(423a) Enabling Transcriptional Control in Saccharomyces Cerevisiae Utilizing a Synthetic Hybrid Promoter Engineering Approach

Well-characterized promoters are essential for pathway engineering and synthetic biology efforts in the model eukaryotic yeast, Saccharomyces cerevisiae.  Specifically, two main types of promoter elements are required: (1) a series of constitutive promoters exhibiting a dynamic range of expression capacities and (2) well-controlled inducible systems with defined expression outputs.  In this talk, we describe recent advances using our synthetic hybrid promoter approach to create a wide range of well-controlled constitutive and inducible promoters in S. 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 the hybrid promoter engineering 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 ability to augment regulation through hybrid promoters.  Specifically, we regulate constitutive expression of endogenous promoters through the use of a galactose-inducible UAS element and decrease the repressive capacity of the native P_GAL promoter through incorporating additional constitutive UAS elements.  Third, we create a library of inducible promoters that enable tunable galactose-inducible gene expression based on the strong P_GAL promoter with nearly a 50-fold dynamic range and likewise increase the transcriptional capacity of this promoter by more than 15%.  Collectively, this work creates the strongest characterized promoters in S. cerevisiae and establishes hybrid promoter construction as a generic, synthetic approach to expand, enhance, and control promoter strength in yeast.