title: Coordinated transcription factor and promoter engineering to establish strong expression elements in Saccharomyces cerevisiae.
abstract: Gene expression requires the coordination of trans-acting factors and cis-DNA elements to initiate transcription. Here we present a coordinated approach that combines cis-acting element engineering with mutant trans-acting factors to engineer yeast promoters. Specifically, we first construct a hybrid promoter based on the ARO9 upstream region that exhibits high constitutive and inducible expression with respect to exogenous tryptophan. Next, we perform protein engineering to identify a mutant Aro80p that affords both high constitutive expression while retaining inducible traits. We then use this mutant trans-acting factor to drive expression and generate ultra-strong promoters with transcriptional output roughly 2 fold higher than TDH3 (GPD), one of the strongest promoters to-date. Finally, we used this element to construct a modular expression system capable of staged outputs resulting in a system with nearly 6-fold, 12-fold and 15-fold expression relative to the off-state. This work further highlights the potential of using endogenous transcription factors (including mutant factors) along with hybrid promoters to expand the yeast synthetic biology toolbox.
authors.affiliation: Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, USA.
authors.forename: John M
authors.initials: JM
authors.lastname: Leavitt
authors.affiliation: Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, USA.|McKetta Department of Chemical Engineering, The University of Texas at Austin, Austin, Texas, USA.
authors.forename: Alice
authors.initials: A
authors.lastname: Tong
authors.affiliation: Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, USA.|McKetta Department of Chemical Engineering, The University of Texas at Austin, Austin, Texas, USA.
authors.forename: Joyce
authors.initials: J
authors.lastname: Tong
authors.affiliation: Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, USA.|McKetta Department of Chemical Engineering, The University of Texas at Austin, Austin, Texas, USA.
authors.forename: Jonathan
authors.initials: J
authors.lastname: Pattie
authors.affiliation: Institute for Cellular and Molecular Biology, The University of Texas at Austin, Austin, Texas, USA. halper@che.utexas.edu.|McKetta Department of Chemical Engineering, The University of Texas at Austin, Austin, Texas, USA. halper@che.utexas.edu.
authors.forename: Hal S
authors.initials: HS
authors.lastname: Alper
mesh_terms: D015966:Gene Expression Regulation, Fungal; D005818:Genetic Engineering / Q000379:methods*; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics; D058615:Synthetic Biology / Q000379:methods; D015534:Trans-Activators; D000637:Transaminases / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics*
chemical_list: D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D000637:Transaminases; C541341:aromatic aminotransferase II, S cerevisiae
keywords: Aro80; Circuits; Hybrid promoters; Inducible; Saccharomyces cerevisiae; traits; trans-acting factors