(139d) Establishing Eubacterium Limosum As a Model Methylotrophic Acetogen

Single-carbon (C1) compounds including carbon monoxide, methanol and formic acid have emerged as promising feedstocks for biofuel and biochemical production. These substrates can be produced renewably from CO₂ through electrocatalysis or hydrogenation with renewable hydrogen, thus bypassing food security and land conversion concerns raised over traditional biofuel feedstocks. Acetogenic microbes are a particularly attractive class of organisms for the biological upgrading of C1 compounds, and use the Wood-Ljungdahl pathway (WLP) to form carbon-carbon bonds between C1 compounds at electron efficiencies greater than 80%. In particular, Eubacterium limosum is capable of robust growth on all three reduced C1 compounds. However, compared to microbes used in traditional bioprocessing, and other acetogens used in the context of industrial syngas-to-ethanol fermentation, a systems-level understanding of E. limosum is lacking, and genetic tools for establishing heterologous product pathways are underdeveloped. We are working on expanding the genetic toolbox for E. limosum by developing tools for high-efficiency recombineering, as well as robust promoter and ribosome binding site (RBS) libraries. We are also employing metabolic flux analysis through isotopic tracer experiments to more fully elucidate the underlying metabolic network topology under both unitrophic and mixotrophic growth conditions. This talk will elaborate on results from both of these efforts. Ultimately, this work will dramatically enhance our ability to engineer E. limosum for the production of biofuels and bioproducts from single-carbon substrates.