Cloning and Identification of a New Repressor of 3,17β-Hydroxysteroid Dehydrogenase of Comamonas Testosteroni

Abstract 3,17β-hydroxysteroid dehydrogenase (3,17β-HSD) is a key enzyme that can degrade steroid compounds in Comamonas testosteroni. Tetracycline repressor (TetR) family, repressors existing in most microorganisms, may play key roles in regulating the expression of 3,17β-HSD. Previous reports showed that three TetR genes were located in the contig58 of C. testosteroni ATCC 11996 (GenBank: AHIL01000049.1), among which the first TetR gene encoded a potential repressor of 3,17β-HSD by sensing environmental signals. However, whether the other proposed TetR genes acts as repressors of 3,17β-HSD is still unknown. In the present study, we cloned the second TetR gene and analyzed the regulation mechanism of the protein on 3,17β-HSD using electrophoretic mobility shift assay (EMSA), gold nanoparticles (AuNPs)-based assay, and loss-of-function analysis. The results showed that the second TetR gene was 660-bp, encoding a 26 kD protein, which could regulate the expression of 3,17β-HSD gene via binding to the conserved consensus sequences located 1100-bp upstream of the 3,17β-HSD gene. Furthermore, the mutant strain of C. testosteroni with the second TetR gene knockout has good biological genetic stability, and the expression of 3,17β-HSD in the mutant strain is slightly higher than that in the wild type under testosterone induction, suggesting the mutant can efficiently degrade steroids as carbon sources. The mutant generated in this study can be used to treat environmental pollution caused by steroid hormones.