Rhodococcal steroid catabolism and responses to environmental stresses

自1995年以来，我一直持有发现基金，支持一个主要关注萜类化合物的微生物分解代谢的研究项目。这一种类繁多的化学物质，包括类固醇，在自然界大量产生，具有重要的生物效应和生态后果。这项研究计划卓有成效，对微生物新陈代谢和生态学知识做出了公认的贡献。该项目也是许多个人资助和成功项目的跳板。这项建议描述了对土壤细菌，乔氏红球菌RHA1的基础研究，调查类固醇分解代谢和对环境压力的反应。本研究的目的是(A)描述类固醇、胆固醇和胆酸的摄取系统，(B)阐明胆酸分解代谢的途径，包括反应、酶和基因，(C)确定对脱水、渗透胁迫和缺氧的耐受机制，以及(D)阐明控制对这些应激反应的遗传调控网络。主要研究方法包括(A)使用微阵列监测8000多个RHA1基因的表达，(B)缺失突变以确定基因功能，(C)蛋白质表达以确定其功能，以及(D)类固醇摄取和酶活性的测量。这项研究将是重要的，因为它将有助于对放线菌代谢、生理和生态关键方面的重要新理解，放线菌是一类在土壤有机质循环中具有关键作用的群体。这项研究还将促进涉及放线杆菌或其酶的应用，用于药物合成、其他工业生物转化(绿色化学)和污染环境的生物修复。

Since 1995, I have held a Discovery Grant supporting a research program focused mainly on microbial catabolism of terpenoids. This diverse family of chemicals, including steroids, is abundantly produced in the natural world and has important biological effects and ecological consequences. This research program has been highly productive and made well-recognized contributions to knowledge of microbial metabolism and ecology. The program has also been the springboard for numerous individually-funded and successful projects. This proposal describes fundamental research on the soil bacterium, Rhodococcus jostii RHA1, investigating steroid catabolism and responses to environmental stresses. Objectives of the research are to (a) characterize uptake systems for the steroids, cholesterol and cholic acid, (b) elucidate the pathway for cholic acid catabolism, including responsible reactions, enzymes and genes, (c) determine mechanisms for tolerance of desiccation, osmotic stress and anoxia and (d) elucidate genetic regulatory networks controlling responses to those stresses. Primary research approaches include (a) use of a microarray to monitor expression of over 8,000 RHA1 genes, (b) deletion mutagenesis to determine gene functions, (c) expression of proteins to determine their functions and (d) measurements of steroid uptake and enzymatic activities. This research will be important, as it will contribute important new understanding of key aspects of the metabolism, physiology and ecology of Actinobacteria, a group with critical roles in the recycling of organic matter in soil. The research will also facilitate applications involving Actinobacteria or their enzymes, for drug synthesis, other industrial biotransformations (green chemistry) and bioremediation of polluted environments.