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

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.

[1]  Fergal O'Gara,et al.  Characterization of Interactions between the Transcriptional Repressor PhlF and Its Binding Site at the phlA Promoter in Pseudomonas fluorescens F113 , 2002, Journal of bacteriology.

[2]  B. Barrell,et al.  Life with 6000 Genes , 1996, Science.

[3]  M. Gossen,et al.  Tetracycline-controlled transcription in eukaryotes: novel transactivators with graded transactivation potential. , 1997, Nucleic acids research.

[4]  Raquel Tobes,et al.  The TetR Family of Transcriptional Repressors , 2005, Microbiology and Molecular Biology Reviews.

[5]  F. O'Gara,et al.  Genome-wide investigation of cellular targets and mode of action of the antifungal bacterial metabolite 2,4-diacetylphloroglucinol in Saccharomyces cerevisiae. , 2013, FEMS yeast research.

[6]  Yizhi Cai,et al.  Multichange isothermal mutagenesis: a new strategy for multiple site-directed mutations in plasmid DNA. , 2013, ACS synthetic biology.

[7]  Ronald W. Davis,et al.  Functional profiling of the Saccharomyces cerevisiae genome , 2002, Nature.

[8]  Dieter Haas,et al.  Autoinduction of 2,4-Diacetylphloroglucinol Biosynthesis in the Biocontrol Agent Pseudomonas fluorescensCHA0 and Repression by the Bacterial Metabolites Salicylate and Pyoluteorin , 2000, Journal of bacteriology.

[9]  Yizhi Cai,et al.  Yeast Golden Gate (yGG) for the Efficient Assembly of S. cerevisiae Transcription Units. , 2015, ACS synthetic biology.

[10]  Jeff Hasty,et al.  A synthetic gene network for tuning protein degradation in Saccharomyces cerevisiae , 2007, Molecular systems biology.

[11]  J. Boeke,et al.  GeneDesign: rapid, automated design of multikilobase synthetic genes. , 2006, Genome research.

[12]  T. Keshavarz,et al.  Microbial metabolism of quorum-sensing molecules acyl-homoserine lactones, γ-heptalactone and other lactones , 2014, Applied Microbiology and Biotechnology.

[13]  Ron Weiss,et al.  Systematic Transfer of Prokaryotic Sensors and Circuits to Mammalian Cells , 2014, ACS synthetic biology.

[14]  Y. Fujita,et al.  Dual Regulation of the Bacillus subtilis Regulon Comprising the lmrAB and yxaGH Operons and yxaF Gene by Two Transcriptional Repressors, LmrA and YxaF, in Response to Flavonoids , 2007, Journal of bacteriology.

[15]  A. H. Wang,et al.  Crystal structure of IcaR, a repressor of the TetR family implicated in biofilm formation in Staphylococcus epidermidis , 2008, Nucleic acids research.

[16]  D. Janssen,et al.  Roles of Horizontal Gene Transfer and Gene Integration in Evolution of 1,3-Dichloropropene- and 1,2-Dibromoethane-Degradative Pathways , 2000, Journal of bacteriology.

[17]  Mark Gerstein,et al.  Biochemical and genetic analysis of the yeast proteome with a movable ORF collection. , 2005, Genes & development.

[18]  J. Vorholt,et al.  Cumate-Inducible Gene Expression System for Sphingomonads and Other Alphaproteobacteria , 2013, Applied and Environmental Microbiology.

[19]  J. Boeke,et al.  Development of a Tightly Controlled Off Switch for Saccharomyces cerevisiae Regulated by Camphor, a Low-Cost Natural Product , 2015, G3: Genes, Genomes, Genetics.

[20]  Christopher A. Voigt,et al.  Genomic Mining of Prokaryotic Repressors for Orthogonal Logic Gates , 2013, Nature chemical biology.

[21]  M. Fussenegger,et al.  A synthetic mammalian gene circuit reveals antituberculosis compounds , 2008, Proceedings of the National Academy of Sciences of the United States of America.

[22]  R. Eaton.  p-Cymene catabolic pathway in Pseudomonas putida F1: cloning and characterization of DNA encoding conversion of p-cymene to p-cumate , 1997, Journal of bacteriology.

[23]  Kunio Yamane,et al.  Bacillus subtilis LmrA Is a Repressor of the lmrAB and yxaGH Operons: Identification of Its Binding Site and Functional Analysis of lmrB and yxaGH , 2004, Journal of bacteriology.

[24]  M. T. Hasan,et al.  Exploring the sequence space for tetracycline-dependent transcriptional activators: novel mutations yield expanded range and sensitivity. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[25]  T. Nihira,et al.  Identification of the varR Gene as a Transcriptional Regulator of Virginiamycin S Resistance inStreptomyces virginiae , 2001, Journal of bacteriology.

[26]  R. Lo,et al.  The cumate gene-switch: a system for regulated expression in mammalian cells , 2006 .

[27]  William D. Richardson,et al.  A short amino acid sequence able to specify nuclear location , 1984, Cell.

[28]  Sangjo Han,et al.  Saccharomyces cerevisiae Genome-Wide Mutant Screen for Sensitivity to 2,4-Diacetylphloroglucinol, an Antibiotic Produced by Pseudomonas fluorescens , 2010, Applied and Environmental Microbiology.

[29]  Judy Qiu,et al.  Total Synthesis of a Functional Designer Eukaryotic Chromosome , 2014, Science.

[30]  Alban Ramette,et al.  Pseudomonas protegens sp. nov., widespread plant-protecting bacteria producing the biocontrol compounds 2,4-diacetylphloroglucinol and pyoluteorin. , 2011, Systematic and applied microbiology.

[31]  M Aldea,et al.  A Set of Vectors with a Tetracycline‐Regulatable Promoter System for Modulated Gene Expression in Saccharomyces cerevisiae , 1997, Yeast.

[32]  Joel S. Bader,et al.  Synthetic chromosome arms function in yeast and generate phenotypic diversity by design , 2011, Nature.

[33]  M. Lewandoski.  Conditional control of gene expression in the mouse , 2001, Nature Reviews Genetics.