Multienzyme complexes of methanogenic archaea

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Methane-producing archaea (methanogens) are “extremophiles”, organisms that thrive under conditions at the limit of life on Earth. Methanogens are unique organisms that grow by producing methane gas which can be harvested for electricity, heat, and transportation fuel. The project will study the biochemistry of a large multi-enzyme complex, akin to a “redox router” switch that has the ability to direct carbon towards either biomass or methane synthesis depending on the energy status of the cell. To the researchers' knowledge this is the first such biological router switch described in any organism that directly integrates energy conservation to biomass synthesis in one enzyme complex. The principal investigator will determine the ratio of enzymes in the complex, identify sites of interaction, and ascertain if the complex changes composition in response to growth substrate. A postdoctoral researcher and graduate students will be trained in the biochemical mechanisms of biological methane production. Research will be shared using hands-on educational modules through the Women in Science and other K-12 outreach activities. This research has the potential to enhance bio-methane production for renewable energy, mitigate methane production in the environment, and engineer methanogens to produce useful chemicals through synthetic biology.Previous work has shown that enzymes in the Wolfe Cycle (CoM-S-S-CoB heterodisulfide reductase, Hdr) and Wood-Ljungdahl CO2 fixation pathways (the carbon monoxide dehydrogenase Cdh, subunit of the acetyl-CoA decarbonylase/synthase, ACDS; and methylene tetrahydromethanopterin reductase, Mer) form a multienzyme complex in the methanogen Methanosarcina acetivorans. The goal of the project is to determine if methanogens adjust Cdh/Hdr/Mer complex stoichiometry in response to growth substrate switching. The hypothesis is that methanogenic growth kinetics of M. acetivorans is dependent on the formation and stoichiometry of the terminal oxidoreductase Hdr with the carbon monoxide dehydrogenase (Cdh) of the ACDS complex. Molecular, biochemical, and biophysical techniques will be used to detect and characterize multienzyme complexes that form in vitro and in vivo. Enzyme complex composition and subunit exchange kinetics will be assessed in relation to growth substrate, and the effect of deletion and overexpression of enzyme complex components on cell physiology will be measured. In vivo and in vitro crosslinking mass spectrometry will be employed to map protein interaction interfaces which will then be used to model the router complex using crystal structures of homologous subunits. Subunit stoichiometries will be manipulated and their effect on growth rate, product yield, and metabolic efficiency of cells will be determined. Graduate students and postdoctoral researchers from underrepresented groups will be trained in anaerobic microbiology, redox biochemistry, and synthetic biology techniques. Research will be disseminated through publications, presentations, and outreach activities such as the popular Women in Science workshop.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.