Glutamate-1-semiadhyde aminotransferase (GSAT) is an enzyme in the upstream biosynthetic pathway of uroporphyrinogen III that is the substrate of uroporphyrinogen III methyltransferase (UPMT), a novel red fluorescent protein. In order to detect the effect of overexpression of GSAT with UPMT on the fluorescent intensity in Escherichia coli, we amplified maize upmt gene by PCR and inserted into the first cistron of pET Duet-1 plasmid to create the vector pETU. The expressed UPMT was fused histidine tag at N terminus. We also amplified E. coli hemL gene encoding GSAT by PCR reaction, eliminated Nco I site within the hemL gene by site-directed mutagenesis and subcloned into pET-51b plasmid. The resultant hemL gene was inserted the second cistron of pETU plasmid to produce the vector pETeGU. The expressed GSAT has the extra Strep-TagII at N terminus. Compared to overexpression upmt gene alone, coexpression both genes did not resulted in the remarkable change in either the amount of the UPMT, as estimated by western blot analysis, or the constitution of red fluorescent materials, as shown by UV/visible light scanning analysis, but increased cellular level of the fluorescent material trimethylpyrrocorphin with the specific absorption at 354 nm. The red fluorescence emitted by the colonies cooverexpressing both enzymes completely disappeared after treated by 2 mmol/L gabaculine, the GSAT inhibitor, suggested that the recombinant GSAT may increase the cellular level of uroporphyrinogen III, and thus enhanced the red fluorescence of the E. coli cells conferred by the recombinant UPMT.
Escherichia coli ; genetics ; metabolism ; Genes, Plant ; Genetic Vectors ; genetics ; Intramolecular Transferases ; biosynthesis ; genetics ; Luminescent Proteins ; biosynthesis ; genetics ; Methyltransferases ; biosynthesis ; genetics ; Mutagenesis, Site-Directed ; Recombinant Proteins ; biosynthesis ; genetics ; Zea mays ; genetics