Elucidation of the Newly Discovered Methane Fermentation Pathway by Systems-Level Approaches

甲烷气体是生物分解产生的副产品，是天然气的主要成分，也是污水处理厂和垃圾填埋场产生的沼气的主要成分。甲烷也是一种强大的温室气体，其效力是二氧化碳的20多倍。因此，重要的是要找到方法和方法，最大限度地减少从这些多个来源排放到大气中的甲烷的数量。但同样重要的是，避免浪费这种宝贵的能源，例如在油井地点产生甲烷时燃烧，因为通过管道运输甲烷是不可行的。以甲烷为主要营养物质的细菌，即所谓的“吃甲烷的人”，或称甲烷营养菌，分布广泛，并消耗大量自然产生的甲烷。可以利用甲烷营养来减少气体的释放。此外，人们越来越有兴趣利用这些细菌通过生物技术应用将废弃的甲烷转化为有价值的燃料和化学品。出于这些原因，了解甲烷氧化菌是如何利用甲烷作为营养物质是很重要的。最近发现的一种由这些生物生长的新模式导致甲烷转化为以前未知的产物。这一新的过程可能会改变甲烷在环境中消耗的整体情况。该项目旨在深入研究这种新的增长模式。然后，这些知识可以用来更好地了解这些细菌在消除自然界中有害甲烷方面的作用，并增加利用它们将废弃的甲烷转化为有价值的产品的可行性；两者都对气候变化和能源可持续性有潜在影响。此外，该项目将至少培训和指导10名学生，其中包括1名研究生，每年2-3名本科生，以及每年1-2名高中生。将利用高中的研讨会和公共活动，如西雅图太平洋科学中心的Paws on Science，广泛传播关于该项目及其结果的信息。将使用多层系统方法来阐明I类甲烷氧化细菌中新发现的基于甲烷的新陈代谢模式的细节：在氧气限制的条件下，甲烷被转化为甲醇，然后通过已建立的氧气依赖路线转化为甲醛。但甲醛随后通过核酮糖单磷酸途径和混酸发酵途径相结合，转化为排泄出来的甲酸盐、醋酸盐、乳酸、琥珀酸和氢气。该项目将解决的问题包括：碳如何在代谢的各个分支中流动，发酵如何平衡，是否发生混合发酵/呼吸代谢过程，如果发生，两者如何平衡，两种代谢模式如何调节，以及哪些成分是必不可少的。因为这些问题是相互关联的，涉及将新陈代谢网络作为一个系统来理解，所以它们特别适合使用系统方法。首先，被选为这些研究的模式生物的甲烷纳豆菌的现有代谢模型将被修改，以纳入新的途径，产生一系列预测。然后，将在呼吸和发酵条件下生长的稳定细胞中测量一组系统级参数，并测量从呼吸到发酵的转换时间点。这些参数包括转录组学、代谢组学和13C-全球通量标记，并结合产量、生长速度、底物消耗速度和产品生成速度的测量。最后，关键预测基因将发生突变，并对突变表型进行评估。在某些情况下，这将包括完整的系统级分析。通过这种方式，将产生一个全面的图像，包括在这种新的代谢发酵模式中所涉及的成分的特性、在其中执行该模式的代谢环境、以及细胞如何从呼吸模式转换到发酵模式。该项目将由芝加哥大学的系统和合成生物学集群和CBET的生物技术、生化和生物质工程项目共同资助。

Methane gas is generated as a byproduct from the decomposition of living things, and it is the main component of both natural gas and in the biogas generated from sewage treatment plants and landfills. Methane is also a powerful greenhouse gas, over 20 times more potent than CO2. Therefore, it is important to find ways and means to minimize the amount of methane being emitted to the atmosphere from these multiple sources. But it is also important to avoid wasting this valuable energy source, such as by flaring off methane when generated at oil well sites because it is not feasible to move it through a pipeline. Bacteria that use methane as a key nutrient, so-called "methane-eaters", or methanotrophs, are widespread, and consume a great deal of naturally-produced methane. Methanotrophy can be exploited to reduce release of the gas. In addition, there is growing interest in using these bacteria to convert wasted methane into valuable fuels and chemicals via biotechnology applications. For these reasons, it is important to understand how the methanotrophs use methane as a nutrient. A recently discovered new mode of growth by these organisms results in conversion of methane into products that were not previously known to be generated. This novel process will likely alter the overall picture of how methane is consumed in the environment. This project aims to thoroughly investigate this new growth mode. Such knowledge can then be used to better understand the role of these bacteria in removing harmful methane in nature, and increases the feasibility of using them to convert wasted methane into valuable products; both have potential impacts for climate change and energy sustainability. In addition, this project will involve training and mentoring of at least 10 students, including a graduate student, 2-3 undergraduates each year, and 1-2 high school students each year. Workshops at high schools and public events such as Paws on Science at the Pacific Science Center in Seattle will be used to widely disseminate information about this project and its outcomes.A multi-tiered systems approach will be used to elucidate the details of a newly discovered mode of methane-based metabolism in Group I methanotrophs: Under conditions of oxygen limitation, methane is converted to methanol and subsequently to formaldehyde via the already established oxygen-dependent route. But the formaldehyde is then converted to excreted formate, acetate, lactate, succinate, and hydrogen via a combination of the ribulose monophosphate pathway and a mixed acid fermentation pathway. Questions that will be addressed by this project include how carbon flows through the various branches of the metabolism, how the fermentation is balanced, whether a hybrid fermentation/respiration metabolic process occurs and if so, how the two are balanced, how the two metabolic modes are regulated, and which components are essential. Because these questions are interconnected and involve understanding the metabolic network as a system, they are especially amenable to using a systems approach. First, existing metabolic models for the methanotroph chosen as a model organism for these studies, Methylomicrobium alcaliphilum, will be modified to incorporate the new pathway, generating a set of predictions. Then a set of systems-level parameters will be measured in steady-state cells grown under respiratory and fermentation conditions and for timepoints in a switchover from respiration to fermentation. These parameters include transcriptomics, metabolomics, and 13C-global flux labeling coupled to measurements of yield, growth rate, substrate consumption rates and product generation rates. Finally, key predicted genes will be mutated and the mutant phenotypes assessed. This will include, in some cases, a full systems-level analysis. In this way a comprehensive picture will be generated to include the identity of the components involved in this new fermentation mode of metabolism, the metabolic context within which this mode is carried out, and how the cell makes the transition from a respiratory mode to a fermentation mode. This project will be funded jointly by the Systems and Synthetic Biology cluster in MCB and the Biotechnology, Biochemical and Biomass Engineering program in CBET.