(259b) Engineering Responsive Hybrid Promoters for Metabolic Engineering in Oleaginous Yeast Yarrowia Lipolytica

Yarrowia lipolytica is a non-conventional, oleaginous yeast that grows on numerous substrates, including glycerol, fats, and other hydrophobic carbon sources. As an efficient producer of citric acid, lipase, and triacylglycerides, Y. lipolytica has gained attention as a bio-production platform. While genetic tools for constitutive Y. lipolytica promoters are available, metabolic engineering of Y. lipolytica would be advanced if inducible promoters of defined strength and inducibility were available. As an oleaginous yeast, engineering fatty acid synthesis and metabolism is of great interest, and there is considerably little information on the molecular details of fatty acid regulated gene expression. Through a series of promoter truncations of two fatty acid responsive genes, acyl-CoA oxidase 2 (POX2), and acetyl-CoA acetyl transferase (PAT1), we identified key elements in fatty acid regulated promoters including upstream activating sequences (UAS) and core promoter elements that confer fatty acid responsiveness. Using GFP as a reporter for promoter activity, we measured cell fluorescence with flow cytometry and quantitative PCR for gene-expression levels. This work further elucidates the substrate dependence on the induction of genes involved in β-oxidation and peroxisome proliferation by identifying consensus binding sites on the promoters and key transcriptional factors. We will describe our newly created fatty acid responsive synthetic hybrid promoters, which have well-defined strength and particular fatty acid inducer profiles. With new well-defined inducible promoters, metabolic engineering of fatty acid regulated pathways will increase yield, productivity, and the scope of molecules that can be produced by Y. lipolytica.