Increased Acetyl-CoA Derived Production in Yeast through the Use of an Optimized Heterologous Phosphoketolase Pathway

Petroleum is a declining resource and easily accessible reserves are assumed to be depleted in a foreseeable future, which is why it is important to develop fuels derived from renewable resources. One alternative is to develop and use cell factories, which can directly convert plant-based material into fuels, of which a successful example is ethanol production by yeast. However, since ethanol cannot cover the full range of applications of liquid fuels today, it is also important to put effort in developing renewable energy dense fuel compounds, such as alkanes or fatty acid ethyl esters. 

This work investigates overproduction of fatty acids - the precursor for energy dense biofuels - in Saccharomyces cerevisiae through utilization of an alternative route for cytosolic acetyl-CoA production in yeast. The aim is to find efficient enzymes that catalyze the formation of acetyl-CoA directly from intermediates in the glycolysis and pentose phosphate pathway (PPP), which potentially could also provide additional NADPH required as a cofactor for fatty acid biosynthesis. Therefore, an in vitro evaluation of several heterologous phosphoketolases and phosphotransacetylases was conducted, and catalytically efficient enzymes were selected for in vivo confirmation. The most effective candidates displayed beneficial effects on growth rate when expressed in a pyruvate decarboxylase negative strain, likely due to the increased capacity of the strain to produce the essential cytosolic acetyl-CoA. Furthermore, selected candidates will be expressed in a strain with an acetyl-CoA carboxylase modified to have decreased posttranslational inactivation - thus yielding more of the fatty acid precursor malonyl-CoA - and the produced fatty acids will be quantified. In summary, the optimized heterologous route is capable of redirecting carbon flux towards C2-synthesis in yeast, and constitutes a promising tool to improve formation of acetyl-CoA derived products.