Genome-Based Analysis of Methylotrophy

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基本代谢系统。许多生物合成反应都需要C1单元，此外，甲醛是在许多代谢反应中产生的，必须解毒。甲基化营养体是一种能够以C1化合物为唯一碳源和能量源生长的微生物，甲基化营养体可以被视为所有生物中发现的C1代谢的特殊版本。我们利用该项目中生成的甲基化菌模型Methylobacterium extorquens AM1的基因组序列作为平台，为这种生物的同化和能量代谢如何实现动态平衡建立了一个新的概念框架。我们建议开始使用生化、遗传、基因组和建模方法的组合来测试这个模型。首先，我们将确定同化代谢的关键部分，乙醛酸盐再生周期的剩余未知细节，特别是关于产生还原等价物的步骤。这一目标解决了甲基营养生长过程中还原性当量如何平衡的问题。其次，我们将评估两种主要同化途径（乙醛酸再生循环和PHB合成）之间13-羟基丁基- cobranchpoint的小分子调节因子。这个目的解决了什么信号是参与调节碳在主要同化分支点的流动的问题。最后，我们将在转录和酶活性水平上评估参与三种主要同化途径的基因的表达，并将这些信息与关键中间体和辅助因子的细胞池的变化联系起来。这个目标解决了碳流如何根据细胞需要在这些途径之间平衡的问题。这项研究的结果将是对甲基化的中枢同化代谢的系统水平的理解。这些方法将为生理水平上的功能基因组学提供一个模型，并将为未来研究甲基化同化和能量代谢的整合以及甲基化和异养之间的转换创造一个平台。

DESCRIPTION (provided by applicant): The production, interconversion, and transfer of C1 units is an important basic metabolic system in all of biology. C1 units are required for a number of biosynthetic reactions and in addition, formaldehyde is produced in a number of metabolic reactions and must be detoxified. 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. We have used the genome sequence of a model methylotroph, Methylobacterium extorquens AM1 generated in this project as a platform to develop a new conceptual framework for how assimilatory and energy metabolism achieve dynamic balance in this organism. We propose to begin to test this model using a combination of biochemical, genetic, genomic, and modeling approaches. First, we will determine the remaining unknown details of a key part of assimilatory metabolism, the glyoxylate regeneration cycle, especially with respect to the steps generating reducing equivalents. This aim addresses the question of how reducing equivalents are balanced during methylotrophic growth. Second, we will assess small molecule regulators of the 13-hydroxybutyryI-CoAbranchpoint between two of the main assimilatory pathways, the glyoxylate regeneration cycle and PHB synthesis. This aim addresses the question of what signals are involved in regulating the flow of carbon at the main assimilatory branchpoint. Finally, we will assess expression of the genes involved in the three main assimilatory pathways at both transcript and enzyme activity levels and correlate this information with alterations in cellular pools of key intermediates and cofactors. This aim addresses the question of how carbon flow is balanced between these pathways according to cell needs. The result of this study will be a systems-level understanding of central assimilatory 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 integration of methylotrophic assimilatory and energy metabolism and the switch between methylotrophy and heterotrophy.