Selective production of L-aspartic acid and L-phenylalanine by coupling reactions of aspartase and aminotransferase in Escherichia coli

With L-aspartate (L-Asp) as the amino donor, L-phenylalanine (L-Phe) can be prepared from phenylpyruvate (PPA) via an amination reaction mediated by a minotransferase (encoded by aspC). On the other hand, L-Asp can be produced by an aspartase (encoded by aspA) -catalyzed reaction using fumaric acid a s substrate. To overproduce aspartase in Escherichia coli, the aspA gene wa s cloned and overexpressed 180 times over the wild-type level. The use of A spA-overproducing E. coli strain for L-Asp production exhibited an 83% conv ersion, approaching to the theoretical yield, whereas the wild-type strain obtained scarcely L-Asp. Furthermore, the recombinant strain overproducing both AspA and AspC was able to produce L-Asp and L-Phe simultaneously by us ing fumaric acid and PPA as substrates. As a result, the conversion yields obtained for L-Asp and L-Phe were 78% and 85%, respectively. In sharp contr ast, the wild-type strain attained a conversion of L-Phe less than 15% and an undetectable level of L-Asp. This result illustrates a potential and att ractive process to yield both L-Asp and L-Phe by coupling AspA and AspC. A further study on the repeated use of the recombinant strain immobilized wit h calcium alginate showed that after eight batch runs L-Asp conversion main tained roughly constant (around 75%), whereas L-Phe conversion dropped to 6 5% from 81%. This result indicates the stability of AspA being superior to AspC. (C) 2000 Elsevier Science Inc. All rights reserved.