Engineered Saccharomyces cerevisiae strain BioS-1, for the detection of water-borne toxic metal contaminants.
Saccharomyces cerevisiae responds to extracellular toxic stimuli by increasing intracellular cyclic AMP levels, leading to activation of a cAMP-dependent protein kinase, protein kinase A (PKA). Activated PKA phosphorylates downstream substrates, including specific DNA-binding proteins, to turn off the expression of most or all of the yeast genes. Such cAMP-PKA-mediated inhibition of gene expression in response to toxic stimuli appears to be unique to S. cerevisiae. For instance, in mammalian cells, the cAMP-PKA signaling pathway is rather responsive to growth factors and hormones in addition to being primarily involved in the activation of gene expression. Activation of gene expression by the cAMP-PKA pathway in mammalian cells is due mainly to the presence of cAMP-response elements (CREs) located in the promoters of many mammalian genes, and the expression of PKA-responsive stimulatory transcription factor CRE-binding protein, commonly referred as CREBP, which binds to the CREs. Thus, activation of the cAMP-PKA signaling pathway results in the phosphorylation of CREBP by PKA, and phosphorylated CREBP transactivates specific gene expression by interacting with the cognate CRE. Based on these findings, we sought to engineer a yeast-based biosensor, in which the stress-sensing cAMP-PKA pathway of yeast is coupled to the mammalian CREBP-CRE-stimulated gene expression pathway, which drives the expression of a reporter protein, such as green fluorescent protein (GFP). As a primary step toward the development of this biosensor, we engineered a yeast strain, BioS-1, by genetically altering YPH 501, a wild-type strain of S. cerevisiae, to express human CREBP and human CRE promoter-driven GFP. Exposure of BioS-1 to varying concentrations of As3+, Fe2+, Pb2+, and Cd2+ elicits concentration-dependent expression of the GFP reporter that can be easily monitored by the fluorescence emitted by GFP. The results also indicate that the engineered BioS-1 yeast cells can detect 2.5 ppm of these toxic metals and report it through the expression of GFP within 3 h. The results presented herein demonstrate that this engineered yeast strain can detect metal toxicants and can validate the use of this prototypic yeast strain to develop a biosensor that can be used to detect and monitor cytotoxic water-borne toxic heavy metals.
Radhika
V
V
Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Milkevitch
Matthew
M
Audigé
Valéry
V
Proikas-Cezanne
Tassula
T
Dhanasekaran
N
N
D001681:Biological Assay / Q000379:methods*; D015374:Biosensing Techniques / Q000379:methods; D050882:CREB-Binding Protein; D006801:Humans; D007700:Kinetics; D008670:Metals / Q000032:analysis* / Q000494:pharmacology*; D009687:Nuclear Proteins / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D015202:Protein Engineering / Q000379:methods; D012441:Saccharomyces cerevisiae / Q000145:classification / Q000187:drug effects* / Q000378:metabolism*; D013050:Spectrometry, Fluorescence / Q000379:methods; D015534:Trans-Activators / Q000032:analysis / Q000096:biosynthesis* / Q000235:genetics; D014874:Water Pollutants, Chemical / Q000032:analysis* / Q000494:pharmacology*
D008670:Metals; D009687:Nuclear Proteins; D015534:Trans-Activators; D014874:Water Pollutants, Chemical; D050882:CREB-Binding Protein; C494363:CREBBP protein, human