Engineered citrate synthase improves citramalic acid generation in Escherichia coli.

Abstract

The microbial product citramalic acid (citramalate) serves as a five-carbon precursor for the chemical synthesis of methacrylic acid. This biochemical is synthesized in Escherichia coli directly by the condensation of pyruvate and acetyl-CoA via the enzyme citramalate synthase. The principal competing enzyme with citramalate synthase is citrate synthase, which mediates the condensation reaction of oxaloacetate and acetyl-CoA to form citrate and begin the tricarboxylic acid cycle. A deletion in the gltA gene coding citrate synthase prevents acetyl-CoA flux into the tricarboxylic acid cycle, and thus necessitates the addition of glutamate. In this study the E. coli citrate synthase was engineered to contain point mutations intended to reduce the enzyme's affinity for acetyl-CoA, but not eliminate its activity. Cell growth, enzyme activity and citramalate production were compared in several variants in shake flasks and controlled fermenters. Citrate synthase GltA[F383M] not only facilitated cell growth without the presence of glutamate, but also improved the citramalate production by 125% compared with the control strain containing the native citrate synthase in batch fermentation. An exponential feeding strategy was employed in a fed-batch process using MEC626/pZE12-cimA harboring the GltA[F383M] variant, which generated over 60 g/L citramalate with a yield of 0.53 g citramalate/g glucose in 132 hr. These results demonstrate protein engineering can be used as an effective tool to redirect carbon flux by reducing enzyme activity and improve the microbial production of traditional commodity chemicals.