Disrupting the mtr-operon in Methanosarcina acetivorans enables methyl-reducing methanogenesis with hydrogen and serine as the alternative electron donors.

Methanosarcina acetivorans is a model methanogen because of its metabolic versatility and genetic tractability. This microbe is not known to natively utilize hydrogen as a catabolic electron donor, despite its genome encoding hydrogenases, and the fact that this microbe can be used to heterologously express functional hydrogenases. Its native hydrogenases are expressed at a very low level and are suggested to have a role in recycling hydrogen that is produced as a byproduct of nitrogen fixation. To explore whether hydrogen can act as a catabolic electron donor, we utilized a previously constructed strain (str. JB-MF) that has the operon encoding for the methyl-H4MPT:CoM methyltransferase (Mtr) disrupted which makes growth dependent on oxidation of electron donors other than methanol. We showed that this M. acetivorans strain can grow by methanol reduction to methane with hydrogen as the sole electron donor. The strain was then used to test the putative electron donors for methyl-reducing methanogenesis: hydrogen, serine, and ethanol. Hydrogen and serine were identified to act as catabolic electron donors for methyl-reducing methanogenesis, which demonstrates the expanded metabolic versatility of M. acetivorans JB-MF. The methanophenazine-reducing hydrogenase (Vht) and the F420-reducing hydrogenase (Frh) were determined to be involved in hydrogen metabolism, because deletion of either made M. acetivorans JB-MF incapable of growing using H2-dependent methyl-reduction. We demonstrate that strain JB-MF is a suitable chassis to screen alternative electron donors for methanogenesis and that this strain may be used for targeted directed evolution of oxidoreductases.