Study On Recombinant Expression Of Ectoine Synthase Gene

Some microorganism synthesizes solute osmotic pressure compensation mechanism under environmental osmotic pressure. Ectoine, (1,4,5,6-tetrahydro-2-methyl-4-pyrimdine carboxylic acid) as one of the most commonly found osmolytes in nature, is not only a highly efficient stabilizing agent of osmotic stress, but also provides energy for cells. And it can protect cells, protein, nucleic acids, and other macromolecule activity under dehydration, radiation, freeze-thawing, and other inverse environment. It can also stabilize the hydration layer of natural protein, and protect enzyme and DNA against freeze-thawing, heating, drying, high saline, radiation and other inverse environment. Nowadays, ectoine has been applied in stabilizing of enzyme, protective of microorganism, moisturizer for skin care products and the protective of healthy cells in chemotherapy. Currently, ectoine is produced by bacterial fermentation. This producing process is the technical bottleneck in the industrial production because ectoine is the intracellular symbiosis and restricted by intracellular concentration threshold resulting to the less output and higher costing.In order to improve the efficiency of ectoine preparation, Cobetia marina ectoine synthase gene ectABC was heterologous expressed by genetic engineering techniques, and the expression of conditions on the expression of ectoine was investigated. The ectABC DNA fragments of C. marina was amplified by genome walking, and then constructed expression vector PpET/ect and pET/ect which were transformed into E.coli BL21. With the selective of antibiotic resistance, PCR identification of ectABC and phenotype of Ectoine synthetic, ectABC recombinant were obtained. The effect of reconstruction method and recombinant induction expression condition on the expression of ectoine was studied.Ectoine synthase gene ectABC of C. marina consisted of three open reading frames of 558 bp,1263 bp and 393 bp which shared one promoter Pect. The ectABC gene of C. marina and expression vector pET43.1a were confirmed by double restriction enzyme digestion and ligated with T4 DNA connection kit and then recombinant gene expression vector PpET/ect and pET/ect which was with and without carrying the ectABC of C. marina were obtained separately. Recombinant PpET/ect and pET/ect were obtained from E. coli BL21 which is transformed from recombinant gene expression vector PpET/ect and pET/ect. Study the impact of recombinant PpET/ect/BL06 and pET/ect/BL22 induced expression on ectoine synthesis conditions, the results showed that:ectABC of C. marina transformed into E. coli BL21 can express ectoine under particular conditions. No ectoine was synthesized when LB medium including 1% NaCl induced expression. It was more appropriate for growth of recombinant PpET/ect/BL06 and pET/ect/BL22 in low-salt environment, but the higher salt concentration was beneficial to expression of ectoine synthesis of recombinant PpET/ect/BL06. C. marina ectoine synthase gene ectABC upstream promoter Pect is necessary in induction expression under high salt environment. When without IPTG, Lac promoter does not work. NaCl as the inducer to induced expression, the upstream promoter Pect of ectABC genes induce ectoine induced expression, but the expression of Lac promoter is lower than the promoter with Pect expression of synergy. NaCl concentration is in 1%-6% ranges, the higher NaCL concentration is, the more favorable recombinants PpET/ect/BL06 the expression of ectoine synthesis.