Construction and characterization of Escherichia coli D-3-phosphoglycerate dehydrogenase mutants with feedback-inhibition relief.
- Author:
Hui DENG
1
;
Cunwu CHEN
1
;
Chuanbo SUN
1
;
Chuanbao WEI
1
Author Information
- Publication Type:Journal Article
- Keywords: Escherichia coli phosphoglycerate dehydrogenase; construction of mutants; relief of feedback inhibition
- MeSH: Electrophoresis, Polyacrylamide Gel; Escherichia coli; enzymology; Escherichia coli Proteins; genetics; Industrial Microbiology; Mutagenesis, Site-Directed; Phosphoglycerate Dehydrogenase; genetics; Serine; biosynthesis
- From: Chinese Journal of Biotechnology 2016;32(4):468-477
- CountryChina
- Language:Chinese
- Abstract: 3-Phosphoglycerate dehydrogenase (PGDH, EC 1.1.1.95) is the key enzyme in L-serine biosynthesis and its coding gene is serA. PGDH is feedback inhibited by L-serine. In order to relieve the feedback-inhibition of PGDH by L-serine, H344 or D346 or D364 were chosen for site directed mutagenesis. The mutants were generated by the standard QuikChange mutagenesis, further subcloned into expression vector pT7-7 and transformed into Escherichia coli BL21 (DE3) cells. The recombinant cells were collected after cultured in LB media post induced by isopropyl beta-Dthiogalactopyranoside. The enzymes were purified by anion exchange chromatography, and SDS-PAGE showed that the purified enzymes were homogenous. Enzyme characterization indicated that the mutant enzyme showed similar activity, optimal temperature, and optimal pH as that of the wild-type enzyme. Moreover, feedback inhibition study showed that the activity of the double mutant (N346A/H344A) could remain 96% in the presence of serine up to 160 mmol/L, whereas the activity of the wild-type enzyme remains only 50% in the presents of serine of 7 μmol/L, thus successfully relieving the feedback inhibition of PGDH with its activity remained.
