Evaluation of precursor and cofactor engineering strategies influencing fatty acid metabolism in Saccharomyces cerevisiae
Doctoral thesis, 2019
A major portion of the thesis is focused around a heterologous metabolic pathway to produce acetyl-CoA based on the activity of a phosphoketolase (XFPK) and a phosphotransacetylase (PTA). This pathway theoretically allows to reduce carbon and energy losses compared to the native yeast system. We identified several efficient XFPK candidates with potential to generate a high flux through the pathway. Furthermore, we show that two endogenous proteins – Gpp1 and Gpp2 – efficiently degrade the XFPK-formed produced acetyl-phosphate (AcP) to acetate, accumulating during cultivation. We show that this limits the benefit of the heterologous pathway, likely due to increased proton decoupling and ATP consumption during acetate activation. When we co-expressed XFPK and PTA, deletion of GPP1 appeared to be required to enable a significant flux towards acetyl-CoA during growth on glucose, reducing acetate accumulation. While a 25% increase in FA production was observed at the end of the glucose phase, the final titer was reduced by 20% compared to the control. We suggest that PTA expression negatively affects FA production during ethanol consumption due to low level of AcP during such conditions, leading to net flux from acetyl-CoA to AcP. Therefore, we propose that ethanol formation should be avoided in order to optimize XFPK/PTA use.
Regarding cofactor supply, we investigated if increasing activity of Stb5 – a transcriptional activator of genes involved in the pentose phosphate pathway (PPP) and NADPH production – could influence FA synthesis positively. STB5 overexpression had a beneficial effect on FA production in the glucose phase, an effect shown to be independent of flux through the PPP. However, final titers were affected negatively, and transcriptomic analysis indicates that mechanisms were activated in cells to counteract a Stb5-imposed redox imbalance. This suggests that an effective drain of NADPH – e.g. during product formation – is required to prevent systemic negative effects of STB5 overexpression.
The results produced within the scope of this thesis will serve as an aid in future metabolic engineering strategies targeting compounds relying on acetyl-CoA or NADPH.
Alexandra Linda Bergman
Chalmers, Biology and Biological Engineering, Systems and Synthetic Biology
Heterologous phosphoketolase expression redirects flux towards acetate, perturbs sugar phosphate pools and increases respiratory demand in Saccharomyces cerevisiae
Microbial Cell Factories,; Vol. 18(2019)
Investigation of putative regulatory acetylation sites in Fas2p of Saccharomyces cerevisiae. Manuscript, available at bioRxiv (https://doi.org/10.1101/430918)
Effects of overexpression of STB5 in Saccharomyces cerevisiae on fatty acid biosynthesis, physiology and transcriptome
FEMS Yeast Research,; Vol. 19(2019)
Functional expression and evaluation of heterologous phosphoketolases in Saccharomyces cerevisiae
AMB Express,; Vol. 6(2016)
However, the establishment of efficient oil crops, such as palm oil plantations in tropical regions, are associated with a large GHG emissions, biodiversity loss and soil degradation. Oleochemical production via microbial catalysts – such as the common baker’s yeast Saccharomyces cerevisiae – can use a considerably broader range of renewable substrates compared to the conventional production processes. Additionally, it allows for precise modification of the catalytic properties of the chosen host by introducing genetic alterations. This is a research field referred to as metabolic engineering.
Oleochemicals are derived from fatty acids (FAs), whose biosynthesis depends on the conversion of a substrate to the molecule acetyl-CoA – the precursor for FA synthesis. Cellular FA synthesis additionally requires large amounts of the reducing cofactor NADPH. The primary aim of this thesis was to develop and evaluate metabolic engineering strategies with potential to positively influence FA production of S. cerevisiae, mediated via an increased supply of acetyl-CoA or NADPH.
In part we evaluated enzyme candidates from bacteria producing acetyl-CoA via a mechanism decreasing energy and CO2 loss compared to the native yeast system. We also investigated if the activity of a positive cellular regulator of NADPH biosynthesis could be increased to enhance FA synthesis. The results produced within this thesis will aid in the future design of yeast cell factories capable of producing FAs at high level.
Biochemistry and Molecular Biology
Chemical Process Engineering
Doktorsavhandlingar vid Chalmers tekniska högskola. Ny serie: 4549
KA-salen, Kemigården 4, Kemihuset, Göteborg
Opponent: Professor Antonius van Maris, Department of Industrial Biotechnology, Royal Institute of Technology, Sverige