Improvement of reporter gene assay for highly sensitive dioxin detection using protoplastic yeast with inactivation of CWP and PDR genes

Kawanishi, Masanobu; Mori, Kentaro; Yamada, Rina; Ito-Harashima, Sayoko; Yagi, Takashi

Improvement of reporter gene assay for highly sensitive dioxin detection using protoplastic yeast with inactivation of CWP and PDR genes

2020

Kawanishi, Masanobu | Mori, Kentaro | Yamada, Rina | Ito-Harashima, Sayoko | Yagi, Takashi

A yeast reporter gene assay system with improved performance for dioxin detection was established. Since yeast reporter gene assays are relatively simple, easy to handle, and inexpensive, they have been used for various assessments of environmental contaminants. We previously constructed a yeast assay strain expressing the aryl hydrocarbon receptor (AhR) and AhR nuclear translocator (Arnt) carrying the lacZ reporter gene, for detection of dioxins. In the present study, genes encoding cell wall mannoproteins and ATP-binding cassette transporters in the yeast assay strains were deleted in order to increase the substance influx and prevent its efflux. We also established an assay procedure for protoplasts of these yeasts. These modifications improved the detection limit 40-fold and reduced the duration of the assay by 40%. By combining the yeast protoplast and a rapid sample preparation technique using disposal multilayer solid-phase extraction columns to remove unintended aryl hydrocarbons, this yeast reporter gene assay system detected the ligand activities of dioxins and related compounds in 1 g of forest soil containing dioxins at a concentration 10 times lower than the Japanese environmental standard for dioxins in soil.

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