Journal
BIOTECHNOLOGY JOURNAL
Volume 11, Issue 7, Pages 866-876Publisher
WILEY-V C H VERLAG GMBH
DOI: 10.1002/biot.201600029
Keywords
Aro80; Circuits; Hybrid promoters; Inducible; Saccharomyces cerevisiae; traits; trans-acting factors
Funding
- DARPA Living Foundries Program [HR0011-14-3-0002]
- Air Force Office of Scientific Research [FA9550-14-1-0089]
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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.
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