Enhanced conversion rate of L-phenylalanine by coupling reactions of aminotransferases and phosphoenolpyruvate carboxykinase in Escherichia coli K-12.
In Escherichia coli, aspartate aminotransferase (encoded by aspC) and aromatic amino acid aminotransferase (encoded by tyrB) share overlapping substrate specificity in the syntheses of aromatic amino acids. Through the transamination reactions catalyzed by AspC or TyrB, L-phenylalanine (L-Phe) can be produced from phenylpyruvate with aspartic acid as the amino donor. To modulate and enhance the production levels of proteins, both aspC and tyrB were subcloned into a runaway-replication vector. As a result, the specific activities of AspC and TyrB obtained showed 65-fold and 50-fold increases, respectively, compared with the wild-type level. Employing resting cells of AspC- and TyrB-overproducing E. coli K-12 strains for L-Phe productions resulted in molar conversion yields of 70% and 55%, respectively. With an additional introduction of phosphoenolpyruvate carboxykinase (encoded by pck) into the transamination reactions, the conversion yields were improved to 93% from 70% and to 75% from 55% in a relatively short time. These results account for more than an 8-fold increase in productivity, as compared to the previous report (Calton et al., 1985). In addition, a four-run reuse of the recombinant cells for L-Phe production gave a total yield of 91 g/L with a 93% conversion.
Chao
Y P
YP
Department of Chemical Engineering, Feng Chia University, 100 Wenhwa Road, Taichung, Taiwan, Republic of China. ypchao@fcu.edu.tw
Lai
Z J
ZJ
Chen
P
P
Chern
J T
JT
D001221:Aspartate Carbamoyltransferase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D001709:Biotechnology; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics / Q000378:metabolism*; D005798:Genes, Bacterial; D010649:Phenylalanine / Q000096:biosynthesis*; D019764:Phosphoenolpyruvate Carboxykinase (ATP) / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D000637:Transaminases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*
D011994:Recombinant Proteins; D010649:Phenylalanine; D001221:Aspartate Carbamoyltransferase; D000637:Transaminases; C019729:aromatic amino acid aminotransferase; D019764:Phosphoenolpyruvate Carboxykinase (ATP)