SRP493773 PRJNA1085037 Nature AND Nurture: Enabling formate-dependent growth in Methanosarcina acetivorans Methanogenic archaea are crucial in global carbon cycling as around 1 Gt of the potent greenhouse gas, methane, is produced annually. Major contributors belong to the order Methanosarcinales, which contain some of the most versatile methanogens that are capable of acetotrophic, 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 an exogenous formate dehydrogenase, the two metabolically engineered M. acetivorans strains acquired the capacity for formate-dependent methanogenesis pathways with one capable of formate-dependent methyl-reduction and the other capable of formate-dependent CO2-reduction. Through nurturing the strain capable of CO2-reduction with adaptive laboratory evolution, we were able to enable growth and methanogenesis of M. acetivorans solely on formate, a metabolism only reported in methanogens without cytochromes which are limited by their versatility. M. acetivorans also showed acetogenic potential where the formate-dependent CO2-reducing strain was able to divert 10% of carbon to acetate instead of methane. Our results show that even though M. acetivorans lacks energy converting hydrogenase and cannot use H2, it has yet-uncharacterized capacity to obtain reduced ferredoxins from oxidizing formate. Our work encourages 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 as well.