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Engineered Saccharomyces cerevisiae strain BioS‐1, for the detection of water‐borne toxic metal contaminants
Author(s) -
Radhika V.,
Milkevitch Matthew,
Audigé Valéry,
ProikasCezanne Tassula,
Dhanasekaran N.
Publication year - 2005
Publication title -
biotechnology and bioengineering
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.136
H-Index - 189
eISSN - 1097-0290
pISSN - 0006-3592
DOI - 10.1002/bit.20344
Subject(s) - saccharomyces cerevisiae , protein kinase a , yeast , signal transduction , gene expression , biology , gene , transcription factor , microbiology and biotechnology , reporter gene , biochemistry , kinase , creb1 , phosphorylation , regulation of gene expression , chemistry , creb
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 As 3+ , Fe 2+ , Pb 2+ , and Cd 2+ 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. © 2005 Wiley Periodicals, Inc.