Bacterial metabolism of steroid compounds: studies on a novel degradation pathway for bile salts within the family Sphingomonadaceae

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This proposal aims at the elucidation of a metabolic pathway in environmental bacteria that is responsible for the aerobic degradation of bile salts. Bile salts are surface-active steroid compounds that are produced in the intestinal tract of all vertebrates and enter the environment via excretions where they are subject to aerobic and anaerobic bacterial metabolism. In previous funding periods, we identified a novel pathway variant that is being used by a distinct family of environmental bacteria, the Sphingomonadaceae, which are well-known for their potential in biodegradation of pollutants. According to our genomic, proteomic and biochemical studies, this pathway variant is particularly characterized by a distinct reaction sequence for the degradation of the carboxylic side chain, which is part of all bile salts and attached to the steroid skeleton at a specific position. The initiating part of this side-chain degradation involves the introduction of a double bond by an acyl-CoA-dehydrogenase at a known position. The further reactions for cleaving the side chain from the steroid skeleton remained unknown since our model organism Sphingobium sp, strain Chol11 and related sphingomonads completely lack the genes required for this process known from other steroid-degrading bacteria. Proteomic and physiological analyses indicate involvement of a hydroxylation in an unknown position by a Rieske monooxygenase. In the proposed project, we want to investigate side chain degradation in two model organisms in parallel approaches by genetic, physiological and biochemical methods. In particular, we aim at deleting candidate genes for side-chain degradation including the aforementioned monooxygenase for analyzing the role of the encoded proteins in the metabolic pathway. In parallel, we will study the degradation pathway with whole cells and cell extracts by submitting intermediates of the proposed pathway as substrates. These intermediates will have to be produced by biotransformation. Finally, we also aim at isolating and characterizing key enzymes of this metabolic pathway. Apart from the elucidation of a novel bacterial pathway this project has relevance for ecology because it addresses the environmental degradation of potentially ecotoxic steroids that enter agricultural areas in relatively high concentration via manure. Furthermore, our project is relevant for biotechnology because it might initiate new and sustainable ways for producing certain bile salts and other steroid compounds, which are important pharmaceuticals.