title: Replacement of the initial steps of ethanol metabolism in Saccharomyces cerevisiae by ATP-independent acetylating acetaldehyde dehydrogenase.
abstract: In Saccharomyces cerevisiae ethanol dissimilation is initiated by its oxidation and activation to cytosolic acetyl-CoA. The associated consumption of ATP strongly limits yields of biomass and acetyl-CoA-derived products. Here, we explore the implementation of an ATP-independent pathway for acetyl-CoA synthesis from ethanol that, in theory, enables biomass yield on ethanol that is up to 40% higher. To this end, all native yeast acetaldehyde dehydrogenases (ALDs) were replaced by heterologous acetylating acetaldehyde dehydrogenase (A-ALD). Engineered Ald(-) strains expressing different A-ALDs did not immediately grow on ethanol, but serial transfer in ethanol-grown batch cultures yielded growth rates of up to 70% of the wild-type value. Mutations in ACS1 were identified in all independently evolved strains and deletion of ACS1 enabled slow growth of non-evolved Ald(-) A-ALD strains on ethanol. Acquired mutations in A-ALD genes improved affinity-Vmax/Km for acetaldehyde. One of five evolved strains showed a significant 5% increase of its biomass yield in ethanol-limited chemostat cultures. Increased production of acetaldehyde and other by-products was identified as possible cause for lower than theoretically predicted biomass yields. This study proves that the native yeast pathway for conversion of ethanol to acetyl-CoA can be replaced by an engineered pathway with the potential to improve biomass and product yields.
authors.affiliation: Department of Biotechnology, Delft University of Technology, 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, Julianalaan 67, 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, 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, 2628 BC Delft, the Netherlands.
authors.forename: Marlous
authors.initials: M
authors.lastname: van Dijk
authors.affiliation: Amyris Inc, 5885 Hollis Street, Ste. 100, Emeryville, CA94608, USA.
authors.forename: Kirsten
authors.initials: K
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, 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, 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, Julianalaan 67, 2628 BC Delft, the Netherlands a.j.a.vanmaris@tudelft.nl.
authors.forename: Antonius J A
authors.initials: AJ
authors.lastname: van Maris
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000445:Aldehyde Oxidoreductases / Q000235:genetics* / Q000378:metabolism*; D003470:Culture Media / Q000737:chemistry; D000431:Ethanol / Q000378:metabolism*; D060847:Metabolic Engineering; D053858:Metabolic Networks and Pathways / Q000235:genetics*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000254:growth & development / Q000378:metabolism*
chemical_list: D003470:Culture Media; D011994:Recombinant Proteins; D000431:Ethanol; D000255:Adenosine Triphosphate; D000445:Aldehyde Oxidoreductases; C027014:acetaldehyde dehydrogenase (acylating)
keywords: acetyl-CoA; energetics; evolutionary engineering; intracellular metabolites; precursor supply; yeast
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