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Abstract
Acetyl-CoA is a key precursor to a variety of industrially relevant compounds, and its availability is thought to limit the formation of biochemicals derived from this intermediate. The largest metabolic drain of acetyl-CoA in microbes such as Escherichia coli is to citrate via citrate synthase (coded by the gltA gene). This study examines the effect of reducing the catalytic activity of citrate synthase on the conversion of acetyl-CoA to acetate, which serves as a model product derived from acetyl-CoA. Activity was reduced by generating point mutations on chromosomal citrate synthase. These variants were assessed in E. coli having a ΔpoxB background in shake flask, batch and continuous experiments. Characterization of purified wild-type GltA and the A267T variant showed a 16-fold decrease in kcat and an increase in KM in A267T. The results obtained provide important insights on improving the production of compounds derived from acetyl CoA.