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Shikimic acid is one of the most important chiral compounds synthesized in the aromaticamino acid pathway of plants and microorganisms. It is mostly used to produce Tamifluto treat avian flu infections. In E. coli, shiA gene is responsible for shikimic acid uptakefrom the culture media. In this work, shA gene has been knocked out in E. coli JM83 andJDL02 to develop new strains of E. coli JMVS and JMC7 which can allow much moreaccumulation of shikimic acid in their culture supernatants. We also amplified theexpression of araG and tktA gene so as to improve the accumulation level. The assay ofshikimic acid by spectrophotometric method indicated that knocked out mutantsaccumulated two times lower shikimic acid than wild types with an intact shA gene. Theexpression of aroG and tktA showed that optimization of shikimic acid accumulation canbe achieved by combining gene knockout and over-expression approaches. Among theknocked out strains; namely JMVS and JMC7, JMC7 accumulated two times moreshikimic acid than the JMVS. It is believed that the high level of accumulation in JMC7strain results from the deletion of aroL gene into the strain. The supplement of shikimicacid into culture medium elucidated that shiA gene was completely knocked out. Theentire shikimic acid did not cross the membrane to enter the cell. Moreover, it was asignificant reduction of shikimic acid flow from inside the cell to the culture medium into shiAgene knocked out mutant. These results suppose that shiA gene may act in both directionsbecause its disruption prevented the flow of shikimic acid to culture medium, meaningthat shiA gene did not improve the accumulation into culture medium. In this study, itwas not possible to detect the in vivo shikimic acid due to too low accumulation.Keywords: Shikimic acid; ShiA gene knockout; Shikimic acid accumulation; E. coli;over-expression.