Angarita-Zapata, V. | bioRxiv Science

bioRxiv2026

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 (McrANME-1) activity for methane and carbon dioxide capture by selecting McrANME-1 variants with improved growth during methane-dependent cultivation. As a result, we discovered two beneficial substitutions in the catalytic -subunit of McrANME-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.

Synthetic Biology↗

bioRxiv2026

Enhancing Reverse Methanogenesis Using RNA-Guided Silencing

Utilizing methane and carbon dioxide before it can enter the upper atmosphere is beneficial for mitigating climate change as well as for producing valuable chemicals. Because anaerobic methanotrophic archaea (ANME) have not yet been cultured in isolation, we previously reversed methanogenesis by cloning the genes encoding methyl-coenzyme M reductase (Mcr) derived from Black Sea ANME-1 into the methanogen Methanosarcina acetivorans. The resulting engineered archaeal strain captures, rather than produces, methane and may be used to convert methane and carbon dioxide into electricity, acetate, L-lactate, and ethanol. However, the engineered M. acetivorans strain also contains a chromosomal locus encoding its native Mcr (McrM.a.), which produces methane from substrates such as methanol, whereas the heterologously expressed ANME-1 Mcr (McrANME-1) promotes methane oxidation. Therefore, we reasoned that McrM.a. may compete with McrANME-1-mediated reversal of methanogenesis. To enhance the reversal of methanogenesis, here we implemented an antisense RNA (asRNA) silencing approach to suppress McrM.a. during growth on methane while still allowing its expression during routine growth on methanol. We found that silencing McrM.a. during McrANME-1-mediated growth on methane increased ethanol and acetate production by more than an order of magnitude. These results were corroborated by both a more than 10-fold increase in methane utilization by McrANME-1 and a greater than 1,000-fold reduction in the McrM.a. mcrBGA transcript levels under methane-grown conditions. Therefore, asRNA-mediated silencing may be used to enhance methane capture by suppressing production of the host McrM.a. for biotechnological applications.

synthetic biology↗