Print Email Facebook Twitter Replacement of the initial steps of ethanol metabolism in Saccharomyces cerevisiae by ATP-independent acetylating acetaldehyde dehydrogenase Title Replacement of the initial steps of ethanol metabolism in Saccharomyces cerevisiae by ATP-independent acetylating acetaldehyde dehydrogenase Author Kozak, B.U. (TU Delft BT/Industriele Microbiologie) Rossum, Harmen M. (TU Delft BT/Industriele Microbiologie) Niemeijer, M.S. (TU Delft BT/Industriele Microbiologie) van Dijk, M. (TU Delft BT/Industriele Microbiologie) Benjamin, Kirsten (Amyris Inc) Wu, Liang (DSM) Daran, J.G. (TU Delft BT/Industriele Microbiologie) Pronk, J.T. (TU Delft BT/Industriele Microbiologie) van Maris, A.J.A. (TU Delft BT/Industriele Microbiologie) Date 2016 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. Subject Acetyl-CoAEnergeticsEvolutionary engineeringIntracellular metabolitesPrecursor supplyYeast To reference this document use: http://resolver.tudelft.nl/uuid:23dcf3a7-2002-4078-bd2e-254758304dd7 DOI https://doi.org/10.1093/femsyr/fow006 ISSN 1567-1356 Source FEMS Yeast Research, 16 (2) Part of collection Institutional Repository Document type journal article Rights © 2016 B.U. Kozak, Harmen M. Rossum, M.S. Niemeijer, M. van Dijk, Kirsten Benjamin, Liang Wu, J.G. Daran, J.T. Pronk, A.J.A. van Maris Files PDF fow006.pdf 1.09 MB Close viewer /islandora/object/uuid:23dcf3a7-2002-4078-bd2e-254758304dd7/datastream/OBJ/view