Ligand Responsive Hybrid Promoters and Bidirectional 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, fine tuning metabolic engineering of Y. lipolytica would benefit greatly from inducible promoters of defined strength. 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. Here, we describe our work create fatty acid responsive hybrid promoters through two means. We first truncated endogenous beta-oxidation promoters that are known to be responsive to fatty acids. 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. GFP reporter expression and quantitative PCR measurements were made to characterize promoter activity. The second method used employs the UAS sequences from the XPR2 gene. We describe how tandem repeats of these UAS sequences act as a fatty acid responsive promoter when combined with transcriptional start site. We subsequently leveraged the bidirectional nature of these UAS sequences to create a more compact dual-expression vector. This dual-expression system enables more genes to be expressed in Y. lipolytica with smaller plasmids. With new well-defined inducible promoters and expression systems, metabolic engineering of fatty acid regulated pathways will increase yield, productivity, and the scope of molecules that can be produced by Y. lipolytica.