands.
authors.forename: Harmen M
authors.initials: HM
authors.lastname: van Rossum
authors.affiliation: Amyris Inc., 5885 Hollis Street, Ste. 100, Emeryville, CA 94608, USA.
authors.forename: Kirsten R
authors.initials: KR
authors.lastname: Benjamin
authors.affiliation: DSM Biotechnology Center, Alexander Fleminglaan 1, 2613 AX Delft, The Netherlands.
authors.forename: Liang
authors.initials: L
authors.lastname: Wu
authors.affiliation: Department of Biotechnology, Delft University of Technology, Kluyver Centre for Genomics of Industrial Fermentation, Julianalaan 67, 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, Kluyver Centre for Genomics of Industrial Fermentation, Julianalaan 67, 2628 BC Delft, The Netherlands.
authors.forename: Jack T
authors.initials: JT
authors.lastname: Pronk
authors.affiliation: Department of Biotechnology, Delft University of Technology, Kluyver Centre for Genomics of Industrial Fermentation, Julianalaan 67, 2628 BC Delft, The Netherlands. Electronic address: A.J.A.vanMaris@TUDelft.nl.
authors.forename: Antonius J A
authors.initials: AJ
authors.lastname: van Maris
mesh_terms: D000106:Acetate-CoA Ligase / Q000096:biosynthesis / Q000235:genetics; D000105:Acetyl Coenzyme A / Q000096:biosynthesis / Q000235:genetics; D000123:Acetyltransferases / Q000096:biosynthesis / Q000235:genetics; D000445:Aldehyde Oxidoreductases / Q000096:biosynthesis / Q000235:genetics; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics; D003600:Cytosol / Q000201:enzymology*; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins / Q000096:biosynthesis / Q000235:genetics
chemical_list: D001426:Bacterial Proteins; D029701:Saccharomyces cerevisiae Proteins; D000105:Acetyl Coenzyme A; D000445:Aldehyde Oxidoreductases; C018699:aldehyde dehydrogenase (NAD(P)+); D000123:Acetyltransferases; C022560:formate C-acetyltransferase; D000106:Acetate-CoA Ligase
keywords: Acetylating acetaldehyde dehydrogenase; Metabolic compartments; Precursor supply; Pyruvate-formate lyase; Transcriptome; Yeast