Shikimate is a key intermediate for the synthesis of the neuraminidase inhibitors. Microbial production of shikimate and related derivatives has the benefit of cost reduction when compared to traditional methods. In this study, an overproducing shikimate Escherichia coli strain was developed by rationally engineering certain metabolic pathways. To achieve this, the shikimate pathway was blocked by deletion of shikimate kinases and quinic acid/shikimate dehydrogenase. EIICBglc protein involved in the phosphotransferase system, and acetic acid pathway were also removed to increase the amount of available phosphoenolpyruvate and decrease byproduct formation, respectively. Thereafter, three critical enzymes of mutated 3-deoxy-D-arabinoheptulosonate-7-phosphate (DAHP) synthase (encoded by aroG(fbr)), PEP synthase (encoded by ppsA), and transketolase A (encoded by tktA) were modularly overexpressed and the resulting recombinant strain produced 1207 mg/L shikimate in shake flask cultures. Using the fed-batch process, 14.6 g/L shikimate with a yield of 0.29 g/g glucose was generated in a 7-L bioreactor. (C) 2014 Elsevier Ltd. All rights reserved.