(472a) Invited Talk: Harnessing Acetogenic Bacteria for Sustainable Chemical Production from Single-Carbon Substrates

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 substrates, and use the Wood-Ljungdahl pathway (WLP) to form carbon-carbon bonds between C1 compounds at electron efficiencies greater than 80%. However, these bacteria present a number of challenges for metabolic engineering efforts, in particular an undeveloped synthetic biology toolbox, an incomplete understanding of their central metabolism, and the energetic limitations imposed by their anaerobic lifestyle. In this talk, I will present my group’s work over the last few years to overcome these challenges. First, I will describe a dramatically expanded set of genetic tools for the acetogen Eubacterium limosum, including CRISPR-recombineering and CRAGE systems that enable rapid generation of genome-wide precise mutations and integration of large biosynthetic pathways, respectively. Next, I will show how the use of ¹³C-metabolic flux analysis applied to methanol-grown E. limosum has uncovered an unexpected reaction and helped us understand bottlenecks during acetogenic growth. Finally, I will describe a novel co-culture approach we are using to extend the product range achievable by gas fermentation. In this system, an acetogen and an aerobic heterotroph are cultured together under microaerobic conditions, establishing a syntrophic relationship where the aerobe protects the anaerobe from oxygen in exchange for fixed carbon, which it upgrades to higher-value products. Ultimately, the new tools and learnings from this work will accelerate the deployment of acetogenic bacteria as part of the sustainable, circular bioeconomy.