Identification of "missing links" in C- and D-ring cleavage of steroids by Comamonas testosteroni TA441

Comamonas testosteroni TA441 is capable of aerobically degrading steroids through the aromatization and cleavage of the A- and B-rings, followed by D- and C-ring cleavage via β-oxidation. While most of the degradation steps have been previously characterized, a few intermediate compounds remained unidentified. In this study, we proposed that the cleavage of the D-ring at C13-17 required the ScdY hydratase, followed by C-ring cleavage via the ScdL1L2 transferase. The anticipated reaction was expected to yield 6-methyl-3,7-dioxo-decane-1,10-dioic acid-coenzyme A (CoA) ester. To confirm this hypothesis, we constructed a plasmid enabling the induction of targeted genes in TA441 mutant strains. Induction experiments of ScdL1L2 revealed that the major product was 3-hydroxy-6-methyl-7-oxo-decane-1,10-dioic acid-CoA ester. Similarly, induction experiments of ScdY demonstrated that the substrate of ScdY was a geminal diol, 17-dihydroxy-9-oxo-1,2,3,4,5,6,10,19-octanorandrost-8(14)-en-7-oic acid-CoA ester. These findings suggest that ScdY catalyzes the addition of a water molecule at C14 of 17-dihydroxy-9-oxo-1,2,3,4,5,6,10,19-octanorandrost-8(14)-en-7-oic acid-CoA ester, leading to D-ring cleavage at C13-17. Subsequently, the C9 ketone of the D-ring cleavage product is converted to a hydroxyl group, followed by C-ring cleavage, resulting in the production of 3-hydroxy-6-methyl-7-oxo-decane-1,10-dioic acid-CoA ester.IMPORTANCEStudies on bacterial steroid degradation were initiated more than 50 years ago primarily to obtain substrates for steroid drugs. In recent years, the role of steroid-degrading bacteria in relation to human health has gained significant attention, as emerging evidence suggests that the intestinal microflora plays a crucial role in human health. Furthermore, cholic acid, a major component of bile acid secreted in the intestines, is closely associated with the gut microbiota. While Comamonas testosteroni TA441 is recognized as the leading bacterial model for aerobic steroid degradation, the involvement of aerobic steroid degradation in the intestinal microflora remains largely unexplored. Nonetheless, the presence of C. testosteroni in the cecum suggests the potential influence of aerobic steroid degradation on gut microbiota. To establish essential information about the role of these bacteria, here, we identified the missing compounds and propose more details of C-, and D-ring cleavage, which have remained unclear until now.