Bacterial metabolism of steroids, from enzymes to ecosystems

目前的建议是继续一个富有成效的发现基金研究项目。该计划的长期目标保持不变，即发现和理解微生物代谢，生理学和生态学的新方面，重点是类固醇分解代谢。我寻求广泛的理解，包括生物化学，遗传，进化和生态的观点。拟议的研究建立在我以前的发现基金支持的重要进展的基础上。在过去的五年里，我们已经阐明了红球菌（Rhodococcus spp）分解胆固醇的新途径，我们分析了8000多个基因组，绘制了微生物类群中各种类固醇分解代谢途径的发生图谱。此外，我们分析了300多个霰弹枪宏基因组，揭示了细菌类固醇分解代谢在许多自然、工程和宿主环境中的重要性，并确定了一些未培养类群的类固醇分解代谢能力。宏基因组分析显示，海绵中存在具有不同类固醇降解酶的细菌。我们将使用培养依赖性和独立方法进一步研究这些生物，重点关注海绵中发现的多种类固醇结构的新型酶转化。我们最近还发现，约氏红球菌可以在磷脂酰胆碱-胆固醇囊泡上生长，这些囊泡模拟了真核细胞膜中这些脂质的自然状态。有趣的是，磷脂酰胆碱只有在这种状态下才被分解代谢。因此，我们将研究参与囊泡生长和脂质摄取的调节机制和蛋白质。我们最近还发现，木犀草既能分解真核细胞中酯化成脂肪酸的胆固醇，也能在自身细胞内产生和储存胆固醇酯。我们将研究参与甾醇酯分解代谢和合成的酶，这些过程的调控及其与R. jostii和其他细菌的生态学相关性。这些研究将采用多种方法，包括宏基因组学、基因组学、转录组学、微生物分离和培养、分子遗传学、蛋白质表达和生物信息学。该研究的应用目标包括发现可用于化学产品合成，环境保护或治疗的新型生物催化剂。

The current proposal is to continue a productive Discovery Grant research program. The long-term goal of the program remains the same, to discover and understand novel aspects of microbial metabolism, physiology and ecology, with a focus on steroid catabolism. I seek a broad understanding encompassing biochemical, genetic, evolutionary and ecological perspectives. The proposed research builds upon important advances supported by my previous Discovery Grant. In the past five years, we have elucidated a novel pathway for catabolism of cholate by Rhodococcus spp. We have analyzed over 8000 genomes, mapping the occurrence of various steroid catabolism pathways among microbial taxa. And, we have analyzed over 300 shotgun metagenomes, revealing the importance of bacterial steroid catabolism in many natural, engineered and host environments as well as identifying the capacity for steroid catabolism in a number of uncultured taxa. The metagenome analysis revealed the presence of bacteria in marine sponges with very divergent steroid degradation enzymes. We will further investigate these organisms using both culture-dependent and independent approaches, focusing on novel enzymatic transformations of the diverse steroid structures found in sponges. We have also recently found that Rhodococcus jostii can grow on phosphatidylcholine-cholesterol vesicles, which model the natural state of these lipids in eukaryotic cell membranes. Interestingly, phosphatidylcholine is catabolized only when in this state. Therefore we will investigate the regulatory mechanisms and proteins involved in growth on the vesicles and uptake of the lipids. We have also recently found that R. jostii can both catabolize cholesterol esterified to fatty acids, which occur in eukaryotic cells, as well as produce and store cholesteryl esters within their own cells. We will investigate the enzymes involved in sterol ester catabolism and synthesis, the regulation of these processes and their ecological relevance to R. jostii and other bacteria. These investigations will employ diverse approaches, including metagenomics, genomics, transcriptomics, microbial isolation and culturing, molecular genetics, protein expression and bioinformatics. Applied goals of the research include the discovery of novel biocatalysts that can be employed for the synthesis of chemical products, environmental protection or therapeutics.