Print Email Facebook Twitter Replacement of the Saccharomyces cerevisiae acetyl-CoA synthetases by alternative pathways for cytosolic acetyl-CoA synthesis Title Replacement of the Saccharomyces cerevisiae acetyl-CoA synthetases by alternative pathways for cytosolic acetyl-CoA synthesis Author Kozak, B.U. Van Rossum, H.M. Benjamin, K.R. Wu, L. Daran, J.G. Pronk, J.T. Van Maris, A.J.A. Faculty Applied Sciences Department BT/Biotechnology Date 2013-11-19 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), acs1? acs2? 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. Subject yeastacetylating acetaldehyde dehydrogenasepyruvate-formate lyasetranscriptomeprecursor supplymetabolic compartments To reference this document use: http://resolver.tudelft.nl/uuid:aee77b3f-2ba2-41d7-9c38-1cdd1aae1e2b DOI https://doi.org/10.1016/j.ymben.2013.11.005 Publisher Elsevier ISSN 1096-7176 Source Metabolic Engineering, 21, 2014 Part of collection Institutional Repository Document type journal article Rights (c) 2013 The Author(s)This is an open-access article distributed under the terms of the Creative Commons Attribution-NonCommercial-No Derivative Works License,which permits non-commercial use, distribution,and reproduction in any medium, provided the original author and source are credited. Files PDF vanMaris_2013.pdf 767.86 KB Close viewer /islandora/object/uuid:aee77b3f-2ba2-41d7-9c38-1cdd1aae1e2b/datastream/OBJ/view