Production of citramalic acid using metabolically engineered Escherichia coli

Citramalic acid (citramalate) is a five carbon hydroxy-dicarboxylic acid and potential renewable resource for the production of methacrylic acid (MAA). The most common approach for MAA synthesis currently involves the hydrolysis of methacrylamine sulfate obtained from acetone cyanohydrin, which is a hazard related and non-sustainable process. In this study we examined citramalate production in Escherichia coli by overexpressing citramalate synthase. This enzyme encoded by the cimA gene catalyzes the specific condensation of pyruvate and acetyl-CoA with the formation of citramalate. Therefore, the investigation focused on the availability of intracellular pyruvate and acetyl CoA. Although knockouts in ldhA coding lactate dehydrogenase and glcB/aceB coding malate synthase did not benefit citramalate accumulation, knockouts in gltA coding citrate synthase and ackA coding acetate kinase significantly increased citramalate accumulation compared to the control strain. In addition, leuC gene coding 3-isopropylmalate dehydratase was deleted to prevent potential citramalate degradation. We compared citramalate production in MG1655, MG1655 gltA leuC, and MG1655 gltA leuC ackA expressing citramalate synthase in a one liter batch fermentor containing defined medium with 20 g/L glucose initially. MG1655 gltA leuC ackA/pZE12-cimA accumulated 14.8 g/L citramalate in 100 h with a yield of 0.74 g/g.  However, the growth rate of MG1655 gltA leuC ackA/pZE12-cimA was greatly reduced in the defined medium.  Therefore, a glucose-feeding process was designed with the medium initially containing 15 g/L peptone. In this case, MG1655 gltA leuC ackA/pZE12-cimA generated about 50 g/L citramalate in 132 h with a yield of 0.6 g/g.