Exploring Genetic Tools for Metabolic Engineering of Yeast Yarrowia lipolytica

Abstract

Abstract The oleaginous yeast Yarrowia lipolytica has several properties that gives it a high potential as a microbial cell factory for production of oleochemicals, proteins, and natural products. Although genetic tools have been developed for the yeast, more are needed to expand microbial fermentation using Y. lipolytica. The purpose of the thesis is to explore two genetic tools for metabolic engineering of Y. lipolytica. First, the functionality of three enzymes repurposed as colorimetric malonyl-CoA biosensors was tested. Second, a set of 18 tunable promoters with differential expression in different nitrogen conditions was investigated. In the biosensor project, a series of shake flasks experiments were performed to explore the properties of polyketide synthases repurposed as a biosensor. The biosensor signal strength was measured through absorbance in the UV-visible spectra. The dose-dependency of the biosensor was tested by employing it in three strain backgrounds, each assumed to have a different malonyl-CoA availability. The biosensor signal varied depending on the strain background, but not always as expected. Overexpression of the biosensor enzyme gene showed that the strain with increased lipid accumulation that was expected to give the lowest signal, actually gave the highest. This could be explained by the strain oxidising lipids, thereby increasing the malonyl-CoA concentration. In the promoter project, a strain for each promoter was constructed. GFP was used to measure the expression strength. The strains were grown in different media in a microbioreactor system which measured the biomass and fluorescence intensity. Some strains were also investigated in a fluorescence microscope. None of the tested promoter strains showed GFP expression, meaning the promoters did not initiate GFP gene transcription. The troubleshooting steps taken could not explain the unexpected results. More research is needed to establish the tools tested in this thesis. Instead of acting as biosensors, the polyketide synthases could be used for polyketide production. The polyketide produced would then need to be identified, followed by metabolic engineering strategies for increased production. To increase the detection range for the tested promoters, a brighter fluorescent protein and a more sensitive instrument could be used.