Characterization of 3,17β-hydroxysteroid dehydrogenase in Comamonas testosteroni

• Cyclizing RT-PCR was used to investigate the TSS and promoter of 3,17β-HSD.
• The growth and steroid degradation of 3,17β-HSD knock-out mutant were impaired.
• The steroid degradation of 3,17β-HSD high expression mutant was increased.
• 3,17β-HSD might be involved in cholesterol degradation pathway.

3,17β-Hydroxysteroid dehydrogenases (3,17β-HSDs) are found in all forms of life which catalyze the 3-position and 17-position reduction/oxidation of steroids. Comamonas testosteroni (C. testosteroni) ATCC11996 is a gram-negative bacterium which can use steroids as carbon and energy source. 3,17β-HSD is an enzyme which is involved in the complete oxidative degradation of the steroid skeleton, induced in the presence of these compounds in the culture medium. Cyclizing RT-PCR (cRT-PCR) was used to investigate the transcription start site (TSS) and promoter of 3,17β-HSD gene. To prove that 3,17β-HSD is involved in the metabolic pathway of steroid compounds, we prepared a 3,17β-HSD gene knock-out mutant and a mutant of C. testosteroni in which 3,17β-HSD was expressed at high level. The results indicate that 3,17β-HSD gene expression was induced by testosterone, but not by estradiol and cholesterol. Compared to the wild type C. testosteroni, degradation ability of testosterone and cholesterol was almost lost, and degradation of estradiol was decreased in the 3,17β-HSD knock-out mutant. Meanwhile degradation of testosterone, cholesterol was obviously increased in the 3,17β-HSD high expression mutant. Furthermore, the growths in the medium with testosterone, cholesterol or estradiol were impaired in 3,17β-HSD knock-out mutant. The results showed that in addition to testosterone and estradiol, 3,17β-HSD might be also involved in cholesterol metabolism. The location of the TSS and promoter of the 3,17β-HSD gene were found in this work.

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