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。C1单位是许多生物合成反应所必需的，此外，甲醛在许多代谢反应中产生，必须解毒。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。我们已经使用了一个模型甲基营养菌，扭脱甲基杆菌AM 1在这个项目中产生的基因组序列作为一个平台，开发一个新的概念框架，同化和能量代谢如何实现动态平衡，在这个有机体。我们建议开始使用生物化学、遗传学、基因组学和建模方法的组合来测试该模型。首先，我们将确定同化代谢的关键部分，乙醛酸再生循环，特别是关于产生还原当量的步骤，其余未知的细节。这一目标解决了在甲基营养生长过程中如何平衡还原当量的问题。其次，我们将评估两个主要同化途径之间的13-羟丁酰-CoA分支点的小分子调节剂，乙醛酸再生循环和PHB合成。这一目标解决的问题是什么信号参与调节碳流的主要同化branchpoint。最后，我们将评估在转录和酶活性水平的三个主要同化途径中涉及的基因的表达，并将此信息与关键中间体和辅因子的细胞库中的改变相关联。这一目标解决了如何根据细胞需要在这些途径之间平衡碳流的问题。本研究的结果将是一个系统水平上的甲基营养中心同化代谢的理解。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营养同化和能量代谢的整合以及甲基营养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.