Development of a recombinant Arxula adeninivorans cell bioassay for the detection of molecules with progesterone activity in wastewater

Abstract

This study describes the development of a bioassay to detect the presence of progesterone and progesterone-like molecules in wastewater samples. The basis of the bioassay is the integration of the human progesterone receptor gene into the yeast Arxula adeninivorans for the constitutive synthesis of the receptor. After incubation, binding of the analyte to the receptor induces the production of a reporter protein. Two reporter proteins were compared for detection parameters such as half-maximal activity (EC50), limit of detection (LoD) and limit of quantification (LoQ). When the extracellular phytase K was used, an EC50 value of 155 ng L(-1) and a LoD of 27 ng L(-1) progesterone were obtained after 4 h incubation, while use of the fluorescent dsRED as the reporter protein, resulted in an EC50 of 320 ng L(-1) and a LoD of 65 ng L(-1) after 20 h incubation. Use of phytase K as the reporter protein offers decreased incubation time and increased sensitivity; however the dsRED reporter system is less labor-intensive. Additionally, the affinity of known agonists and antagonists of the human progesterone receptor was determined. The utility of this bioassay was confirmed by measuring total progesterone equivalent concentration of samples from a wastewater treatment plant. The A. adeninivorans-based transactivation assay was able to measure concentrations of about 311 ng L(-1) in the influent stream but could not detect progesterone activity in effluent. One key feature of the assay is the robustness of A. adeninivorans, which allows sample measurement without any sample preparation.

Simultaneous detection of three sex steroid hormone classes using a novel yeast‐based biosensor

“…Cloning of G1212/hPR-CFP, G1212/hER-DsRed2, and G1212/hAR-GFP Construction of yeast strains was based on the Xplor 1 2 transformation/expression platform as described in previous works (Chamas et al, 2015;Pham et al, 2016). Constitutive expression of each receptor gene was under the control of a TEF1 promoter and a PHO5 terminator.…”

“…In two earlier publications (Chamas et al, ; Pham et al, ), the use of the fluorescent protein DsRed in A. adeninivorans was described. To accelerate the maturation of the DsRed protein, substitution of three amino acids (H41T, N42Q, and A145P) were introduced using a two‐step polymerase chain reaction as described in the work of Bevis and Glick ().…”

“…To overcome this limitation, the replacement of the enzyme reporter with fluorescent reporter proteins was attempted. The use of fluorescent proteins as reporters in yeast (Beck et al, 2005(Beck et al, , 2008b and in A. adeninivorans in particular (Chamas et al, 2015;Pham et al, 2016) has been demonstrated.…”

“…Arxula adeninivorans has already been demonstrated to be able to produce recombinant human hormone receptors and is the biological host of the commercially available biosensors, Arxula Yeast Estrogen Screen (A‐YES), Arxula Yeast Androgen Screen (A‐YAS), and Arxula Yeast Progesterone Screen (A‐YPS) (Chamas et al, ; Gerlach et al, ; Kaiser et al, ). In these three assays, a similar strategy is used to transform the receptor‐ligand binding into a measurable signal.…”

Blastobotrys (Arxula) adeninivorans: a promising alternative yeast for biotechnology and basic research

“…Sensors based on transgenic A. adeninivorans strains for detection of other molecules, such as omeprazole, lansoprazole (Pham Thi et al ., ) and progesterone (Chamas et al ., ), have also been developed. Two bioassays for photometric and spectrophotometric detection of glucocorticoids, A‐YGS and A‐YGFS, are operational but not yet commercialized (Pham Thi et al ., ).…”