A plasmid-based expression system wherein mekB was fused to a constitutive Methanosarcina acetivorans promoter was used to express MekB, a broad-specificity esterase from Pseudomonas veronii, in M. acetivorans. The engineered strain had 80-fold greater esterase activity than wild-type M. acetivorans. Methyl acetate and methyl propionate esters served as the sole carbon and energy sources, resulting in robust growth and methane formation, with consumption of >97% of the substrates. Methanol was undetectable at the end of growth with methyl acetate, whereas acetate accumulated, a result consistent with methanol as the more favorable substrate. Acetate was consumed, and growth continued after a period of adaptation. Similar results were obtained with methyl propionate, except propionate was not metabolized. IMPORTANCE The fragile interactions of multispecies food chains converting complex biomass to methane are easily disrupted, a major impediment to efficient and reliable conversion of renewable biomass as an alternative to fossil fuels. The hybrid pathway, derived by combining catabolic pathways from a methanogen of the domain Archaea and a strictly aerobic species of the domain Bacteria, catalyzes the complete conversion of an industrial solvent that is also a naturally occurring compound to methane and carbon dioxide. The engineered pathway expands the exceptionally narrow range of substrates utilized by methanogens, exemplifying the simplification of food chains leading to the more-efficient conversion of complex biomass to methane.
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