Citramalic acid (citramalate) is a five carbon hydroxy-dicarboxylic acid and potential precursor for the production of methacrylic acid from renewable resources. We examined citramalate production in Escherichia coli expressing the citramalate synthase gene cimA. 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. A fed-batch process in a controlled fermenter using a glucose feed resulted in 46.5g/L citramalate in 132h with a yield of 0.63g/g, over 75% of the theoretical maximum yield from glucose of 0.82g/g. Biotechnol. Bioeng. 2016;113: 2670-2675. (c) 2016 Wiley Periodicals, Inc.