Engineering central carbon metabolism with phosphoketolase pathways in Saccharomyces cerevisiae
Doctoral thesis, 2021

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.

precursor supply



non-oxidative glycolysis


metabolic engineering

platform strain



Konferensrummet 10’an, Forskarhus 1, Kemigården 4, Göteborg
Opponent: Jack Pronk, Delft Technical University, Netherlands


John Hellgren

Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology

Hellgren, J., Qi, Q., Nielsen, J. & Siewers, V. Proteome and transcriptome analysis of a yeast strain expressing a cyclic phosphoketolase pathway for improved acetyl-CoA supply

Over the last centuries, the excessive release of greenhouse gases into the atmosphere by mankind has hastened global climate change. To mitigate climate change, we must reduce our dependency on fossil fuels. Therefore, we need to produce fuels and chemicals from renewable sources instead. Many industrially relevant chemicals can be produced naturally by microorganisms from sugars. We discovered how they accomplish this when we started to decipher the genetic code, the instruction book of life. The yeast Saccharomyces cerevisiae, commonly known as Baker’s yeast, has been used for thousands of years by humans for its great capacity to make alcohol and raise bread. By changing the genetic code of S. cerevisiae, we can add traits from different organisms and construct a yeast cell factory that can convert sugars to biofuels and chemicals in a sustainable way.

In this thesis, I have engineered S. cerevisiae to convert sugars more efficiently, with the end goal of making cell factories a competitive alternative to fossil-based sources of chemicals and fuels. This was achieved by enabling yeast to use reaction pathways that do not involve loss of carbon as CO2. I implemented a carbon-conserving pathway into yeast and investigated how this pathway affected the physiology of the yeast cell. To conserve more carbon, I expanded this linear carbon-conserving pathway to an artificial cyclic pathway that does not exist in nature. This new cycle shows great potential in increasing product yields, but one of its current limitations involves by-product formation, which needs to be addressed first. In conclusion, this work sets the foundation for the use of a novel carbon-conserving cycle and will aid in the future development of efficient cell factories.

Subject Categories

Biochemistry and Molecular Biology

Bioinformatics and Systems Biology

Areas of Advance




Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 5047



Konferensrummet 10’an, Forskarhus 1, Kemigården 4, Göteborg


Opponent: Jack Pronk, Delft Technical University, Netherlands

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