Construction of an expression system of shikimate kinase II and preparation of shikimic acid 3-phosphate

Shikimic acid 3-phosphate (S-3-P) is needed as a substrate to detect the en zyme activity of 5-enolpyruvylshikimic acid 3-phosphate synthase (CP4EPSPS) , which is expressed in genetically modified soybeans. Therefore, we attemp ted the over-expression of shikimate kinase II (SK-II) in E. coli to biosyn thetically obtain S-3-P, The aroL gene encoding SK-II was constructed in th e expression vector pET22b(+). The aroL expression vector was then transfec ted into E. coli strain BL21 using the electroporation method. The activity of the obtained aroL protein was confirmed by HPLC using an amino-silica c olumn and incorporation of [P-32] from labeled ATP to shikimic acid. The de termination of the reaction product was performed by LC/MS using a carbon c olumn 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 cru de reaction mixture of SK-II.