(485f) Genome Engineering of Clostridium Acetobutylicum for Carbon-Neutral Isopropanol Production in a Synthetic Microbial Coculture

Amidst the ever-increasing global greenhouse gas emissions and the ever-decreasing supply of fossil fuel resources, it is important to develop a carbon-neutral or carbon-negative chemical production platform. Clostridium acetobutylicum (Cac) has a long-standing history of biological ABE (Acetone-Butanol-Ethanol) fermentation from renewable sugar substrates, however, a major barrier to its implementation in large-scale fermentations is the loss of carbon sugar from glycolysis. To overcome this barrier, we constructed a mixotrophic coculture of solventogenic Cac with acetogen Clostridium ljungdahlii (Clj) that uses sugars, as well as CO₂ and H₂ gases released during glycolysis, to synthesize value-added chemicals. Exchange of acetone produced by Cac to Clj produces isopropanol, a novel product possible only by the coculture system. What remains key in the success of microbial chemical production is how to improve the yield of the desired product over competing by-products.

Genome engineering of Cac, including the deletion of competing pathways and enzymes to favor production of acetone, is our strategy. We employ several methods, including screening enzymes for acetone pathway production from multiple-copy plasmids to stable integration of constitutively expressed copies of the acetone operon in the Cac genome using CRISPR/Cas9. Through strong constitutive expression of key acetone pathway enzymes, particularly CoA transferase, we have seen marked increases in the acetone production in Cac monocultures. Our work demonstrates both synthetic biology and metabolic engineering strategies to maximize the reassimilation of CO₂ to favor isopropanol production. While our work on this microbial coculture serves as an example of carbon-neutral production of isopropanol, these same strategies can be more broadly applied to carbon-neutral production of other desirable chemicals.

Supported by the U.S. Department of Energy ARPA-E project under contract AR0001505.