(516c) Engineering Methanogenesis Pathway of Methanogenic Archaea for Biofuel Production

Maryam Raeeszadeh-Sarmazdeh, Jacqueline Gonzalez, Wilfred Chen

Department of Chemical and Biomolecular Engineering, University of Delaware, Newark, DE 19716

Methane gas is the second most prevalent greenhouse gas and an abundant source of energy in the United States. Methane is both produced and consumed in marine sediments through anaerobic methanogenesis. Methanogenic archaea primarily use one-carbon substrates such as CO₂, acetate, or methanol for growth and methanogenesis through CO₂ reduction, aceticlastic, or methylotrophic pathways, respectively. The key step in the methanogenesis pathway, reducing methyl-coenzyme M (CH₃-S-CoM) and coenzyme B (HS-CoB) to methane, is catalyzed by Methyl-coenzyme M reductase (MCR). This reaction, which leads to the formation of a heterodisulfide substrate (CoM-S-S-CoB), is exergonic. Therefore, for reverse methanogenesis to occur, the methane oxidation reaction needs to be coupled with an endergonic reaction that provides a thermodynamically favorable situation.

The genetic toolbox in methanogenic archaea is limited. Therefore, we have investigated Nanoluciferase expression, as a sensitive reporter, through episomal expression and integration into the genome in Methanosarcina acetivorans . In order to engineer anaerobic Methanogenesis pathway to produce biofuel, we have expressed functional Methyl-coenzyme M reductase (MCR) from Methanothermobacter marburgensis in Methanosarcinals. The recombinant strains showed improvement in methane formation yield compared to the wild-type strain. Furthermore, the effect of soluble electron acceptors such as Fe(III), and the regeneration of a heterodisulfide substrate on methane oxidation were investigated.