Engineering Methyl-Coenzyme M Reductase for Enhanced Methane and Carbon Dioxide Capture through Biofilm Growth

Methane is a potent greenhouse gas, nearly half of which is consumed anaerobically by anaerobic methanotrophic archaea (ANME) through methyl-coenzyme M reductase (Mcr). However, ANME cannot be grown as pure cultures, and obtaining active ANME Mcr in vitro remains extremely challenging, preventing previous efforts to engineer this key enzyme. Here, we used directed evolution in the methanogen Methanosarcina acetivorans to enhance ANME-1 Mcr (Mcr _ANME-1 ) activity for methane and carbon dioxide capture by selecting Mcr _ANME-1 variants with improved growth during methane-dependent cultivation. As a result, we discovered two beneficial substitutions in the catalytic α-subunit of Mcr _ANME-1 , S60P and I154V, that increased biofilm growth as well as acetate production and methane capture. AlphaFold structural predictions suggest possible mechanistic explanations for these beneficial substitutions. These findings demonstrate that Mcr can be engineered to enhance methane and carbon dioxide capture, establishing a foundation for biological greenhouse gas mitigation and carbon utilization technologies.