Engineering cytosolic acetyl-CoA metabolism in Saccharomyces cerevisiae

Doctoral thesis, 2015

A Saccharomyces cerevisiae strain carrying deletions in all three pyruvate decarboxylase genes (also called Pdc negative yeast) represents a non-ethanol producing platform strain for biochemical production. However, it cannot grow on glucose as the sole carbon source due to the lack of cytosolic acetyl-CoA for lipid biosynthesis. Its growth inability on glucose could be restored through directed evolution, which was explained by an in-frame internal deletion in MTH1 (MTH1-∆T). The MTH1-∆T allele resulted in reduced glucose uptake, which may attenuate the repression of respiratory metabolism. However, it was not clear what mechanism could provide the cells with sufficient precursors for cytosolic acetyl-CoA. Here we investigated this using a Pdc negative strain with MTH1-∆T, IMI076. Our results identified a route relying on Ach1 that could transfer acetyl units from mitochondria to the cytoplasm. Based on the results a new model was proposed, in which acetyl units are shuttled from the mitochondria to the cytoplasm in the form of acetate. In addition, a collection of Pdc negative strains was constructed and one of them was adaptively evolved on glucose via serial transfer. Three independently evolved strains were obtained, which can grow on glucose as the sole carbon source at maximum specific rates of 0.138 h-1, 0.148 h-1, 0.141 h-1, respectively. Several genetic changes were identified in the evolved Pdc negative strains by genome sequencing. Among these genetic changes, 4 genes were found to carry point mutations in at least two of the evolved strains: MTH1, HXT2, CIT1, and RPD3. Reverse engineering of the non-evolved Pdc negative strain through introduction of the MTH181D allele restored its growth on glucose at a maximum specific rate of 0.05 h-1 in minimal medium with 2% glucose. The non-synonymous mutations in HXT2 and CIT1 may function in the presence of mutated MTH1 alleles and could be related to an altered central carbon metabolism in order to ensure production of cytosolic acetyl-CoA in the Pdc negative strain. In connection with biobased chemical production, it is necessary to engineer the metabolism of cell factories such that the raw material, typically sugars, can be efficiently converted to the product of interest. Although IMI076 could grow on glucose, it was still inefficient at conversion of pyruvate to cytosolic acetyl-CoA. To increase cytosolic acetyl-CoA supply from pyruvate, pyruvate formate lyase and its activating enzyme from Escherichia coli were expressed with two different cofactors, ferredoxin or flavodoxin, and their reductase, respectively, and it was found that the co-expression of either of these cofactors had a positive effect on growth under aerobic conditions, indicating increased activity of PFL. The positive effect on growth was manifested as a higher final biomass concentration and a significant increase in transcription of formate dehydrogenase genes (FDHs). Among the two cofactors reduced flavodoxin was found to be a better electron donor than reduced ferredoxin.