Carbon Flow through the Etylmalonyl-CoA Pathway

通过乙基丙二酰辅酶A途径的碳流

• 批准号: 0842892
• 负责人: Birgit Alber
• 金额: --
• 依托单位: Ohio State University Research Foundation -DO NOT USE
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-06-15 至 2012-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=0842892&HistoricalAwards=false
• 关键词: Carbon; Flow; through; Etylmalonyl; CoA

This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).Recently, there has been a paradigm shift in microbial metabolism: the focus is no longer solely on the elucidation of how substrates are converted to products (inputs and outputs), but rather also includes a concerted effort to understand the connectivity of pathways in the context of the whole organism's metabolic activities. This project provides an example of what necessitates this new thinking. The well-known glyoxylate cycle and the recently described ethylmalonyl-CoA pathway are apparently equivalent ways to convert acetate to precursor metabolites used for cell carbon biosynthesis. However, the two pathways differ drastically. The glyoxylate cycle provides a simple strategy for converting acetyl-CoA to malate relying on enzymes of the citric acid cycle in addition to two specialized enzymes, isocitrate lyase and malate synthase. The ethylmalonyl-CoA pathway of isocitrate lyase-negative bacteria is much more complex. It requires at least five unique enzymes and novel metabolic intermediates are involved. The question then arises why one pathway is used over the other by a given organism or even by the same organism under different growth conditions. The hypothesis is that the distinct intermediates for the two pathways and/or differential regulatory events necessitate carbon flux through either pathway. The ultimate goal of this research is to place the ethylmalonyl-CoA pathway in the context of central carbon metabolism of R. sphaeroides, a photosynthetic purple non-sulfur bacterium used as a model organism.Specifically, several steps of the ethylmalonyl-CoA are not yet understood and will be elucidated. Comparative genomic studies, mutant analysis, 14C-tracer studies, in vitro detection of enzymatic activity, heterologous gene expression and characterization of recombinant proteins will be used to identify further intermediates and genes/proteins of the ethylmalonyl-CoA pathway. It is possible that the ethylmalonyl-CoA pathway replaces the glyoxylate cycle in certain organisms because various substrates may enter the pathway at different points through peripheral routes. Wild type R. sphaeroides and mutants of the ethylmalonyl-CoA pathway will be examined for photoheterotrophic and chemoheterotrophic growth on a variety of organic compounds. The mechanism by which the key enzyme of the ethylmalonyl-CoA pathway, crotonyl-CoA carboxylase/reductase, is regulated in R. sphaeroides will be elucidated using a genetic approach. Furthermore, other genes that may be part of the same regulatory circuit will be identified.Broader impacts: These studies will provide further insights into the control of central carbon metabolism in bacteria and should enhance our understanding of transformations underlying the carbon cycles in a given habitat. Graduate and undergraduate students involved in the project will be sensitized to this emerging aspect of microbial metabolism and will be able to contribute towards solutions in their own research. A student with a strong background in modern microbial metabolism will realize the seemingly unlimited potential of biological transformations and will subsequently be able to apply this knowledge in an educational, academic, or industrial setting.

该奖项是根据2009年美国复苏和再投资法案资助的（公法111-5）.最近，微生物代谢发生了范式转变：重点不再仅仅是阐明底物如何转化为产物（投入和产出），而且还包括在整个有机体的代谢活动的背景下，共同努力了解途径的连接性。这个项目提供了一个需要这种新思维的例子。众所周知的乙醛酸循环和最近描述的乙基丙二酰辅酶A途径显然是将乙酸盐转化为用于细胞碳生物合成的前体代谢物的等效途径。然而，这两种途径截然不同。乙醛酸循环提供了一种将乙酰辅酶A转化为苹果酸的简单策略，除了两种专门的酶，异柠檬酸裂解酶和苹果酸合酶外，还依赖于柠檬酸循环的酶。异柠檬酸裂解酶阴性细菌的乙基丙二酰辅酶A途径要复杂得多。它需要至少五种独特的酶和新的代谢中间体。那么问题就来了，为什么一种途径被给定的生物体使用而不是另一种途径，甚至在不同的生长条件下被相同的生物体使用。该假说是，不同的中间体的两个途径和/或差异的监管事件需要通过任何一个途径的碳通量。本研究的最终目的是将乙基丙二酰辅酶A途径置于R.具体地，乙基丙二酰辅酶A的几个步骤尚未被理解，将被阐明。比较基因组研究、突变体分析、14 C-示踪剂研究、酶活性的体外检测、异源基因表达和重组蛋白的表征将用于鉴定乙基丙二酰-CoA途径的进一步中间体和基因/蛋白。在某些生物体中，乙基丙二酰辅酶A途径可能取代乙醛酸循环，因为各种底物可能通过外周途径在不同点进入该途径。野生型R.将检查类球形菌和乙基丙二酰辅酶A途径的突变体在各种有机化合物上的光异养和化能异养生长。乙基丙二酰辅酶A途径的关键酶巴豆酰辅酶A羧化酶/还原酶在R. sphaeroides将使用遗传方法来阐明。更广泛的影响：这些研究将为控制细菌中的中央碳代谢提供进一步的见解，并应增强我们对特定栖息地碳循环的基础转换的理解。参与该项目的研究生和本科生将对微生物代谢的这一新兴方面敏感，并将能够在自己的研究中为解决方案做出贡献。在现代微生物代谢方面具有强大背景的学生将意识到生物转化的看似无限的潜力，并随后能够在教育，学术或工业环境中应用这些知识。