(632f) Modular and Syntrophic Cocultures of Clostridia to Produce Isopropanol and C6-C8 Alcohols and Carboxylic Acids

Synthetic syntrophic cocultures provide several advantages for the renewable production of target biofuels and chemicals. Engineering all desired metabolic pathways into one organism is difficult, but in a coculture, each organism can specialize according to their natural and engineered capabilities while sharing metabolites and proteins. This work explores cocultures of C. acetobutylicum (Cac), an industrial solventogen that produces ethanol, butanol, and acetate; C. kluyveri (Ckl), which can elongate acetate into longer-chain carboxylic acids; C. ljungdahlii (Clj), an acetogen that can capture carbon dioxide to produce ethanol and acetate; and C. saccharolyticum (Csac), which can quickly produce large amounts of ethanol and acetate. Butanol, hexanol, and octanol, as well as their respective carboxylic acids, are common industrial chemicals, but they are currently produced from petroleum-based processes. Isopropanol is an additional valuable industrial chemical that can be produced biologically, but current modes of production are inefficient. Syntrophic cocultures of Clostridia promise a sustainable and green replacement.

A key benefit of cocultures is their modularity. If the objective is high production of C6-C8 chemicals, Ckl will be needed to perform chain elongation. In these cocultures, an engineered high-ethanol-producing strain of Cac consumes glucose and produces ethanol, acetate, carbon dioxide, butyrate, and butanol. The ethanol and acetate are elongated by Ckl to create valuable hexanoate and octanoate, which can be converted by either Cac or Clj into their respective alcohols, which are easier to extract from the media. Cocultures of Cac and Ckl and Csac and Ckl have been studied. Clj can be added to these co-cultures to capture the carbon dioxide produced by the coculture organisms, which raises carbon efficiency and lowers costs. Notably, this work has resulted in the highest-observed hexanoate production from Ckl without concurrent in-reactor capture. Cocultures of Csac and Ckl produce more hexanoate more quickly than any published coculture (14 g/L in 42 hours). This work also provides more insight into coculture biology, as both flow cytometry and fluorescent microscopy have provided evidence of cell contact and protein sharing between coculture members of different species. To quickly evaluate the impacts of different coculture parameters, a custom rig of eight miniature bioreactors was designed and constructed. Modular and easy to adapt, this system has been key in tuning various coculture conditions and providing for high-throughput strain testing.

Our lab earlier found that in Cac and Clj cocultures, the cells come together and share proteins and metabolites, leading to the emergence of coculture phenomena that are unobserved in monocultures. Isopropanol is produced when the acetone natively produced by Cac is converted into isopropanol by a secondary alcohol dehydrogenase in Clj. Direct cell contact in coculture leads to lower acetone titers in the media as well as a faster onset of isopropanol production. The presence of Clj also enables complete utilization of the carbon and electrons in the sugar substrate due to its use of the Wood-Ljungdahl pathway. Our new work incorporates these insights to maximize the production of isopropanol with a Cac/Clj coculture that utilizes an engineered Cac M5 strain that produces mostly acetone. This coculture method will fully utilize the sugar substrate, maximizing the culture productivity and production of isopropanol. This coculture utilizes the same custom miniature bioreactor setup as the Ckl cocultures, which leads to quick parameter determination and optimization. Altogether, this work shows how Clostridia cocultures can expand the metabolic space by unlocking and better utilizing the capabilities of all coculture members so that more sustainable means of chemical production can be deployed.

Acknowledgement: ARPA-E Award DE-AR0001505: “Bioenergy production based on an engineered mixotrophic consortium for enhanced CO₂ fixation”