KMID : 0545120100200111563
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Journal of Microbiology and Biotechnology 2010 Volume.20 No. 11 p.1563 ~ p.1570
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Construction of high sensitive detection system for endocrine disruptors with yeast n-alkane-assimilating Yarrowia lipolytica
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Cho Eun-Min
Lee Haeng-Seog Eom Chi-Yong Akinori Ohta
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Abstract
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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-1¥á 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 ¥â- galactosidase assay for lacZ and Western blot analysis for hER¥á. 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 ¥â-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 hER¥á 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 pTEF1phER¥á and CXAU1-2XERE was the most effective system for the E2-dependent induction of the ¥â-galactosidase
activity. This system showed the highest ¥â-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.
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KEYWORD
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Endocrine disruptors detection system, Yarrowia lipolytica, hER¥á, ERE
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