Construction of High Sensitive Detection System for Endocrine Disruptors with Yeast n-Alkane-assimilating Yarrowia lipolytica

To construct a highly sensitive detection system for endocrine disruptors (EDs), we have compared the activity of promoters with the n-alkane-inducible cytochrome P450 gene (ALK1), isocitrate lyase gene (ICL1), ribosomal protein S7 gene (RPS7), and the translation elongation factor-1α gene (TEF1) for the heterologous gene in Yarrowia lipolytica. The promoters were introduced into the upstream of the lacZ or hERa reporter genes, respectively, and the activity was evaluated by β-galactosidase assay for lacz and Western blot analysis for hERα. The expression analysis revealed that the ALK1 and ICL1 promoters were induced by n-decane and by EtOH, respectively. The constitutive promoter of RPS7 and TEF1 showed mostly a high level of expression in the presence of glucose and glycerol, respectively. In particular, the TEF1 promoter showed the highest β-galactosidase activity and a significant signal by Western blotting with the anti-estrogen receptor, compared with the other promoters. Moreover, the detection system was constructed with promoters linked to the upstream of the expression vector for the hERα gene transformed into the Y. lipolytica with a chromosome-integrated lacZ reporter gene under the control of estrogen response elements (EREs). It was indicated that a combination of pTEF1p-hERα and CXAU1-2XERE was the most effective system for the E₂-dependent induction of the β-galactosidase activity. This system showed the highest β-galactosidase activity at 10∨-6 M E₂, and the activity could be detected at even the concentration of 10∨-10 M E₂. As a result, we have constructed a strongly sensitive detection system with Y. lipolitica to evaluate recognized/suspected ED chemicals, such as natural/synthetic hormones, pesticides, and commercial chemicals. The results demonstrate the utility, sensitivity, and reproducibility of the system for identifying and characterizing environmental estrogens.