Fatty Acid Responsive Hybrid Promoter Design in Oleaginous Yeast Yarrowia lipolytica

Murtaza Shabbir Hussain1, Samuel Williams1, Mark Blenner1

1Department of Chemical & Biomolecular Engineering, Clemson University, Clemson, SC 29634.

Yarrowia lipolytica is a non-conventional oleaginous yeast that grows on numerous substrates, including glycerol, fats, and other hydrophobic carbon. As an efficient producer of citric acid, lipase, and fatty acids, Y. lipolytica has gained attention as a bioproduction organism. Well- controlled metabolic engineering of this yeast requires a set of inducible promoters that specifically regulate gene expression levels in response to specific molecules within the cell as well as in the extracellular environment. As an oleaginous yeast, the engineering of fatty acid synthesis and metabolism is of great interest, and considerably little information is available on the molecular details of fatty acid regulated gene expression in Y. lipolytica. Through a series of promoter truncations of two fatty acid responsive genes, acyl-CoA oxidase 2 (POX2), and acetyl-CoA acetyl transferase (PAT1), we have identified key elements in fatty acid regulated promoters including upstream activating sequences (UAS) and core promoter elements that confer fatty acid responsiveness. We measured gene expression levels using real-time PCR and GFP expression, and studied the effect of different fatty acids on gene expression. We provide a quantitative relationship between fatty acid concentration and gene expression. Furthermore, we describe the uncoupling of fatty acid responsive promoters from catabolite repression. Combining this information, we are creating new synthetic hybrid promoters of well-defined strength that have well-defined fatty acid response profiles. With new well-defined inducible promoters, metabolic engineering of fatty acid regulated pathways will increase yield, productivity, and the scope of molecules produced by Y. lipolytica.