Metabolic engineering of lipid yeast using new genome editing techniques

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Kemian tekniikan korkeakoulu | Master's thesis
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Master's Programme in Chemical, Biochemical and Materials Engineering
In this work the aim was to produce medium chain dicarboxylic acids with metabolically engineered Y. lipolytica. The demand for sustainable raw materials is continuously growing and there is a need to find alternatives to petroleum based raw materials. In the future, the usage of microbes for production of different valuable chemicals is a growing field. One suitable organism is the oleaginous yeast Yarrowia lipolytica, which can be engineered for efficient and economical production of bio-based chemicals such as dicarboxylic acids. Dicarboxylic acids are useful for example as monomers for plastics such as nylon. To achieve this the metabolism of Y. lipolytica has to be engineered, which can be done with the genomic engineering tool CRISPR/Cas9, which is a globally growing method to make site specific gene deletions fast and efficiently. In this work a markerless CRISPR/Cas9 system was created for Y. lipolytica. Especially the expression of guide RNA (gRNA) under different promoters and in vitro gRNA was studied. As a result, deletions were successfully made with gRNA under a synthetic promoter SCR1 and Cas9 in the same plasmid, but the efficiency was not as high as expected. In this study, dicarboxylic acids were produced from glucose, with a genetically modified Y. lipolytica (deletion of β-oxidation and overproduction of ω-oxidation enzymes). Thioesterases were added to this modified strain to increase production of specifically medium chain length dicarboxylic acids, but these thioesterases did not make a difference to the product dicarboxylic acid chain length. As a result, we were able to produce long chain (16-C18) dicarboxylic acids directly from sugars. There is definitely potential in producing dicarboxylic acids from sugars, but further modifications to production strains are needed to make the process more efficient.
Frey, Alexander
Thesis advisor
Vartiainen, Eija
metabolic engineering, oleaginous yeasts, Yarrowia lipolytica, dicarboxylic acids, CRISPR/Cas9, free fatty acids
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