Utilizing a Wastewater-Based Medium for Engineered Saccharomyces cerevisiae for the Biological Production of Fatty Alcohols and Carboxylic Acids to Replace Petrochemicals

Personal care items, pharmaceutical formulations, food additives, detergents, plasticizers, and industrial solvents manufactured from artificially synthesized acids or petrochemicals can be produced with more sustainable methods. The metabolic engineering of microbial hosts, specifically the heterotrophic yeast Saccharomyces cerevisiae, using a cost-effective wastewater-based growth medium could potentially provide a solution. Upregulation of the fatty acid biosynthesis (FAS) pathway present in this species is key to increasing all fatty acid-derived products. Three plasmids containing genes responsible for the production of the enzymes acetyl-CoA carboxylase (ACC1), fatty acid synthase (FAS), and fatty acid reductase (Far1), were transformed into S. cerevisiae cells using the PEG-LiOAc method. Transformation was conducted separately and together to optimize efficiency. We hypothesized that transformed cells would display more varied fatty acid and fatty alcohol profiles, and an increased ability to grow in a modified wastewater-based medium while degrading dissolved organics. A spectrophotometric assay predicated on the oxidation of NADPH to NADP+ was performed to determine the activity of the overexpressed enzymes. High performance liquid chromatography (HPLC) analysis underscored the presence of C-16 and C-18 fatty alcohols and fatty acids present in the yeast. Finally, a gas chromatography-mass spectrometry (GC-MS) analysis portrayed a reduction in organic compounds in wastewater media that was metabolized by S. cerevisiae while evidence of ethanol production via fermentation was seen. The study adds to research on renewable energy alternatives, but more importantly, demonstrates an effective method in which S. cerevisiae can biologically produce valuable specialty and commodity chemicals to mitigate petroleum use while removing organic content from wastewater.