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

这一建议旨在阐明环境细菌中负责胆盐好氧降解的代谢途径。胆盐是一种表面活性的类固醇化合物，在所有脊椎动物的肠道中产生，并通过排泄进入环境，在那里它们受到有氧和厌氧细菌代谢的影响。在之前的资助期间，我们发现了一种新的途径变体，它正被一种独特的环境细菌家族--鞘氨醇单胞菌科--所使用，这种细菌以其在污染物的生物降解方面的潜力而闻名。根据我们的基因组、蛋白质组和生化研究，这种途径变体的特征是降解羧基侧链的反应序列不同，羧基侧链是所有胆盐的一部分，在特定位置附着在类固醇骨架上。这种侧链降解的起始部分包括在已知位置通过酰基-辅酶A-脱氢酶引入双键。从类固醇骨架上切割侧链的进一步反应尚不清楚，因为我们的模式生物Sphingobium sp、菌株Chol11和相关的鞘氨醇单胞菌完全缺乏从其他类固醇降解菌那里获得的这一过程所需的基因。蛋白质组学和生理学分析表明，Rieske单加氧酶参与了未知位置的羟化。在拟议的项目中，我们希望通过遗传、生理和生化方法以并行的方法研究两种模式生物的侧链降解。特别是，我们的目标是删除包括上述单加氧酶在内的侧链降解候选基因，以分析编码蛋白在代谢途径中的作用。同时，我们将通过提交所提出的途径的中间体作为底物来研究整个细胞和细胞提取物的降解途径。这些中间体必须通过生物转化来生产。最后，我们还旨在分离和鉴定这一代谢途径的关键酶。除了阐明一种新的细菌途径外，该项目还与生态学有关，因为它解决了通过粪便以相对较高的浓度进入农业区的潜在生态毒性类固醇的环境退化问题。此外，我们的项目与生物技术相关，因为它可能启动生产某些胆盐和其他类固醇化合物的新的和可持续的方法，这些都是重要的药物。

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.