Engineering central carbon metabolism with phosphoketolase pathways in Saccharomyces cerevisiae

Abstract: We need more efficient biocatalysts to make sustainable microbial production of chemicals and fuels more profitable before they can replace petroleum-based sources. Rewiring the metabolic pathways in the biocatalysts to avoid the loss of carbon as CO2 can aid in improving product yields and thereby the profitability of the process. In this thesis, I investigated the use of phosphoketolase (PK) pathways in the yeast Saccharomyces cerevisiae to produce the precursor metabolite acetyl-CoA without loss of carbon as CO2. Firstly, we investigated the effect of acetyl-phosphate (AcP) accumulation from the linear PK pathway when downstream product formation is limited. Accumulated AcP was degraded to acetate, which limited the benefit of the PK pathway. Furthermore, we investigated a combinatorial strategy to supply acetyl-CoA and NADPH for fatty acid (FA) production. We combined the PK strategy with overexpression of the transcription factor Stb5 to activate NADPH generating pathways. This strategy increased the FA titer in the glucose phase, but with a counteractive response that possibly arose from the lack of an effective NADPH sink. Secondly, we expanded the linear PK pathway to a novel configuration of the cyclic non-oxidative glycolysis (NOG) that can recycle all the carbon from glucose into acetyl-CoA, thus potentially increasing product yields even further. We showed through kinetic modeling that the new configuration resolves potential bottlenecks in the previous configuration. We verified both in vitro and in vivo functionality of the cycle in S. cerevisiae . Furthermore, we demonstrated increased titers of an acetyl-CoA-derived product in the glucose phase compared to the linear PK pathway, indicating increased precursor supply from the cycle. Finally, we further characterized the S. cerevisiae strain with the cycle, using omics. Most notably, the cycle strain yielded respiro-fermentative growth in chemostat cultures with acetate as the main overflow metabolite. This points to a metabolic imbalance and extensive AcP degradation to acetate, which needs to be resolved before the cycle can be efficiently utilized. This thesis highlights the status of this novel NOG configuration and will aid in the further development of cell factories with high-yield production of acetyl-CoA-derived products.