title: New Orthogonal Transcriptional Switches Derived from Tet Repressor Homologues for Saccharomyces cerevisiae Regulated by 2,4-Diacetylphloroglucinol and Other Ligands.
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.
authors.affiliation: High Throughput Biology Center and Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine , Baltimore, Maryland 21205, United States.|Central Laboratories for Key Technologies, KIRIN Company Limited , Yokohama, Kanagawa 236-0004, Japan.
authors.forename: Shigehito
authors.initials: S
authors.lastname: Ikushima
authors.affiliation: High Throughput Biology Center and Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine , Baltimore, Maryland 21205, United States.|Institute for Systems Genetics and Department of Biochemistry & Molecular Pharmacology, NYU Langone Medical Center , New York, New York 10016, United States.
authors.forename: Jef D
authors.identifier: 0000-0001-5322-4946
authors.initials: JD
authors.lastname: Boeke
mesh_terms: D000818:Animals; D001565:Benzoates / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D017930:Genes, Reporter / Q000187:drug effects / Q000235:genetics; D008024:Ligands; D008322:Mammals / Q000235:genetics; D010696:Phloroglucinol / Q000031:analogs & derivatives* / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000187:drug effects / Q000235:genetics; D011549:Pseudomonas / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics*; D012441:Saccharomyces cerevisiae / Q000235:genetics*; D014158:Transcription, Genetic / Q000187:drug effects* / Q000235:genetics*
chemical_list: D001565:Benzoates; D008024:Ligands; D012097:Repressor Proteins; C034811:tetracycline resistance-encoding transposon repressor protein; C055607:cumic acid; C059817:2,4-diacetylphloroglucinol; D010696:Phloroglucinol
keywords: 2,4-diacetylphloroglucinol; CymR; PhlF; TetR homologue; transcriptional switch; yeast