Integrated Metabolism in Methylobacterium extroquens AM1

描述（由申请人提供）：细菌的代谢是通过一系列复杂的、动态的、相互关联的代谢步骤（代谢模块）发生的。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起作用是综合代谢研究的目标。基于全球“组学”的方法与代谢模型和生理学洞察力相结合，为理解代谢网络如何作为一个综合系统发挥作用奠定了基础。勒索甲基细菌是兼性甲基营养细菌，它具有两种截然不同的生长模式：在单碳化合物上生长是还原力受限的，而在多碳化合物上生长是能量受限的。此外，通过过去美国国立卫生研究院资助的对这种细菌的研究，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型系统，通过对不同代谢条件的比较研究，可以提出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综合系统在敲诈勒索鼠中发挥作用。我们迄今为止的数据表明，在稳定状态下，细胞处于平衡状态，而在扰动期间，代谢网络偏离平衡，细胞在转录，通量和代谢物水平上做出显着反应。这种反应在不同的水平上“重置”了代谢状态，细胞回到了平衡状态。在下一个项目阶段，我们将研究这种反应是如何在代谢模块水平上发生的，并进行一组操作来探测特定的代谢状态和反应场景。本项目的具体目的如下：1。测定细胞的代谢状态。中枢甲基营养代谢与其他已确定的核心代谢功能之间的联系将被详细描述，包括生长速度、应激反应、铁获取和脂肪酸代谢。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。将确定一组标准来定义每种代谢状态。2. 分析代谢状态如何随扰动而变化。使用与具体目标1中相同的一组测量，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，例如通过特定代谢模块改变减少能量平衡和通量。在特定目标1和2中产生的数据将被分析，以确定代谢和遗传回路是如何整合的。本研究有望产生代谢整合的新原理，以及对C1代谢的新认识。

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.