(87b) Engineering Fatty Acid Responsive Elements for Metabolic Engineering in the Oleaginous Yeast, Yarrowia Lipolytica

In the past decade, Y.lipolytica has emerged as an excellent example of a microorganism with several biotechnology applications such as the production of citric acid, lipases and an assortment of lipids. One of the most interesting facets of Y.lipolytica is that its native metabolism is wired towards superior de-novo lipogenesis and an ability to accumulate considerable amounts of lipids. This native oleaginous and lipolytic potential has been further explored to engineer a strain capable of accumulating upwards of 90% lipid content. Most of these feats, however, have been accomplished with a handful of genetic tools to direct metabolic pathway engineering. To tap into the full potential of lipid engineering in Y.lipolytica for efficient and cost effective ways of producing value added lipid products, there is a need for the development of more lipid responsive genetic tools such as promoter systems that are inducible and have varying degrees of fatty acid responsiveness. To date, the only fatty acid inducible promoter system used is the acyl CoA oxidase, POX2, promoter. This native promoter spans 2000 bps and confers weak fatty acid responsiveness relative to engineered constitutive hybrid promoters. In this study, we have identified the fatty acid responsive motifs in the POX2 promoter. Initially, a series of conventional 5â?? truncations of the long promoter was performed. Oleic acid induced transcriptional strength of the truncated promoters was characterized using qPCR at a transcriptional level and using a GFP reporter at the translational level. DNA fragments in between regions of promoter truncations that demonstrated considerable decrease in strength were further screened for transcriptional binding motifs using EMSA coupled with DNase I foot printing. A library of â??protectedâ? sequences was created and alignments were performed to identify testable fatty acid responsive motifs. Here, we have demonstrated an effective methodology to identify substrate inducible elements that can be used to engineer tunable substrate responsive hybrid promoter switches to advance the toolkit for metabolic engineering applications in Y.lipolytica.