Microbial catabolism of steroids, from enzymes to ecosystems

微生物的类固醇分解代谢在生物量分解、废水净化、药物合成和发病机制等方面发挥着重要作用。我的发现资助研究项目在我们对类固醇分解代谢的理解方面取得了非常重大的进展。该计划的下一阶段目标是(1)阐明鲜为人知的类固醇降解的初始步骤，以及(2)探索类固醇分解代谢的多样性、进化和生态学。我假设，最初的分解代谢步骤根据生态位的不同而不同，并可能在微生物与宿主的相互作用中发挥作用。我将研究胆固醇氧化酶和羟基类固醇脱氢酶的作用，包括它们对约氏红球菌使用膜结合类固醇和游离类固醇的相对重要性。这项研究将包括基因表达的分析，突变菌株的生产和特性，以及酶表达和活性测定。更广泛地说，微生物基因组和元基因组学方法为探索微生物代谢和生态的多样性提供了令人兴奋的新潜力，但这些方法由于缺乏功能特征的蛋白质而受到严重阻碍，这导致对基因的错误注释。我将在迭代过程中使用生物信息学和生化分析的独特组合来开发和扩展类固醇分解代谢基因/蛋白质的参考集合。有了这个知识库，我将绘制出跨越微生物类群和不同环境的类固醇分解代谢途径的发生图。因此，在微生物菌株和环境DNA中，我将识别值得进行生物化学研究的新基因和途径。总体而言，这项研究计划将在我们对微生物类固醇分解代谢的机制理解以及对这些途径的进化和生态学的理解方面取得重要进展。这一认识将最大限度地提高我们利用微生物类固醇转化用于工业和环境过程的能力，并可能为微生物-宿主相互作用的新范式打开大门。

Microbial steroid catabolism plays important roles in biomass decomposition, decontamination of wastewaters, synthesis of drugs and pathogenesis. My Discovery Grant research program has made very significant advances in our understanding of steroid catabolism. The next phase of the program has the goals of (1) elucidating the poorly-understood initial steps of steroid degradation and (2) exploring the diversity, evolution and ecology of steroid catabolism. I hypothesize that the initial catabolic steps vary according to ecological niches and potentially play roles in microbe-host interactions. I will investigate the roles of cholesterol oxidases and hydroxysteroid dehydrogenases, including their relative importance for the use of membrane-associated versus free steroids by Rhodococcus jostii. This investigation will involve analysis of gene expression, production and characterization of mutant strains as well as enzyme expression and activity assays. More broadly, microbial genomic and metagenomic approaches offer exciting new potential to explore the diversity of microbial metabolism and ecology, but these approaches are severely hampered by the lack of functionally-characterized proteins, which leads to mis-annotation of genes. I will use a unique combination of bioinformatic and biochemical analyses in an iterative process to develop and expand a reference set of steroid catabolism genes/proteins. With this knowledge base, I will map the occurrence of steroid catabolism pathways across microbial taxa and different environments. Thusly, in microbial strains and in environmental DNA, I will identify novel genes and pathways that warrant biochemical investigation. Overall, this research program will make important advances in our mechanistic understanding of microbial steroid catabolism and in our understanding of the evolution and ecology of the pathways. This understanding will maximize our ability to exploit microbial steroid transformations for industrial and environmental processes and may open the door to a new paradigm for microbe-host interactions.