The effect of 3-ketosteroid-Δ(1)-dehydrogenase isoenzymes on the transformation of AD to 9α-OH-AD by Rhodococcus rhodochrous DSM43269.

Rhodococcus rhodochrous DSM43269 is well known for its 3-ketosteroid-9α-hydroxylases. However, the function of its 3-ketosteroid-Δ(1)-dehydrogenases (KSDD) remains unknown. This study compared the involvement of ksdds in the strain's androst-4-ene-3,17-dione (AD) transformation via gene deletion. The conversion was performed using AD as substrate or directly with 9α-hydroxyandrost-4-ene-3,17-dione (9α-OH-AD). The single deletion of ksdd1 or ksdd3 did not appear to result in the accumulation of 9α-OH-AD, whereas the single mutant △ksdd2 could preserve this compound to some extent. To further compare the role of ksdds in this strain, double mutants were constructed. All ksdd2 mutants combined with ksdd1 and/or ksdd3 resulted in the accumulation of 9α-OH-AD, among which the double mutant △ksdd2,3 behaved similarly to the single mutant △ksdd2 in this process. The mutant that lacked both ksdd1 and ksdd3 was still displayed, with no effect on the degradation of 9α-OH-AD. The triple mutant △ksdd1,2,3 was then constructed and exhibited the same capability as △ksdd1,2, accumulating more 9α-OH-AD than △ksdd2,3 and △ksdd2. The transcription of KSDD1 and KSDD2 increased, whereas that of KSDD3 seemed to exhibit no change, despite the use of the inducer AD or 9α-OH-AD. Thus, only ksdd1 and ksdd2 were involved in the transformation of AD to 9α-OH-AD. ksdd2 had the main role, ksdd1 had a minor effect on 9α-OH-AD degradation, and ksdd3 did not exhibit any action in this course.