Bile salts are amphiphilic steroids with digestive functions in vertebrates. Upon excretion, bile salts are degraded by environmental bacteria. Degradation of the bile salt steroid skeleton resembles the well-studied pathway for other steroids, like testosterone, while specific differences occur during side chain degradation and the initiating transformations of the steroid skeleton. Of the latter, two variants via either Δ^1,4- or Δ^4,6-3-ketostructures of the steroid skeleton exist for 7-hydroxy bile salts. While the Δ^1,4 variant is well known from many model organisms, the Δ^4,6 variant involving a 7-hydroxysteroid dehydratase as a key enzyme has not been systematically studied. Here, combined proteomic, bioinformatic, and functional analyses of the Δ^4,6 variant in Sphingobium sp. strain Chol11 were performed. They revealed a degradation of the steroid rings similar to that of the Δ^1,4 variant except for the elimination of the 7-OH as a key difference. In contrast, differential production of the respective proteins revealed a putative gene cluster for the degradation of the C₅ carboxylic side chain encoding a CoA ligase, an acyl-CoA dehydrogenase, a Rieske monooxygenase, and an amidase but lacking most canonical genes known from other steroid-degrading bacteria. Bioinformatic analyses predicted the Δ^4,6 variant to be widespread among the Sphingomonadaceae, which was verified for three type strains which also have the predicted side chain degradation cluster. A second amidase in the side chain degradation gene cluster of strain Chol11 was shown to cleave conjugated bile salts while having low similarity to known bile salt hydrolases. This study identifies members of the Sphingomonadaceae that are remarkably well adapted to the utilization of bile salts via a partially distinct metabolic pathway. IMPORTANCE This study highlights the biochemical diversity of bacterial degradation of steroid compounds, in particular bile salts. Furthermore, it substantiates and advances knowledge of a variant pathway for degradation of steroids by sphingomonads, a group of environmental bacteria that are well known for their broad metabolic capabilities. Biodegradation of bile salts is a critical process due to the high input of these compounds from manure into agricultural soils and wastewater treatment plants. In addition, these results may also be relevant for the biotechnological production of bile salts or other steroid compounds with pharmaceutical functions.

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