Cloning, Functional Characterization And Spectroscopy Analysis Of The Dunaliella Salina 5-enolpyruvylshikimate-3-phosphate Synthase Gene

5-enolpyruvylshikimate-3-phosphate (EPSP) synthase is a critical enzyme in the shikimate pathway for the biosynthesis of aromatic amino acids and other essential aromatic compounds in bacteria, fungi and plants. It catalyzes the formation of EPSP and the inorganic phosphate from shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP).The enzyme is widely researched because it has been identified as the primary target of the nonselective herbicide glyphosate. Glyphosate shows competitive inhibition for PEP but no inhibition for S3P. It binds to the EPSP synthase in place of PEP and forms a stable enzyme-S3P-glyphosate ternary complex. Therefore, glyphosate can block the synthesis of aromatic amino acids and result in the death of organism. Expression of a foreign glyphosate-tolerant EPSP synthase by gene engineering or amplification of EPSP synthase has been demonstrated to confer tolerance to the glyphosate.Furthermore, the shikimate pathway is completely absent in mammals, fish, birds, reptiles and insects, so it is an attractive target for novel antibiotics. It was reported that bacterial mutants lacking EPSP synthase can not survive and cause infection and interaction with other enzymes in shikimate pathway can also induce the antibacterial effect.In a word, EPSP synthase is very important in both glyphosate-resistant crops and the design of new antibiotic. Therefore, it is very significant to research the nature, the catalytic mechanism and the relationship between structure and function of the enzyme. Studies focused on new EPSP synthase will provide more valuable experimental data to this field. Although EPSP synthase have been purified in both prokaryotic and eukaryotic and the gene(aroA) encoding EPSP synthase have been cloned from many species, the enzyme has never been cloned in algae. As the aroA gene of Dunaliella salina has never been reported the paper carried out the following research:1) Cloning of the aro A gene of Dunaliella salina.We have got the EPSP synthase expression sequence tag (EST) from D. salina in our previous works. Based on the EST, DNA fragments about 900 and 1200 bp were cloned by 3' RACE and 5' RACE respectively. Finally, compared with the EST, a cDNA sequence of 2341 bp was obtained. It contained an ORF of 1545 bp flanked by 30 and 766 bp of 5' UTR and 3' UTR regions respectively. The 3' UTR contained the polyA. Sequence data have been deposited at GenBank under accession number EF051488.2) By bioinformatics analysis, we found that the cloned cDNA contains the complete open reading frame(ORF) of D. salina EPSP synthase gene, the encoded amino acid sequences showed high similarity with other EPSP synthases; meanwhile, we also forecasted the EPSP synthase of D. salina has chloroplast transit peptide on its N-terminal which is consistent with the plant EPSP synthase; phylogenetic analysis showed that the divergence between the D.salina and bacteria EPSP synthases occurred some time later than the D.salina and higher plant; we also found that the protein has the unique motif of EPSP synthase family and the 3D-structure between D. salina and E. coli EPSP synthase enzyme are very consistent. These results all showed that the cDNA we cloned belongs to EPSP synthase gene.3) Expression and purification of D. salina EPSP synthase.D. salina EPSP synthase gene was constructed in pGEX-4T-1 vector and transformed into E.coli JM109; when the E. coli was induced by 0.03mM IPTG at 30℃, the recombinant protein was maximum in soluble formation; the recombinant fusion protein was purified using GST affinity chromatography; Finally, the the GST labeling protein was removed by thrombin.4) Functional characterization of the D. salina EPSP synthase in vivo.The recombinant plasmid with D. salina aroA gene was transformed into the E. coli ER2799 (with the aroA gene deleted from its genome); The result indicated that the recombinant D. salina EPSP synthase can catalyze the formation of EPSP in E. coli and sustain the synthesis of aromatic amino acid; Glyphosate-resistant experiments showed high expression of the recombinant EPSP synthase could increase the tolerance to glyphosate. All these showed that the cloned aroA gene was in active form in E. coli.5) By fluorescence spectroscopy, we have analyzed the conformational change of EPSP synthase on the binding of substrates. The results showed that there is tiny fluoroscopy change on binding of S3P. The fluoroscopy changed enormously when S3P and PEP bind to the enzyme. Therefore, we believe that the binding with S3P and PEP induces the macroconformational change from the "open" to the "close" form. Moreover, we found that phosphate ions might occupy parts of the PEP or glyphosate binding site and lead to the conformational change.6) The culture of glyphosate-resistance D. salina and the expression analysis.We cultivated glyphosate-resistant D. salina by adding glyphosate to the mediagradually. Real-Time Quantitative PCR was used to investigate the mRNA level of aroA gene among these species. The result showed that the mRNA level in 2.0mM glyphosate-resistant D. salina was almost 12 times than the normal D. salina. Finally, we cloned the aroA gene of the resistant D. salina. The alignment with normal gene showed no mutated site was found. Therefore, we conclude that high expression of EPSP synthase results in the resistance to glyphosate. Further experiments confirmed that it is a long time for the D. salina to over-express its EPSP synthase to adapt the glyphosate.