Replacement of endogenous ethanol metabolism in Saccharomyces cerevisiae by an ATP-independent acetylating acetaldehyde dehydrogenase pathway

In Saccharomyces cerevisiae and other eukaryotes, ethanol dissimilation is initiated by its oxidation and activation to cytosolic acetyl-CoA. The associated consumption of ATP strongly limits yields of biomass and acetyl-CoA-derived products. Here, we explore implementation of an ATP-independent pathway for acetyl-CoA synthesis from ethanol that, in theory, enables superior biomass or product yields. To this end, all native yeast acetaldehyde dehydrogenases (ALDs) were replaced by heterologous acetylating acetaldehyde dehydrogenase (A-ALD), which can theoretically increase the biomass yield on ethanol by up to 40%. Engineered Ald- strains expressing different A-ALDs did not immediately grow on ethanol, but serial transfer in ethanol-grown batch cultures yielded growth rates of up to 70% of the wild-type value. Mutations in ACS1 were identified in all independent evolved strains and deletion of ACS1 enabled slow growth of Ald- A-ALD strains on ethanol. Acquired mutations in A-ALD genes improved VMAX/KM for acetaldehyde of the encoded enzymes. One of five evolved strains showed a significant 5% increase of its biomass yield in ethanol-limited chemostat cultures. Increased production of acetaldehyde and other by-products was identified as possible cause for lower than theoretically predicted biomass yields. This study proves that the native yeast pathway for conversion of ethanol to acetyl-CoA can be replaced by an engineered pathway that has the potential to strongly improve biomass and product yields. Based on metabolic and evolutionary engineering, whole-genome resequencing, reverse engineering and physiological analysis of evolved strains, we identify intracellular acetaldehyde levels and provision of intramitochondrial acetyl-CoA as key targets for further optimization of ethanol conversion by eukaryotic cell factories.