Shikimic acid (SA) is an important metabolic intermediate with diverse commercial applications. In this work, antisense RNA interference and gene deletion were carried out to inactivate the aroK gene in an SA-producing Escherichia coil strain, DHPYA-T7. In this strain, the aroL, ptsHIcrr and ydiB genes are deleted, and the tktA, glk, aroE and aroB genes are overexpressed. Flask cultivations of the DHPYA-T7 derivative strains showed that the accumulation of SA increased 2.69-fold after aroK gene deletion (DHPYAAS-T7) and 1.29-fold after antisense RNA interference (DHPYAS-T7). Furthermore, the activity of shikimate kinase in DHPYAAS-T7 was 0.21-fold of that in strain DHPYAS-T7. In a 10-L fermentation, SA accumulation increased to 1850 mg L-1 in strain DHPYAAS-T7, which is a 1.5-fold increase over that in strain DHPYAS-T7. These results demonstrate that aroK gene inactivation in DHPYA-T7 leads to high SA accumulation, especially when this inactivation is caused by chromosomal deletion. (C) 2012 Elsevier Ltd. All rights reserved.