Regulation of C1 Metabolism in Methylotrophs

描述（由申请人提供）：C1单位的产生、相互转化和转移是所有生物学中重要的代谢系统。甲基营养菌是能够在C1化合物上生长作为唯一碳源和能源的微生物，并且甲基营养可以被视为在所有生物体中发现的C1代谢的专门版本。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，这是所有代谢的起点。我们对扭脱甲基杆菌AM1中消耗甲醛的途径获得了重要的新见解，并开发了一种细胞如何控制甲醛通量以实现碳和能量代谢动态平衡的模型，避免甲醛毒性。在这个项目中，我们将专注于我们的工作模型预测的三个途径或模块，这三个途径或模块对于理解甲醛和能量代谢的通量至关重要，即H4MPT途径，H4F途径和3种甲酸脱氢酶。我们建议开始使用生物化学、遗传学、基因组学和计算方法的组合来测试我们在甲基营养中与甲醛相关的中心代谢的概念模型，最初侧重于了解细胞如何响应甲醛产生速率的变化。这个复杂的系统有两个基本的电路，将被分析，遗传电路，由转录和翻译元件和相关的信号成分，输出是转录本和蛋白质，和代谢电路，由酶，辅因子，中间体，和相关的信号化合物，输出是代谢流。由于测量所有组件及其特性的难度，我们将采取模块化方法并测量每个模块的输出，整合结果以创建对响应和结果影响的系统级理解。具体目标是：1.用微阵列和蛋白质组学分析基因电路的输出。2.通过酶分析、代谢物测量和直接流量测量分析代谢回路的输出。3.使用将遗传回路和代谢回路的功能相关联的计算模型来整合结果。本研究的结果将是一个系统水平的了解甲醛代谢的甲基营养。这些方法将在生理水平上为功能基因组学提供一个模型，并将为未来研究正常和应激代谢之间的相互作用以及遗传和代谢回路之间相互作用的机制理解创造一个平台。

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.