Coordinated transcription factor and promoter engineering to establish strong expression elements in Saccharomyces cerevisiae.

by Leavitt, JM; Tong, A; Tong, J; Pattie, J; Alper, HS

Biotechnol Journal. 2016 Jul;11(7):866-76. doi: 10.1002/biot.201600029. Epub 2016 May 19.

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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