Methanogenic archaea are crucial in global carbon cycling as they produce around 1 Gt of the potent greenhouse gas methane annually. Major contributors belong to the order Methanosarcinales, represented by versatile methanogens that are capable of acetoclastic, methylotrophic, and CO2-reducing methanogenesis. The genetically tractable model methanogen Methanosarcina acetivorans by its nature shows versatility in substrate utilization and energy conservation pathways but cannot utilize formate. In this study, we expanded the primary metabolism of M. acetivorans to include formate-dependent methanogenesis. By introducing formate dehydrogenase, two metabolically engineered M. acetivorans strains acquired the capacity for formate-dependent methanogenesis. One catabolic pathway relies on formate-dependent methyl-reduction, and the other relies on formate-dependent CO2-reduction. Nurturing the M. acetivorans strain capable of CO2-reduction with adaptive laboratory evolution enabled growth and methanogenesis solely on formate, a metabolism only reported in methanogens without cytochromes. M. acetivorans also showed acetogenic potential where the formate-dependent CO2-reducing strain was able to divert ≈ 10% of carbon from the substrates provided to acetate instead of methane. Even though M. acetivorans lacks energy-converting hydrogenase and cannot use H2, we show that M. acetivorans has a yet-uncharacterized capacity to obtain reduced ferredoxins from oxidizing formate. Our work encourages a reevaluation of our understanding of formate utilization in Methanosarcinales. By enabling formate-dependent methanogenesis, we have expanded the substrate spectrum of a versatile model methanogen with cytochromes to include formate.

2025