Screening, characterization and utilization of D-amino acid aminotransferase to obtain D-phenylalanine

Abstract

D-phenylalanine (D-Phe) is an important side-chain building block for semi-synthetic penicillins and cephalosporins. To synthesize D-Phe, D-amino acid aminotransferases (Dat) from Bacillus subtilis WB600, Bacillus licheniformis ATCC 14580, and Bacillus amyloliquefaciens were firstly compared. The theoretical 3 dimensional models of Dat were constructed, which showed appropriate configuration for synthesis of D-Phe in virtual screening. The comparison 3 different Dat on D-Phe formation was tested. All the heterogenous Dat showed high thermostability and pH stability, while Dat from B. subtilis demonstrated a better potential for Phe production than those from B. licheniformis and B. amyloliquefaciens. Then the dat from B. subtilis was expressed in an L-Phe producing chassis of Escherichia coli W14 (pR15ABK) to obtain D-Phe and different factors affecting D-Phe accumulation were tested. The engineered strain E. coli BCEA (pR15ABKApRdatBS) accumulated 1.72 g/L D-Phe in a 15-L jar fermenter which is the highest fermentation concentration that had been reported.