Saccharomyces cerevisiae BLYAS, a new bioluminescent bioreporter for detection of androgenic compounds

Abstract

A Saccharomyces cerevisiae strain, capable of autonomous bioluminescence, was engineered to respond to androgenic chemicals. The strain, S. cerevisiae BLYAS, contains the human androgen receptor in the chromosome and was constructed by inserting a series of androgen response elements between divergent yeast promoters GPD and ADH1 on pUTK401 that constitutively expressed luxA and luxB to create pUTK420. Cotransformation of this plasmid with a second plasmid (pUTK404), containing the genes required for aldehyde synthesis (luxCDE) and FMN reduction (frp), yielded a bioluminescent bioreporter responsive to androgenic chemicals. Using dihydrotestosterone (DHT) as a standard, the response time and the 50% effective concentration values were 3 to 4 h and (9.7 +/- 4.6) x 10(-9) M, respectively. The lower limit of detection in response to DHT was 2.5 x 10(-9) M, and in response to testosterone it was 2.5 x 10(-10) M. This strain is suitable for high-throughput screening of chemicals with potential for remote environmental monitoring systems because of the assay speed, sensitivity, and self-containment.

FIG. 1.

Cloning strategy for pUTK416 to -420. Sequential amplifications were used to generate GPDAREADH1. This construct was directionally cloned into the BamHI (B) and SpeI (Sp) site of pUTK401, generating pUTK416. A series of AREs (one to four) was directionally cloned into the BglII (Bg) and KasI (Ks) sites on pUTK416, yielding pUTK417 (one ARE), pUTK418 (two AREs), pUTK419 (three AREs), and pUTK420 (four AREs).

FIG. 2.

Three-dimensional plot of bioluminescence versus time for DHT. Initial bioluminescence was observed within 2 hours for 1.0 × 10⁻⁸ M and reached a maximum at approximately 6 hours.

FIG. 3.

EC₅₀-response profile of select chemicals using S. cerevisiae BLYAS. Data were obtained 4 hours postinduction and represent the means and standard deviations of 17 replicates for DHT and 3 replicates for all other chemicals.