Developing synthetic, minimal promoters in Saccharomyces cerevisiae
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Promoters enable synthetic biologists to manipulate protein expression at the DNA level. For this reason, promoters are essential for almost all applications aiming to engineer an organism. Unfortunately, promoters available for eukaryotic organisms are derived directly from the genome. Such promoters are large and result in substantial and therefore, difficult DNA insertions to express a heterologous multi-gene pathway. Furthermore, their high sequence homology provides the organism the opportunity to perform homologous recombination resulting in undesirable gene deletions. For these reasons, there is a critical demand for short promoters with low sequence homology to the organism’s genome to continue synthetic biology advancements in eukaryotic hosts. This work addresses the need for yeast promoters by engineering the promoter’s two distinct DNA regions– the upstream activating sequence (UAS) and the downstream 3ˋ area comprised of the promoter’s core. The modularity of these regions is demonstrated in a non-conventional yeast, Yarrowia lipolytica by assembling multiple native UAS in tandem with a core. In doing so, the strongest promoters ever reported for Y. lipolytica were created. Drawing from these lessons, the length of promoters in the popular host strain Saccharomyces cerevisiae was minimized. The core region is first addressed by establishing promoter libraries with minimized de novo cores. Synthetic cores are isolated from a short promoter library and are evaluated in six DNA contexts to establish nine minimal cores with modularity, robustness, and context independence. Second, the UAS region was minimized. To do so, a randomized region of DNA was hybridized upstream of a synthetic minimal core to construct 18 de novo libraries of promoters. From these libraries, 26 short constitutive and inducible UAS elements were isolated. Collectively, this work highlights the utility of hybrid promoter engineering to increase the number of promoters available for host organisms, Y. lipolytica and S. cerevisiae. More importantly, it establishes a highly desirable set of 81 synthetic, minimal promoters of inducible and constitutive function that provides a 70-fold range of expression in S. cerevisiae. Furthermore, the workflow presented herein is generic enough for application in other eukaryotic host organisms to build their synthetic biology toolboxes.