Living cells (bacteria, yeast and mammalian cell lines) have been widely exploited for biosensing as they provide useful information about the bioavailability of target analyte. Nonetheless, one of the main drawbacks of cell-biosensors based on reporter gene technology is the long assay time (hours) due to the slow RNA production of the reporter enzyme upon induction.

Here we report the development of an improved bioluminescent yeast-based whole-cell bioassay which provides rapid (15 min) and sensitive detection of estrogenic compounds.

In particular, S. Cerevisiae cells were genetically engineered to express the human estrogen receptor (hERα or hERβ) and the Nanoluc luciferase under the regulation of Estrogen Response Elements (ERE). The two strains were named NanoYES-Alpha and NanoYES-Beta.

Nanoluc luciferase was used as BL reporter since it represents the brightest luciferase available to date (150-fold brighter than firefly luciferase). In addition, its small size (only 19KDa) and the absence of post-translational modifications, enable rapid synthesis and folding of the reporter thus reducing response time.

The Nanoluc luciferase coding sequence was further optimized to increase its expression efficiency in yeast cells, and the use of a destabilized variant, allowed to reduce the background expression obtaining a rapid response upon induction by target analyte.

The developed bioassay responds to 17β-estradiol (E2) with a limit of detection of 0.035±0.004 nM and EC50 of 0.6nM for NanoYES-Alpha, and LOD of 0.60±0.04 pM and an EC50 of 0.3nM with NanoYes-Beta. This performance was obtained with 15 min incubation time (compared to 2.5h of previously published yeast estrogen assays), making them the fastest BL yeast-estrogen bioassays developed to date.

In the perspective to use the developed biosensor for actual on-field analysis, we also integrated immobilized yeast(NanoYEs-Alpha) cells with a smartphone-controlled GoPro Hero5 camera as light sensor.