New Orthogonal Transcriptional Switches Derived from Tet Repressor Homologues for Saccharomyces cerevisiae Regulated by 2,4-Diacetylphloroglucinol and Other Ligands

Shigehito Ikushima, Jef D. Boeke

    Research output: Contribution to journalArticle

    Abstract

    Here we describe the development of tightly regulated expression switches in yeast, by engineering distant homologues of Escherichia coli TetR, including the transcriptional regulator PhlF from Pseudomonas and others. Previous studies demonstrated that the PhlF protein bound its operator sequence (phlO) in the absence of 2,4-diacetylphloroglucinol (DAPG) but dissociated from phlO in the presence of DAPG. Thus, we developed a DAPG-Off system in which expression of a gene preceded by the phlO-embedded promoter was activated by a fusion of PhlF to a multimerized viral activator protein (VP16) domain in a DAPG-free environment but repressed when DAPG was added to growth medium. In addition, we constructed a DAPG-On system with the opposite behavior of the DAPG-Off system; i.e., DAPG triggers the expression of a reporter gene. Exposure of DAPG to yeast cells did not cause any serious deleterious effect on yeast physiology in terms of growth. Efforts to engineer additional Tet repressor homologues were partially successful and a known mammalian switch, the p-cumate switch based on CymR from Pseudomonas, was found to function in yeast. Orthogonality between the TetR (doxycycline), CamR (d-camphor), PhlF (DAPG), and CymR (p-cumate)-based Off switches was demonstrated by evaluating all 4 ligands against suitably engineered yeast strains. This study expands the toolbox of “On” and “Off” switches for yeast biotechnology.

    Original languageEnglish (US)
    Pages (from-to)497-506
    Number of pages10
    JournalACS Synthetic Biology
    Volume6
    Issue number3
    DOIs
    StatePublished - Mar 17 2017

    Keywords

    • 2,4-diacetylphloroglucinol
    • CymR
    • PhlF
    • TetR homologue
    • transcriptional switch
    • yeast

    ASJC Scopus subject areas

    • Biomedical Engineering
    • Biochemistry, Genetics and Molecular Biology (miscellaneous)

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