Genome-Based Analysis of Methylotrophy

描述(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单位，此外，甲醛是在许多代谢反应中产生的，必须解毒。甲基营养细菌是一种能够以氯化合物为唯一碳源和能源生长的微生物，甲基营养可以看作是在所有生物体中发现的一种特殊的氯代谢物。我们利用本项目中产生的一种模式甲硫杆菌的基因组序列作为平台，为该生物体的同化和能量代谢如何实现动态平衡开发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再生循环的剩余未知细节，特别是关于产生还原当量的步骤。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13-羟基丁酸-辅酶A在两个主要同化途径，乙醛再生循环和PHB合成之间的分支点的小分子调节。这一目标解决了在主要的同化支点调节碳流所涉及的信号的问题。最后，我们将在转录和酶活性水平上评估三个主要同化途径中涉及的基因的表达，并将这些信息与关键中间体和辅因子细胞池的变化相关联。这一目标解决了如何根据细胞需求在这些途径之间平衡碳流的问题。这项研究的结果将是对甲基营养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的功能基因组学提供一个模型，并将为未来研究甲基营养的同化和能量代谢的整合以及甲基营养和异养之间的转换提供一个平台。

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.