(499c) Engineering a Mixotrophic Clostridium Consortium Directly Exchanging Cellular Materials Via Cell Fusion for Biochemical Production with CO2 Fixation.

Engineering microbial consortia provides insights into syntrophic microbial interactions and expands metabolic capability for complicated functions that individual population is unable to perform. Here, we present a synthetic Clostridium consortium that enables biosynthesis of biofuels, chemicals, and materials with transformative yields from sugar catabolism. As a case study, we targeted production of isopropanol, which serves as biofuel component or a valuable chemical with a market size of $2.65B (2019) & a CAGR of 8.2% (2020-27). We engineered a non-sporulating Clostridium acetobutylicum M5 strain which lacks the megaplasmid pSOL1 that encodes for the solventogenic pathways (e.g., acetone, butanol) in wild type C. acetobutylicum. Several genes of C. acetobutylicum M5 were successfully deleted using CRISPR/Cas9 and mazF toxin-antitoxin system, minimizing undesirable byproduct formation. To optimize acetone production, a key precursor for isopropanol, acetone biosynthesis pathway was redesigned and constructed under the control of strong and constitutive promoters and computationally designed ribosome binding sites. Engineered C. acetobutylicum M5 chassis expressing the synthetic acetone pathway produced acetone without butyrate formation. The acetone producing C. acetobutylicum M5 strain was paired with C. ljungdalhii, which consumes CO₂ and H₂ and converts acetone to isopropanol using endogenous dehydrogenases. Through heterologous cell fusion, the Clostridium strains efficiently exchanged cellular materials, enabling isopropanol production with supra-theoretical yields. We examined the cell fusion between the engineered C. acetobutylicum M5 and C. ljungdalhii using anaerobic fluorescence reporters, visualizing exchange of cellular materials between the two strains. The Clostridium consortium platform represents an engineered syntrophic microbial consortium for carbon neutral or negative production of biofuels, chemicals, and materials.