Bacterial metabolism of steroids, from enzymes to ecosystems

目前的提议是继续开展一项富有成效的发现资助研究计划。该计划的长期目标保持不变，即发现和了解微生物代谢、生理学和生态学的新方面，重点是类固醇分解代谢。我寻求一种广泛的理解，包括生化、遗传、进化和生态角度。这项拟议的研究建立在我之前的探索基金支持的重要进展的基础上。在过去的五年里，我们阐明了红球菌分解胆酸代谢的一条新途径。我们已经分析了8000多个基因组，绘制了各种类固醇分解代谢途径在微生物类群中的发生。并且，我们已经分析了300多个鸟枪式元基因组，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，以及在一些未培养的类群中确定类固醇分解代谢的能力。后基因组分析显示，在具有非常不同的类固醇降解酶的海绵中存在细菌。我们将使用培养依赖和独立的方法进一步研究这些生物，重点是海绵中发现的各种类固醇结构的新的酶转化。我们最近还发现，Jostii红球菌可以生长在磷脂酰胆碱-胆固醇小泡上，这是真核细胞膜中这些脂类的自然状态的模拟。有趣的是，磷脂酰胆碱只有在这种状态下才会分解代谢。因此，我们将对囊泡生长和脂质摄取的调控机制和蛋白进行研究。我们最近还发现，R.jostii既可以将真核细胞中存在的胆固醇分解为脂肪酸，也可以在自己的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，这些过程的调节以及它们与约氏杆菌和其他细菌的生态相关性。这些研究将采用不同的方法，包括元基因组学、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合成化学产品、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.