TitleSynthetic biology tools for programming gene expression without nutritional perturbations in Saccharomyces cerevisiae.
Publication TypeJournal Article
Year of Publication2014
AuthorsR McIsaac, S, Gibney, PA, Chandran, SS, Benjamin, KR, Botstein, D
JournalNucleic Acids Res
Volume42
Issue6
Paginatione48
Date Published2014 Apr
Keywords5' Untranslated Regions, Binding Sites, Estradiol, Galactokinase, Gene Expression Regulation, Fungal, Genetic Engineering, Ligands, Promoter Regions, Genetic, Receptors, Estrogen, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Synthetic Biology, Transcription Factors
Abstract

A conditional gene expression system that is fast-acting, is tunable and achieves single-gene specificity was recently developed for yeast. A gene placed directly downstream of a modified GAL1 promoter containing six Zif268 binding sequences (with single nucleotide spacing) was shown to be selectively inducible in the presence of β-estradiol, so long as cells express the artificial transcription factor, Z3EV (a fusion of the Zif268 DNA binding domain, the ligand binding domain of the human estrogen receptor and viral protein 16). We show the strength of Z3EV-responsive promoters can be modified using straightforward design principles. By moving Zif268 binding sites toward the transcription start site, expression output can be nearly doubled. Despite the reported requirement of estrogen receptor dimerization for hormone-dependent activation, a single binding site suffices for target gene activation. Target gene expression levels correlate with promoter binding site copy number and we engineer a set of inducible promoter chassis with different input-output characteristics. Finally, the coupling between inducer identity and gene activation is flexible: the ligand specificity of Z3EV can be re-programmed to respond to a non-hormone small molecule with only five amino acid substitutions in the human estrogen receptor domain, which may prove useful for industrial applications.

Alternate JournalNucleic Acids Res.