Microbial catabolism of steroids, from enzymes to ecosystems

微生物类固醇分解代谢在生物质分解、废水净化、药物合成和发病机理等方面发挥着重要作用。我的发现基金研究项目在我们对类固醇分解代谢的理解方面取得了重大进展。该项目的下一阶段的目标是(1)阐明尚不清楚的类固醇降解的初始步骤，(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因生态位而异，并可能在微生物-宿主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相关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分析，突变菌株的生产和特征，以及酶表达和活性测定。更广泛地说，微生物基因组学和宏基因组学方法为探索微生物代谢和生态学的多样性提供了令人兴奋的新潜力，但这些方法受到缺乏功能特征蛋白的严重阻碍，这导致基因的错误注释。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。有了这个知识库，我将绘制出类固醇分解代谢途径在微生物分类群和不同环境中的发生。因此，在微生物菌株和环境DNA，我将确定新的基因和途径，保证生化研究。总的来说，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我们对途径的进化和生态学的理解方面取得重要进展。这种理解将最大限度地提高我们在工业和环境过程中利用微生物类固醇转化的能力，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.