Construction of a biosensor mutant of Comamonas testosteroni for testosterone determination by cloning the EGFP gene downstream to the regulatory region of the 3,17β-HSD gene

Comamonas testosteroni (C. testosteroni) is able to catabolize a variety of steroids and polycyclic aromatic hydrocarbons. 3,17β-Hydroxysteroid dehydrogenase (3,17β-HSD) from C. testosteroni is a testosterone-inducible protein and a key enzyme in steroid degradation. 3,17β-HSD is a member of the short-chain dehydrogenase/reductase (SDR) superfamily. We found that a 2.4 kb regulatory DNA fragment upstream of the 3,17β-HSD gene (βhsd) responds to steroids and triggers βhsd gene induction. To exploit this cis-acting regulatory element for a steroid determination system, plasmids pK2.4-EGFP-4 and pBB2.4-EGFP-8 were constructed. Both plasmids contain the EGFP gene fused downstream to a 2.4 kb DNA fragment from C. testosteroni. However, whereas pK2.4-EGFP-4 could integrate into the chromosomal DNA of C. testosteroni and knock out the βhsd gene promoter, pBB2.4-EGFP-8 could replicate in C. testosteroni cells as a free plasmid DNA. After integration of pK2.4-EGFP-4 into the βhsd gene promoter, 3,17β-HSD expression could not be induced such that EGFP expression in the mutant cells was at low levels. In contrast, in C. testosteroni cells transformed with pBB2.4-EGFP-8 the expression of EGFP was induced with testosterone. Our results showed that fluorescence counts (relative fluorescence units; RFU) increased in parallel with testosterone concentrations. Of note, estradiol and cholesterol could not induce the EGFP reporter gene. In summary, this new biosensor system might be used for the specific determination of testosterone.