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Coordinated transcription factor and promoter engineering to establish strong expression elements in Saccharomyces cerevisiae
Author(s) -
Leavitt John M.,
Tong Alice,
Tong Joyce,
Pattie Jonathan,
Alper Hal S.
Publication year - 2016
Publication title -
biotechnology journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.144
H-Index - 84
eISSN - 1860-7314
pISSN - 1860-6768
DOI - 10.1002/biot.201600029
Subject(s) - promoter , mutant , transcription factor , saccharomyces cerevisiae , biology , transcription (linguistics) , gene , synthetic biology , gene expression , genetics , computational biology , microbiology and biotechnology , linguistics , philosophy
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.