ETHOX – Reveal the missing steps in archaeal ethane oxidation – the ETHyl-CoM OXidation to acetyl-CoA

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The archaea Ca. Argoarchaeum and Ca. Ethanoperedens catalyze the complete anaerobic oxidation of ethane to derive their cellular energy, and the electrons generated during this process are transferred to a partner bacterium. Therefore, these anaerobic microorganisms control the fluxes of the second most abundant gas component in the seafloor, avoiding its release in the atmosphere. We showed that ethane is activated by a novel divergent variant of methyl-coenzyme M reductases, described as ethyl-coenzyme M reductase (ECR). This enzyme captures ethane as the thiol adduct ethyl-CoM that will be progressively transformed into acetyl-CoA, the turntable of the metabolism. The following reactions leading to acetyl-CoA are unknown and will require uncharted biochemistry. Our recent cultivation success of the fast-growing ethane oxidizer Ca. Ethanoperedens will provide biological material to decipher which enzymes and chemical reactions operate the ethyl-CoM oxidation into acetyl-CoA. Here we propose to (i) reveal the unknown intermediates, (ii) identify the enzymes catalyzing the pathway and (iii) decipher their molecular mechanisms underlying these reactions. This 3-year project will be guided by the two Pl, a postdoc, a PhD student and an expert in metabolite analysis. We will cultivate the microbial consortium containing a majority of Ca. Ethanoperedens in a fermenter allowing us to produce large amounts of biomass metabolically active. Using a combination of stable isotope labelling and metabolite analysis we will identify the intermediates formed during the oxidation of ethyl-CoM to acetyl-CoA. Comparative analyses of genomes, transcriptomes and proteomes will allow us to identify candidate enzymes catalyzing the formation of such intermediates. The proposed reaction catalyzed by these enzymes will be assayed and confirmed in cell extracts and in different protein fractions using ethyl-CoM, different electron acceptors and additional intermediates. The enzymes will be natively isolated from the microbial consortia because they might contain unforeseen cofactors, a challenging task already achieved for the ECR characterization. Their structures will be obtained by crystallization and x-ray diffraction. The elucidation of this transformation in ethane degraders will complete the knowledge of this catabolic pathway and might be extended to some common principles for other short- and long-chain alkane oxidizing archaea. This overall work package is a prerequisite to pave avenues towards the biotechnological development of ethane biological production based on CO2 reduction, a process that would answer the urgent needs of our modern society to capture the greenhouse gas and store energy from renewable sources into a stable molecule.