A dynamic range of well-controlled constitutive and tunable promoters are essential for metabolic engineering and synthetic biology applications in all host organisms. Here, we apply a synthetic hybrid promoter approach for the creation of strong promoter libraries in the model yeast, Saccharomyces cerevisiae. Synthetic hybrid promoters are composed of two modular components-the enhancer element, consisting of tandem repeats or combinations of upstream activation sequences (UAS), and the core promoter element. We demonstrate the utility of this approach with three main case studies. First, we establish a dynamic range of constitutive promoters and in doing so expand transcriptional capacity of the strongest constitutive yeast promoter, P(GPD) , by 2.5-fold in terms of mRNA levels. Second, we demonstrate the capacity to impart synthetic regulation through a hybrid promoter approach by adding galactose activation and removing glucose repression. Third, we establish a collection of galactose-inducible hybrid promoters that span a nearly 50-fold dynamic range of galactose-induced expression levels and increase the transcriptional capacity of the Gal1 promoter by 15%. These results demonstrate that promoters in S. cerevisiae, and potentially all yeast, are enhancer limited and a synthetic hybrid promoter approach can expand, enhance, and control promoter activity.

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.