Evaluation of precursor and cofactor engineering strategies influencing fatty acid metabolism in Saccharomyces cerevisiae

Abstract: If humanity is to reduce the rate of climate change, it is essential that our societies switch to a more sustainable production of fuels and chemicals, which in turn depends on technological development. Oleochemical production via microbial catalysts – such as the yeast Saccharomyces cerevisiae – can use a considerably broader range of renewable substrates compared to the conventional production processes. Additionally, it enables optimization of the catalytical properties of the chosen host via metabolic engineering. Oleochemicals are derived from fatty acids (FAs), whose biosynthesis depends on the conversion of a substrate to cytosolic acetyl-CoA – the precursor for FA synthesis. 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.      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.