title: Development of a Tightly Controlled Off Switch for Saccharomyces cerevisiae Regulated by Camphor, a Low-Cost Natural Product.
abstract: Here we describe the engineering of a distant homolog of the Tet repressor, CamR, isolated from Pseudomonas putida, that is regulated by camphor, a very inexpensive small molecule (at micromolar concentrations) for use in Saccharomyces cerevisiae. The repressor was engineered by expression from a constitutive yeast promoter, fusion to a viral activator protein cassette, and codon optimization. A suitable promoter responsive to the CamR fusion protein was engineered by embedding a P. putida operator binding sequence within an upstream activating sequence (UAS)-less CYC1 promoter from S. cerevisiae. The switch, named the Camphor-Off switch, activates expression of a reporter gene in camphor-free media and represses it with micromolar concentrations of camphor.
authors.affiliation: High Throughput Biology Center and Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Central Laboratories for Key Technologies, KIRIN Company Limited, Yokohama, Kanagawa 236-0004, Japan.
authors.forename: Shigehito
authors.initials: S
authors.lastname: Ikushima
authors.affiliation: Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, New York University Langone Medical Center, New York, New York 10011.
authors.forename: Yu
authors.identifier: http://orcid.org/0000-0003-4659-9832
authors.initials: Y
authors.lastname: Zhao
authors.affiliation: High Throughput Biology Center and Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205 Institute for Systems Genetics and Department of Biochemistry and Molecular Pharmacology, New York University Langone Medical Center, New York, New York 10011 jef.boeke@nyumc.org.
authors.forename: Jef D
authors.identifier: http://orcid.org/0000-0001-5322-4946
authors.initials: JD
authors.lastname: Boeke
mesh_terms: D001688:Biological Products / Q000494:pharmacology*; D002164:Camphor / Q000494:pharmacology*; D015870:Gene Expression; D005786:Gene Expression Regulation; D023061:Gene Order; D017930:Genes, Reporter; D005818:Genetic Engineering; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000235:genetics* / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D001688:Biological Products; D012097:Repressor Proteins; D002164:Camphor
keywords: CamR; Pseudomonas putida; Tet system; TetR homolog