Construction of an Expression System of Shikimate Kinase II and Preparation of Shikimic Acid 3-Phosphate

Abstract

Shikimic acid 3-phosphate (S-3-P) is needed as a substrate to detect the enzyme activity of 5-enolpyruvylshikimic acid 3-phosphate synthase (CP4EPSPS), which is expressed in genetically modified soybeans. Therefore, we attempted the over-expression of shikimate kinase II (SK-II) in E. coli to biosynthetically obtain S-3-P. The aroL gene encoding SK-II was constructed in the expression vector pET22b(+). The aroL expression vector was then transfected into E. coli strain BL21 using the electroporation method. The activity of the obtained aroL protein was confirmed by HPLC using an amino-silica column and incorporation of [32P] from labeled ATP to shikimic acid. The determination of the reaction product was performed by LC/MS using a carbon column and periodate treatment.HPLC using a carbon column does not use a non-volatile buffer as the mobile phase. Thus, this method should be useful for preparing S-3-P from the crude reaction mixture of SK-II.