Replacement of the Saccharomyces Cerevisiae Acetyl-CoA Synthetases By Acetylating Acetaldehyde Dehydrogenase for Cytosolic Acetyl-CoA Synthesis

Cytosolic acetyl-coenzyme A is a precursor for many compounds whose production from sugars is already implemented or under investigation by industry. The robustness of Saccharomyces cerevisiae combined with fast developments in yeast synthetic biology and systems biology have made this microorganism a popular metabolic engineering platform for the production of biotechnologically relevant compounds [1]. 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 ATP-independent pathway for acetyl-CoA synthesis [2]. The functional expression of different bacterial genes encoding acetylating acetaldehyde dehydrogenase (A-ALD) was studied in strains, in which native route of cytosolic acetyl-CoA formation was disrupted by deletion of all five known genes coding for acetaldehyde dehydrogenases (ALD). In the next step, Acs- Ald- S. cerevisiae strain was constructed in which A-ALD from Escherichia coli - eutE successfully replaced the two step reaction of cytosolic acetyl-CoA synthesis. In this strain, aerobic growth rate of 0.27 h-1 was observed, which was equal to
79% of the Ald+ Acs+ reference strain. In glucose-limited chemostat cultures biomass yield on
glucose of A-ALD-dependent strain was lower than that of the reference strain. Subsequently, a systems biology approach was used to investigate the physiological impact of these interventions in cytosolic acetyl-CoA metabolism. Transcriptome analysis suggested that reduced biomass yield was caused by acetaldehyde. This hypothesis was further supported by the increased levels of acetaldehyde in A-ALD-dependent strain. 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 [3]. Despite these interventions in
acetyl-CoA metabolism, the intracellular acetyl-CoA concentration did not change significantly between strains, which suggest a strong homeostatic regulation of its concentration [4]. Nonetheless, the levels of some compounds that have cytosolic acetyl-CoA as a precursor (such as lysine) were elevated, suggesting an increased availability of this molecule. Although further modifications are
needed to achieve optimal in vivo performance of the alternative reaction for supply of cytosolic acetyl-CoA as a product precursor, this study demonstrates for the first time that yeast ACS can be fully replaced.
References

1. Hong, K. K., Nielsen, J., 2012. Metabolic engineering of Saccharomyces cerevisiae: a key cell factory platform for future biorefineries. Cell Mol Life Sci. 69, 2671-2690.

2. Kozak, B. U., van Rossum, H. M., Benjamin, K. R., Wu, L., Daran, J. M., Pronk, J. T., van Maris, A. J., 2014. Replacement of the Saccharomyces cerevisiae acetyl-CoA synthetases by alternative pathways for cytosolic acetyl-CoA synthesis. Metab Eng. 21, 46-59.

3. Takahashi, H., McCaffery, J. M., Irizarry, R. A., Boeke, J. D., 2006. Nucleocytosolic acetyl-coenzyme a synthetase is required for histone acetylation and global transcription. Mol Cell. 23, 207-217.

4. Cai, L., Sutter, B. M., Li, B., Tu, B. P., 2011. Acetyl-CoA induces cell growth and proliferation by promoting the acetylation of histones at growth genes. Mol Cell. 42, 426-437.