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

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 pg/ml, estradiol between 1.6 and 12.8 pg/mland cholesterol between 19.3 and 15.4 pg/ml.. With the resulting cell-free system we could determine testosterone in a range between 28 and 219 pg/ml, estradiol between 0.029 and 0.430 pg/mg and cholesterol between 9.7 and 77.2 pg/ml [DOSAGE ERROR CORRECTED].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 3 alpha-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.