Integrated Metabolism in Methylobacterium extroquens AM1

描述(由申请人提供)：细菌中的新陈代谢通过一系列复杂、动态和相互关联的代谢步骤(代谢模块)进行。这种代谢系统的输出取决于细胞中产生蛋白质的遗传回路和产生通量和代谢物池的代谢回路之间的相互作用。了解这个复杂的系统如何在细胞中发挥作用是综合新陈代谢研究的目标。以全球“组学”为基础的方法，结合代谢模型和生理洞察，正在为理解代谢网络如何作为一个综合系统发挥作用奠定基础。Extorquens是一种兼性甲基营养细菌，它具有两种截然不同的生长模式：在一碳化合物(C1)上生长是还原能力受限的，而在多碳化合物上生长是能量受限的。此外，通过过去由美国国立卫生研究院资助的这种细菌的工作，一套计算和遗传工具以及多变量“组学”数据集是可用的。因此，这种细菌正在成为一个有吸引力的模式系统，在其中使用不同代谢条件的比较研究来询问有关代谢整合的基本问题。我们建议分析甲基营养代谢如何作为一个完整的系统在Extorquens中发挥作用。到目前为止，我们的数据表明，在稳定状态下，细胞处于平衡状态，而在扰动期间，代谢网络失去平衡，细胞在转录、通量和代谢物水平上做出戏剧性的反应。这种反应使新陈代谢状态“重置”到不同的水平，细胞恢复到平衡状态。在下一个项目期间，我们将讨论这种反应是如何在代谢模块水平上发生的，并进行一系列操作来探测特定的代谢状态和反应情景。本项目的具体目标如下：1.确定细胞的代谢状态。将详细介绍中枢甲基营养代谢和已确定的其他核心代谢功能之间的联系，包括生长速度、应激反应、铁的获取和脂肪酸代谢。所有已知模块的代谢状态将通过测量转录本、蛋白质、酶活性、通量和代谢物池来确定。将确定一套定义每种代谢状态的标准。2.分析代谢状态如何在扰动下发生变化。使用与特定目标1相同的一组测量，我们将确定代谢模块如何响应底物生长速度和通量的变化，以及当基因修饰导致代谢状态改变时，它们如何变化，例如改变降低功率平衡和改变通过特定代谢模块的通量。将分析在特定目标1和2中产生的数据，以确定新陈代谢和遗传电路是如何整合的。这项研究有望在代谢整合方面产生新的原则，以及对C1代谢的新见解。

DESCRIPTION (provided by applicant): Metabolism in bacteria occurs via a set of complex, dynamic, and interconnected metabolic steps (metabolic modules). The outputs of such metabolic systems depend on the interplay between the genetic circuits in the cell, which generate proteins, and the metabolic circuits, which generate flux and metabolite pools. Understanding how this complex system functions in the cell is the goal of integrated metabolism studies. Global "omics"-based approaches coupled to metabolic models and physiological insight are setting the stage for understanding how the metabolic network functions as an integrated system. Methylobacterium extorquens is a facultative methylotrophic bacterium, which has the property of two dramatically different modes of growth: growth on one-carbon (C1) compounds is reducing-power limited, while growth on multi-carbon compounds is energy-limited. In addition, through past NIH-funded work on this bacterium, a suite of computational and genetic tools and multivariate "omics" datasets are available. Therefore, this bacterium is becoming an attractive model system in which to ask fundamental questions regarding metabolic integration, using comparative studies of different metabolic conditions. We propose to analyze how methylotrophic metabolism functions as an integrated system in M. extorquens. Our data to date show that at steady-state, the cells are in balance, while during perturbations, the metabolic network is shifted out of balance and the cells respond dramatically at the transcriptional, flux and metabolite levels. The response "resets" the metabolic state at a different level, and the cells return to a balanced state. In this next project period, we will address how this response occurs at the metabolic module level, and carry out a set of manipulations to probe specific metabolic states and response scenarios. The specific aims of this project are as follows: 1. Determine metabolic states of the cell. Linkages between central methylotrophic metabolism and other core metabolic functions that have been identified will be detailed, including growth rate, stress response, iron acquisition, and fatty acid metabolism. Metabolic states for all known modules will be determined by measuring transcripts, proteins, enzyme activities, fluxes, and metabolite pools. A set of criteria will be identified that define each metabolic state. 2. Analyze how the metabolic state changes in response to perturbations. Using the same set of measurements as in specific aim 1, we will determine how metabolic modules respond to changes in growth rate and flux of substrates and how they change when genetic modifications cause altered metabolic states, such as altered reducing power balance and altered flux through specific metabolic modules. The data generated in specific aim 1 and 2 will be analyzed to determine how the metabolic and genetic circuits are integrated. This study is expected to result in new principles in metabolic integration, as well new insights into C1 metabolism.