Generalizing a hybrid synthetic promoter approach in Yarrowia lipolytica.

Both varied and strong promoters are essential for metabolic and pathway engineering applications in any host organism. To enable this capacity, here we demonstrate a generalizable method for the de novo construction of strong, synthetic hybrid promoter libraries. Specifically, we demonstrate how promoter truncation and fragment dissection analysis can be utilized to identify both novel upstream activating sequences (UAS) and core promoters-the two components required to generate hybrid promoters. As a base case, the native TEF promoter in Yarrowia lipolytica was examined to identify putative UAS elements that serve as modular synthetic transcriptional activators. Resulting synthetic promoters containing a core promoter region activated by between one and twelve tandem repeats of the newly isolated, 230 nucleotide UASTEF#2 element showed promoter strengths 3- to 4.5-fold times the native TEF promoter. Further analysis through transcription factor binding site abrogation revealed the GCR1p binding site to be necessary for complete UASTEF#2 function. These various promoters were tested for function in a variety of carbon sources. Finally, by combining disparate UAS elements (in this case, UASTEF and UAS1B), we developed a high-strength promoter with for Y. lipolytica with an expression level of nearly sevenfold higher than that of the strong, constitutive TEF promoter. Thus, the general strategy described here enables the efficient, de novo construction of synthetic promoters to both increase native expression capacity and to produce libraries for tunable gene expression.