Improving Succinate Production in E. coli through Substrate Channeling

Current industrial production of petrochemicals releases CO2 as a byproduct into the atmosphere, contributing to climate change. The sustainable alternative, microbial carbon capture, has primarily focused on phototrophs that have naturally occurring carbon fixation pathways, but are slow-growing, difficult to genetically engineer, and require sunlight, which limits their large-scale production capacity. Using a heterotroph such as Escherichia coli allows for chemical production at high titers, rates, and yields (TRY) while being fast growing and easy to genetically engineer. Under fermentation conditions, the carboxylases in E. coli fix inorganic carbon in the reductive branch of the TCA cycle, producing industrially relevant chemical precursors such as succinate. However, the carboxylase’s access to CO2 is limited by the conditions surrounding it; most of the inorganic carbon inside the cell is in the form of bicarbonate. Increasing the local concentration of CO2 near the carboxylase may improve the kinetics of the pathway. To do this, a fusion protein that colocalizes carbonic anhydrase and phosphoenolpyruvate carboxykinase (Pck) was created. However, since strains expressing this fusion protein did not grow above OD600 = 1 under fermentation conditions, further design optimization and investigation is needed.