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

该建议旨在阐明环境细菌的代谢途径，该途径负责胆汁盐的有氧降解。胆盐是一种具有表面活性的类固醇化合物，在所有脊椎动物的肠道中产生，并通过排泄物进入环境，在那里它们受到好氧和厌氧细菌代谢的影响。在之前的资助期内，我们发现了一种新的途径变体，它被一个独特的环境细菌家族所使用，Sphingomonadaceae，以其生物降解污染物的潜力而闻名。根据我们的基因组学、蛋白质组学和生化研究，这种途径变体的特点是羧基侧链降解的独特反应序列，羧基侧链是所有胆汁盐的一部分，并附着在类固醇骨架的特定位置。侧链降解的起始部分涉及酰基辅酶a脱氢酶在已知位置引入双键。由于我们的模式生物Sphingobium sp，菌株Chol11和相关鞘单胞菌完全缺乏从其他类固醇降解细菌中已知的这一过程所需的基因，因此从类固醇骨架上切割侧链的进一步反应仍然未知。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基化。在拟议的项目中，我们希望通过遗传，生理和生化方法并行研究两种模式生物的侧链降解。特别是，我们的目标是删除侧链降解的候选基因，包括前面提到的单加氧酶，以分析编码蛋白在代谢途径中的作用。同时，我们将研究整个细胞和细胞提取物的降解途径，通过提交所提出的途径的中间体作为底物。这些中间体必须通过生物转化来生产。最后，我们还旨在分离和表征该代谢途径的关键酶。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高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，因为它可能开创新的和可持续的方法来生产某些胆盐和其他类固醇化合物，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.