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，是天然气和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，比二氧化碳强20倍以上。 因此，重要的是找到方法和手段来最小化从这些多种来源排放到大气中的甲烷的量。 但同样重要的是要避免浪费这种宝贵的能源，例如在油井产生甲烷时燃烧甲烷，因为通过管道输送甲烷是不可行的。 利用甲烷作为关键营养物的细菌，即所谓的“甲烷食用菌”或甲烷营养菌，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。 由于这些原因，了解甲烷氧化菌如何将甲烷作为营养物是很重要的。 最近发现的一种新的生长模式，这些生物体的结果是甲烷转化成产品，以前不知道会产生。 这一新的过程可能会改变甲烷在环境中消耗的整体情况。 本项目旨在深入研究这种新的增长模式。 这些知识可用于更好地了解这些细菌在去除自然界中有害甲烷方面的作用，并增加利用它们将废弃甲烷转化为有价值产品的可行性;两者都对气候变化和能源可持续性具有潜在影响。 此外，该项目将涉及培训和指导至少10名学生，包括一名研究生，每年2-3名本科生，每年1-2名高中生。 将利用高中讲习班和公共活动，如西雅图太平洋科学中心的Paws on Science，广泛传播有关该项目及其成果的信息。将采用多层次系统方法阐明第I组甲烷氧化菌中新发现的甲烷代谢模式的细节：在氧限制的条件下，甲烷转化为甲醇，随后通过已经建立的氧依赖性途径转化为甲醛。 但是甲醛随后通过核酮糖单磷酸途径和混合酸发酵途径的组合转化为排泄的甲酸盐、乙酸盐、乳酸盐、琥珀酸盐和氢。 该项目将解决的问题包括碳如何流经代谢的各个分支，发酵如何平衡，混合发酵/呼吸代谢过程是否发生，如果是这样，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，并且涉及将代谢网络理解为一个系统，所以它们特别适合使用系统方法。 首先，现有的代谢模型的甲烷氧化菌选择作为这些研究的模式生物，嗜碱甲基微菌，将被修改，以纳入新的途径，产生一组预测。 然后，将在呼吸和发酵条件下生长的稳态细胞中测量一组系统水平参数，并测量从呼吸到发酵的时间点。 这些参数包括转录组学、代谢组学和13 C-全局通量标记，以及产量、生长速率、底物消耗速率和产物生成速率的测量。 最后，将突变关键预测基因并评估突变表型。 在某些情况下，这将包括全面的系统级分析。 以这种方式，将产生一个全面的画面，包括在这种新的代谢发酵模式中涉及的组分的身份，在其中进行这种模式的代谢背景，以及细胞如何从呼吸模式过渡到发酵模式。该项目将由MCB的系统和合成生物学集群以及CBET的生物技术，生物化学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.