Fatty alcohols are long-chain aliphatic hydrocarbons with one hydroxyl group usually attached to their terminal carbon. These important oleaginous chemicals are widely used in detergent, lubricant, personal care, and pharmaceutical industries. The use of oleaginous yeasts as a cell factory to produce fatty alcohols from renewable resources is a sustainable and promising alternative to traditional approaches relying on plant oils or petrochemicals. In this thesis project, the expression levels of multi-copy insertion of tafar1 gene in a model oleaginous yeast Yarrowia lipolytica was quantitatively measured first. Then the possibility of knocking out negative regulators of INO1 in this yeast to increase its fatty alcohol productivity was explored. Through NCBI protein BLAST, gene deletion targets, mot1, pah1, rpd3, and isw2, were selected, and each gene was then deleted separately from yeast genome by homologous recombination. Engineered strains were cultivated in shake flasks for 5 days using the YPD medium with 40g/L glucose (YPD4). Every 24 hours, the OD600 value of each strain was measured with a UVspectrophotometer, and concentrations of fatty alcohols produced by engineered strains were detected using GC-FID. The growth curve showed that the deletion of RPD3 gene significantly inhibited cell growth while no obvious change was observed for other gene deletions. Among the knockout strains, the RPD3 knockout strain produced the highest titer of hexadecanol 278.5 mg/L. However, no single gene knockout was found to enhance fatty alcohol production in comparison to the control strain. The results highlight the complexity and uncertainty of manipulating both structural and regulatory genes. Finally, based on these findings, future metabolic engineering strategies to increase fatty alcohol production in Y. lipolytica were proposed
