Figure 5
Principle of A. adeninivorans yeast estrogen sensor (A-YES). A. adeninivorans G1211 transformed with the plasmids pAL-HPH-hERα and pAL-ALEU2m-GAA(2xERE-107)-phyK (G1211/pAL-HPH-hERα – pAL-ALEU2m-GAA(2xERE-107)-phyK) was the bio component of the A-YES. It expresses the estrogen receptor gene (TEF1 promoter – hERα gene – PHO5-terminator) constitutively and produces a relatively constant level of recombinant hERα independent of the estrogen concentration. In the presence of estrogen or estrogen analogues, however, hERα forms a hERα-estrogen-dimer complex, which binds to the ERE-region of the GAA promoter located in the second reporter gene expression cassette. The cassette (GAA-ERE- promoter – phyK gene – PHO5 terminator) is activated, the phyK gene is expressed and phytase is synthesized. Since this enzyme contains a native signal sequence it is secreted and accumulates extracellularly. The recombinant phytase level is then quantified using a simple biochemical method.(C) Specificity of the A-YES based on A. adeninivorans G1211/pAL-HPH-hERα – pAL-ALEU2m-GAA(2xERE-107)-phyK for a range of steroids and steroid metabolites. The graphs depict the log concentration of 17α-ethynylestradiol (□), 17β-estradiol (■), estrone (▲), estriol (●), coumestrol (○) and bisphenol A (+) plotted against the recombinant phytase activity of the medium after 30 h incubation.