Cis- and trans-regulatory elements of 3a-hydroxysteroid dehydrogenase/carbonyl reductase as biosensor system for steroid determination in the environment

摘要

3alpha-Hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni is a key enzyme in the degradation of steroids in the environment. The encoding gene, hsdA, is expressed only at very low levels in the absence of steroids, but undergoes a several fold induction in the presence of steroid substrates. In previous investigations, we have elucidated the mechanism of hsdA regulation that involves several activators and repressors. In the present study, the hsdA gene was replaced by the green fluorescent protein (GFP) gene which was inserted downstream from the hsdA regulatory region. By homologous integration into the chromosomal DNA, the C testosteroni mutant strain CT-GFP5-1 was generated and used as fluorescence based biosensor system for steroid determination. With this cell-based system we could determine testosterone in a range between 57 and 450 ng/ml, estradiol between 1.6 and 12.8 ng/ml, and cholesterol between 19.3 and 154.4 ng/nl. Interestingly, the sensitivity of this bioassay could be further increased by using only the cytosol of the mutant. With the resulting cell-free system we could determine testosterone in a range between 28 and 219pg/ml, estradiol between 0.029 and 0.430 fg/ml, and cholesterol between 9.7 and 77.2 fg/ml. The recovery ratio of the extraction was around 95% and the maximum fluorescence signals were obtained as early as after 30 min. Limitations of the established steroid biosensor system were quenching at higher steroid concentrations and the relatively high background of fluorescence, which are currently being improved in our lab. Combined, by exploiting the regulatory region of the gene hsdA that codes for the enzyme 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase we have constructed a mutant C testosteroni strain that can be used as a sensitive biosensor system for steroid determination in the environment.