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Metab Eng. 2014 Jan;21:46-59. doi: 10.1016/j.ymben.2013.11.005. Epub 2013 Nov 19.

Replacement of the Saccharomyces cerevisiae acetyl-CoA synthetases by alternative pathways for cytosolic acetyl-CoA synthesis.

Author information

1
Department of Biotechnology, Delft University of Technology, Kluyver Centre for Genomics of Industrial Fermentation, Julianalaan 67, 2628 BC Delft, The Netherlands.
2
Amyris Inc., 5885 Hollis Street, Ste. 100, Emeryville, CA 94608, USA.
3
DSM Biotechnology Center, Alexander Fleminglaan 1, 2613 AX Delft, The Netherlands.
4
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.

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.

KEYWORDS:

Acetylating acetaldehyde dehydrogenase; Metabolic compartments; Precursor supply; Pyruvate-formate lyase; Transcriptome; Yeast

PMID:
24269999
DOI:
10.1016/j.ymben.2013.11.005
[Indexed for MEDLINE]
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