Carbon Flow through the Etylmalonyl-CoA Pathway

该奖项是根据2009年美国复苏和再投资法案（公法111-5）资助的。最近，微生物代谢的范式发生了转变：重点不再仅仅是阐明底物如何转化为产物（输入和输出），而是包括协调一致的努力，以理解整个生物体代谢活动背景下途径的连通性。这个项目提供了一个需要这种新思维的例子。众所周知的乙基丙二醇-辅酶a途径和乙基丙二醇-辅酶a途径显然是将乙酸酯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。乙醛酸循环为乙酰辅酶a转化为苹果酸提供了一种简单的策略，它依赖于柠檬酸循环中的酶以及两种特殊的酶，异柠檬酸裂解酶和苹果酸合成酶。异柠檬酸裂解酶阴性菌的乙基丙二醇-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参与。那么问题来了，为什么一个给定的有机体，甚至是同一个生物体在不同的生长条件下，会使用其中一种途径而不是另一种途径。假设是两个途径的不同中间体和/或不同的调节事件需要通过任一途径的碳通量。本研究的最终目标是将乙基丙二醇-辅酶a途径置于作为模式生物的光合紫色非硫细菌球孢霉中心碳代谢的背景下。具体来说，乙基丙二醇辅酶a的几个步骤尚不清楚，将被阐明。比较基因组研究、突变体分析、14c示踪研究、体外酶活性检测、外源基因表达和重组蛋白表征将用于鉴定乙基丙二醇-辅酶a途径的进一步中间体和基因/蛋白。乙基丙二酰辅酶a途径可能在某些生物体中取代乙醛酸循环，因为各种底物可能通过外周途径在不同的点进入该途径。研究了野生型球藻和乙基丙二酰辅酶a途径突变体对多种有机化合物的光异养和化学异养生长。本文将从遗传学的角度阐明球藻中乙基丙二酰辅酶a途径的关键酶——crotonyl-CoA羧化酶/还原酶的调控机制。此外，其他可能属于同一调控回路的基因也将被识别出来。更广泛的影响：这些研究将提供对细菌中心碳代谢控制的进一步见解，并应增强我们对特定栖息地中碳循环转化的理解。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，并能够在他们自己的研究中为解决方案做出贡献。具有现代微生物代谢强大背景的学生将认识到生物转化的看似无限的潜力，并随后能够将这些知识应用于教育，学术或工业环境中。

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.