Generalizing a hybrid synthetic promoter approach in Yarrowia lipolytica
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APPLIED GENETICS AND MOLECULAR BIOTECHNOLOGY
Generalizing a hybrid synthetic promoter approach in Yarrowia lipolytica John Blazeck & Ben Reed & Rishi Garg & Rachelle Gerstner & Annie Pan & Vaibhav Agarwala & Hal S. Alper
Received: 23 June 2012 / Revised: 3 August 2012 / Accepted: 6 September 2012 # Springer-Verlag Berlin Heidelberg 2012
Abstract 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 highElectronic supplementary material The online version of this article (doi:10.1007/s00253-012-4421-5) contains supplementary material, which is available to authorized users. J. Blazeck : B. Reed : R. Garg : R. Gerstner : A. Pan : V. Agarwala : H. S. Alper Department of Chemical Engineering, The University of Texas at Austin, 1 University Station, C0400, Austin, TX 78712, USA H. S. Alper (*) Institute for Cellular and Molecular Biology, The University of Texas at Austin, 1 University Station, C0400, Austin, TX 78712, USA e-mail: [email protected]
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. Keywords Hybrid promoter . Promoter engineering . Yarrowia lipolytica . TEF
Introduction High, tunable levels of gene expression are necessary for metabolic and pathway engineering applications in model host organisms. However, newly isolated or poorly understood organisms often lack well-characterized promoters required for high transcription rates and tunable gene expression. Prior attempts to modulate gene expression include altering endogenous promoter strength through point mutations (Alper et al. 2005; Nevoigt et al. 2006), tuning intergenic regions in
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