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

University Of Washington, Seattle WA

Investigators

Abstract

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.