Cytosolic Acetyl-CoA Synthesis By Pyruvate-Formate Lyase in Yeast

Many industrially relevant products, like farnesene, artemisinin and 1-butanol have acetyl-CoA as a precursor. The yeast Saccharomyces cerevisiae is a widely used metabolic engineering platform because of its robustness and genetic accessibility. This makes acetyl-CoA produced in the cytosol of yeast an important building block for the formation of many (heterologous) products. Therefore fast and efficient production of cytosolic acetyl-CoA in brewer’s yeast is of industrial interest.

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 these enzymes consume the equivalent of 2 ATP, it constrains maximum yields of acetyl-CoA-derived products. Therefore, the replacement of ACS by the ATP-independent pathway of pyruvate-formate lyase (PFL) for cytosolic acetyl-CoA synthesis was explored.

After evaluating expression of five different PFL genes, acs1Δ acs2Δ S. cerevisiae strains were constructed in which PFL successfully replaced ACS. In PFL-dependent strains, anaerobic growth rates were stoichiometrically coupled to formate production. The biomass yield on glucose was lower than those of the reference strain. Transcriptome analysis suggested that this reduced biomass yield was caused by formate. 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, further modifications are needed to achieve optimal in vivo performance of the PFL pathway for supply of cytosolic acetyl-CoA as a product precursor.

See also Kozak, B. U., Van Rossum, H.M. et al. Metabolic engineering 21C, 46–59 (2013).