Studies on the Anoxic Steroid Degradation by the Denitrifying Bacteria

The microbial degradation of steroids is challenging because of its complex chemical structure and low solubility in water. The intermediates involved in oxic steroid degradation have been studied in some detail, and a central aerobic pathway for steroid degradation was established twenty years ago. As a result, the oxic metabolism includes some oxygenase-catalyzed reactions which require molecular oxygen as a co-substrate. However, little is known about the mechanisms that operate in anoxic steroid metabolism. It is clear that anoxic metabolism involves unprecedented, oxygen-independent reactions. In my previous Ph. D. work, S. denitrificans was used as the model organism, and the initial steps involved in the anoxic cholesterol degradation have been proposed. This anoxic pathway includes a novel anaerobic hydroxylation at C-25 of cholesterol. However, nothing is known about the gene(s) and enzyme participated in this anoxic reaction. To understand further about how cholesterol is degraded by S. denitrificans under anoxic conditions, the chemical, biochemical, and molecular biological approaches will be applied. The specific aims are (1) To isolate and identify the intermediates involved in the following anoxic cholesterol-catabolizing pathway. (2) To find out more genes involved in anoxic cholesterol metabolism by screening a cosmid gene library of S. denitrificans. (3) To purify and characterize cholest-4-en-3-one hydroxylase that catalyzes the novel anaerobic hydroxylation at C-25 of cholesterol. In addition, some C18 steroid hormones will also be used as the substrate to isolate the denitrifying bacteria that can mineralize these steroids in the absence of dioxygen. Consider that some steroids are used as drugs, and most steroids are produced industrially by the process of biotransformation. The study on anoxic microbial steroid metabolism may unravel novel enzymes and transformation processes, and its potential impact on many biotechnological, pharmaceutical, and clinical applications can be expected.

Project ID:PA9801-1971
External Project ID:NSC98-2312-B182-003-MY3