Cloning and identification of a new repressor of 3,17β-Hydroxysteroid dehydrogenase of Comamonas testosteroni

• Published in: Molecular biology reports 2021-11, Vol.48 (11), p.7067-7075
• Main Authors: Xie, Weiqi, Xia, Qin, Chen, Ling, Xiong, Guangming, Gao, Yuwei, Yu, Yuanhua, He, Xiuxia
• Format: Article
• Language: English
• Subjects: 17-Hydroxysteroid Dehydrogenases - antagonists & inhibitors; Animal Anatomy; Animal Biochemistry; Antibiotics; Bacterial Proteins - biosynthesis; Bacterial Proteins - chemistry; Bacterial Proteins - genetics; Biomedical and Life Sciences; Bioremediation; Cloning, Molecular; Comamonas testosteroni; Comamonas testosteroni - chemistry; Comamonas testosteroni - genetics; Comamonas testosteroni - metabolism; Dehydrogenases; Electrophoretic mobility; Enzyme Inhibitors - chemistry; Enzyme Inhibitors - metabolism; Genes; Histology; Life Sciences; Metabolic pathways; Morphology; Mutants; Nanoparticles; Original Article; Recombinant Proteins - biosynthesis; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; Repressors; Steroid hormones; Testosterone; TetR gene; Trans-Activators - biosynthesis; Trans-Activators - chemistry; Trans-Activators - genetics
• ISSN: 0301-4851; 1573-4978
• DOI: 10.1007/s11033-021-06566-9

Background 3,17β-hydroxysteroid dehydrogenase (3,17β-HSD) is a key enzyme in the metabolic pathway for steroid compounds catabolism 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 are 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 act as repressors of 3,17β-HSD are still unknown. Methods and Results In the present study, we cloned the second tetR gene and analyzed the regulatory 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 knocked-out mutant expresses 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. Conclusions The second tetR gene acts as a negative regulator in 3,17β-HSD expression, and the mutant has potential application in bioremediation of steroids contaminated environment.