(605c) Cell-Free Succinic Acid Production: Engineering E. coli

Succinic acid (SA) is a value-added chemical that can be derived from biomass, but its accumulation in the fermentation medium can inhibit bacterial growth and decrease SA production. To address these challenges, cell-free biosynthesis of SA has been explored as a carbon-negative process that fixes one mole of CO₂ per mole of SA produced. Using the BW25113 strain of E. coli, flux balance analysis (FBA) was performed to analyze the effects of deleting branches diverting flux from the SA biosynthetic pathway on cell growth. Gene targets were identified for knockouts (≤ 10% reduction in growth rate) or affinity pulldown (> 10% reduction), upon cell lysis. The desired modifications were introduced into the target strain using Cas12a-based CRISPR recombineering. Strains were evaluated for their growth rates with different levels of genomic modifications to assess their tolerance. Most modifications were well-tolerated, with no significant reduction (< 9%) in growth rates, consistent with FBA results. The crude lysate from different strains will also be processed to enrich SA biosynthetic machinery and subsequently analyzed for SA accumulation. Generating a robust strain to maximize the flux in the SA biosynthetic pathway will improve carbon-utilization efficiency, increase SA yields, and provide an economically competitive route for SA production.