Construction and expression of mutagenesis strain of aroG gene from Escherichia coli K-12.
3-Deoxy-d-arabino-heptulonate-7-phosphate (DAHP) synthase 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 (Tyr), tryptophan (Typ), and phenylalanine (Phe). 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 overlap extension PCR (SOE-PCR) technique. The feedback-resistant DAHP synthase encoded by aroG was achieved by replacing the residue Pro150 of aroG with Leu as to increase net carbon flow down the common pathway. SDS-PAGE and Western blots were used to assess the protein expression level of aroGM which showed the strain harboring the mutated aroGM150 gene achieving over-expression compared to the strain containing an empty plasmid pET-28b((+)).
Laboratory of Nutrition and Functional Food, Jilin University, Changchun 130062, PR China.
Songyi
S
Lin
Laboratory of Nutrition and Functional Food, Jilin University, Changchun 130062, PR China.
Rong
R
Liang
Laboratory of Nutrition and Functional Food, Jilin University, Changchun 130062, PR China.
Xiujuan
X
Meng
College of Animal Sciences, Jilin University, Changchun 130062, PR China.
Hongsheng
H
OuYang
Laboratory of Nutrition and Functional Food, Jilin University, Changchun 130062, PR China; College of Animal Sciences, Jilin University, Changchun 130062, PR China. Electronic address: yanhy@jlu.edu.cn.
Haiyang
H
Yan
Laboratory of Nutrition and Functional Food, Jilin University, Changchun 130062, PR China. Electronic address: jinkuang8499@163.com.
Ying
Y
Wang
Department of Food, Nutrition and Packaging Sciences, Clemson University, Clemson, SC 29634, USA.
Gregory S
GS
Jones
D015153:Blotting, Western; D003001:Cloning, Molecular / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D048168:Escherichia coli K12 / Q000235:genetics*; D005798:Genes, Bacterial; D005822:Genetic Vectors / Q000378:metabolism; D016296:Mutagenesis / Q000235:genetics*
Over-expression; Site-directed mutagenesis; aroG