Regulation of C1 Metabolism in Methylotrophs

甲基营养菌 C1 代谢的调节

• 批准号: 6723471
• 负责人: Mary E Lidstrom
• 金额: $ 21.8万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 1987
• 资助国家: 美国
• 起止时间: 1987-08-01 至 2008-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6723471
• 关键词: 1 carbon compound; Methanobacteriaceae; bacterial genetics; biochemical evolution; enzyme activity; formaldehyde; formates; gene expression; gene targeting; genetic regulation; methane; microarray technology; microorganism culture; microorganism metabolism; molecular cloning; oxidation; proteomics; transfection

DESCRIPTION (provided by applicant): The production, interconversion, and transfer of C1 units is an important metabolic system in all of biology. Methylotrophs are microorganisms capable of growth on C1 compounds as sole carbon and energy sources, and methylotrophy can be viewed as a specialized version of the C1 metabolism found in all organisms. A distinguishing feature of methylotrophic metabolism is the generation and consumption of formaldehyde as a central intermediate, the starting point for all of metabolism. We have gained major new insights into the pathways that consume formaldehyde in Methylobacterium extorquens AM1, and have developed a model for how the cell controls formaldehyde flux to achieve a dynamic balance of carbon and energy metabolism, avoiding formaldehyde toxicity. In this project, we will focus on the three pathways, or modules, that our working model predict are central to understanding flux of formaldehyde and energy metabolism, the H4MPT pathway, the H4F pathway, and the 3 formate dehydrogenases. We propose to begin to test our conceptual model of formaldehyde-related central metabolism in methylotrophy using a combination of biochemical, genetic, genomic, and computational approaches, focused initially on understanding how the cell responds to changes in the formaldehyde production rate. This complex system has two fundamental circuits that will be analyzed, the genetic circuit, consisting of the transcriptional and translational elements and the associated signaling components, with the output being transcripts and proteins, and the metabolic circuit, consisting of enzymes, cofactors, intermediates, and the associated signaling compounds, with the output being metabolic flux. Because of the difficulty of measuring all of the components and their characteristics, we will take a modular approach and measure outputs for each of the modules, integrating the results to create a systems-level understanding of response and resultant effects. The specific aims are: 1. Analyze the output of the genetic circuit with microarrays and proteomics. 2. Analyze the output of the metabolic circuit with enzyme assays, metabolite measurements, and direct flux measurements. 3. Integrate the results using computational models that correlate the functioning of the genetic circuit and the metabolic circuit. The result of this study will be a systems-level understanding of formaldehyde metabolism in methylotrophy. These approaches will provide a model for functional genomics at the physiological level, and will create a platform for future studies of the interaction between normal and stressed metabolism, and the mechanistic understanding of the interplay between genetic and metabolic circuits.

描述(申请人提供)：C1单位的产生、相互转化和转移是整个生物学中一个重要的代谢系统。甲基营养细菌是一种能够以氯化合物为唯一碳源和能源生长的微生物，甲基营养可以看作是在所有生物体中发现的一种特殊的氯代谢物。甲基营养代谢的一个显著特征是甲醛作为中心中间体的产生和消耗，它是所有新陈代谢的起点。我们对甲基extorquens AM1中消耗甲醛的途径有了重大的新见解，并开发了一个细胞如何控制甲醛通量的模型，以实现碳和能量代谢的动态平衡，避免甲醛毒性。在这个项目中，我们将重点关注我们的工作模型预测的三个通路或模块，它们是理解甲醛和能量代谢的关键，H4MPT途径，H4F途径和3个甲酸脱氢酶。我们建议开始使用生化、遗传、基因组和计算方法的组合来测试我们的概念模型，即在甲基营养中与甲醛相关的中央代谢，最初的重点是了解细胞如何对甲醛生成率的变化做出反应。这个复杂的系统有两个将要分析的基本电路，一个是遗传电路，由转录和翻译元件以及相关的信号成分组成，输出是转录本和蛋白质；另一个是代谢电路，由酶、辅因子、中间体和相关的信号化合物组成，输出是代谢通量。由于很难衡量所有组成部分及其特点，我们将采取模块化方法，衡量每个模块的产出，综合结果，以建立对反应和所产生影响的系统层面的理解。具体目的是：1.用基因芯片和蛋白质组学方法分析基因电路的输出。2.用酶测定、代谢物测定和直接通量测定分析代谢回路的输出。3.使用将遗传回路和新陈代谢回路的功能关联起来的计算模型来整合结果。这项研究的结果将是一个系统水平的了解甲醛代谢在甲基营养。这些方法将为生理水平的功能基因组学提供一个模型，并将为未来研究正常代谢和应激代谢之间的相互作用以及从机制上理解遗传和代谢回路之间的相互作用创造一个平台。