Engineering Yeast Central Metabolism for Improved Yields of Fuels and Chemicals

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Breath-taking advances in synthetic biology, including the recent addition of CRISPR-Cas9 to the metabolic engineering toolbox, greatly accelerate introduction and  optimization of pathways towards native and non-native products in the yeast Saccharomyces cerevisiae.

Especially for transport fuels and commodity chemicals, economic constraints require that product yields on the carbohydrate substrate approach the theoretical limits set by conservation laws and thermodynamics. For many products, achieving such near-theoretical yields represents a formidable challenge that not only requires ‘tuning’ of product pathways but also a reconfiguration of central metabolism, which plays pivotal roles in precursor supply, energy conservation and redox-cofactor regeneration.

With the aim to improve ethanol yields on sugar substrates, we have explored new engineering strategies to engineer redox metabolism in anaerobic S. cerevisiae cultures. Implementation of acetic-acid reduction pathways based on a bacterial acetylating acetaldehyde dehydrogenase enabled improved ethanol yields on lignocelllosic hydrolysates, in which acetic acid is a key inhibitor of yeast performance. Functional expression of Calvin-cycle enzymes enables increased ethanol yields by the reduction of carbon dioxide, which is abundantly present in all yeast-based anaerobic fermentation processes.

Cytosolic acetyl-coenzyme A is required for the formation of many heterologous products made by engineered S. cerevisiae strains, including lipids, isoprenoids and flavonoids. In S. cerevisiae, formation of this precursor is notoriously expensive in terms of ATP requirements due to the involvement of acetyl-CoA synthetase. We have demonstrated functional expression and assembly of a bacterial pyruvate-dehydrogenase complex in the yeast cytosol, which offers a new and attractive pathway towards cytosolic acetyl-CoA. The mitochondrial carnitine shuttle in another pathway that, theoretically, could lead to ATP-efficient provision of cytosolic acetyl-CoA. However, previous research indicated that, in vivo, the yeast carnitine shuttle operates unidirectionally and does not allow for export of acetyl-CoA from the mitochondrial matrix to the cytosol. By a combination of metabolic engineering, laboratory evolution and reverse engineering of laboratory-evolved strains, we identified mutations that enable the ‘reversal’ of this shuttle mechanism in glucose-grown yeast cultures.