title: Engineering an NADPH/NADP+Redox Biosensor in Yeast.
abstract: Genetically encoded biosensors have emerged as powerful tools for timely and precise in vivo evaluation of cellular metabolism. In particular, biosensors that can couple intercellular cues with downstream signaling responses are currently attracting major attention within health science and biotechnology. Still, there is a need for bioprospecting and engineering of more biosensors to enable real-time monitoring of specific cellular states and controlling downstream actuation. In this study, we report the engineering and application of a transcription factor-based NADPH/NADP+redox biosensor in the budding yeast Saccharomyces cerevisiae. Using the biosensor, we are able to monitor the cause of oxidative stress by chemical induction, and changes in NADPH/NADP+ratios caused by genetic manipulations. Because of the regulatory potential of the biosensor, we also show that the biosensor can actuate upon NADPH deficiency by activation of NADPH regeneration. Finally, we couple the biosensor with an expression of dosage-sensitive genes (DSGs) and thereby create a novel tunable sensor-selector useful for synthetic selection of cells with higher NADPH/NADP+ratios from mixed cell populations. We show that the combination of exploitation and rational engineering of native signaling components is applicable for diagnosis, regulation, and selection of cellular redox states.
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Kongens Lyngby, 2800, Denmark.
authors.forename: Jie
authors.initials: J
authors.lastname: Zhang
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Kongens Lyngby, 2800, Denmark.
authors.forename: Nikolaus
authors.initials: N
authors.lastname: Sonnenschein
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Kongens Lyngby, 2800, Denmark.
authors.forename: Thomas P B
authors.initials: TP
authors.lastname: Pihl
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Kongens Lyngby, 2800, Denmark.
authors.forename: Kasper R
authors.initials: KR
authors.lastname: Pedersen
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Kongens Lyngby, 2800, Denmark.
authors.forename: Michael K
authors.initials: MK
authors.lastname: Jensen
authors.affiliation: The Novo Nordisk Foundation Center for Biosustainability, Technical University of Denmark , Kongens Lyngby, 2800, Denmark.|Joint BioEnergy Institute , Emeryville, California 94608, United States.|Biological Systems & Engineering Division, Lawrence Berkeley National Laboratory , Berkeley, California 94720, United States.
authors.forename: Jay D
authors.initials: JD
authors.lastname: Keasling
mesh_terms: D015374:Biosensing Techniques; D005818:Genetic Engineering / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D009249:NADP / Q000032:analysis* / Q000378:metabolism; D010084:Oxidation-Reduction; D011401:Promoter Regions, Genetic; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism
chemical_list: D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D014157:Transcription Factors; C086677:YAP1 protein, S cerevisiae; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D009249:NADP
keywords: biosensor; dosage-sensitive genes; redox; yeast