Site-directed mutagenesis and over expression of aroG gene of Escherichia coli K-12

3-Deoxy-D-arabino-heptulonate-7-phosphate (DAHP) synthetase is one of the key enzymes, which catalyzes the first step in the aromatic amino acid biosynthetic pathway and yields the three amino acids tyrosine, tryptophan, and phenylalanine. In Escherichia coli (E. coli), three differently regulated DAHP synthases carry out the first regulated step in the aromatic amino acid biosynthetic pathway. The three DAHP synthases encoded by the genes aroG, aroF and aroH are inhibited by phenylalanine, tyrosine and tryptophan, respectively. In this work, the aroG gene was cloned and mutated by site-directed mutagenesis using splicing overlap extension PCR (SOE-PCR) technique. The feedback-resistant DAHP synthase encoded by aroG was achieved by replacing the residue Leu175 of aroG with Asp as to increase net carbon flow down the common pathway. SOS-PAGE which was used to access the protein expression level of aroGM showed the strain harbored mutated aroGM gene achieve over-expression compared to strain contain empty plasmid pET-28b (+). (C) 2012 Elsevier B.V. All rights reserved.