Strategy for Stable Overexpression of Recombinant β-agarase Gene in Yeast
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In this study, we compared that optimal production system of β-agarase encoded by the AGAG1 gene derived from Alteromonas sp. The pRS-TG, pRS-TDG, pRS-AG, and pRS-PG plasmids containing TEF1, TDH3, ADH1, and PGK1 promoters, respectively, were constructed to select a suitable promoter for the expression of the AGAG1 gene. To investigate the effects of promoters on the expression of the AGAG1 gene, each plasmid was introduced into a Saccharomyces cerevisiae FY834 strain. Each transformant was cultivated in rich medium and β-agarase was successfully produced and secreted into medium. The agarase activity reached 0.3-0.8 unit/ml and the FY834/pRS-TG strain showed highest agarase activity (0.83 unit/ml). The agarase activity in the FY834/pRS-TG strain was about 2 folds higher than compared with activity of agarase expressed by inducible promoter (GAL10 promoter). Furthermore, the AGAG1 gene integrated by YIp system was stably expressed in 112 generations and activity of recombinant agarase was maintained.
Abstract
In this study, we compared that optimal production system of β-agarase encoded by the AGAG1 gene derived from Alteromonas sp. The pRS-TG, pRS-TDG, pRS-AG, and pRS-PG plasmids containing TEF1, TDH3, ADH1, and PGK1 promoters, respectively, were constructed to select a suitable promoter for the expression of the AGAG1 gene. To investigate the effects of promoters on the expression of the AGAG1 gene, each plasmid was introduced into a Saccharomyces cerevisiae FY834 strain. Each transformant was cultivated in rich medium and β-agarase was successfully produced and secreted into medium. The agarase activity reached 0.3-0.8 unit/ml and the FY834/pRS-TG strain showed highest agarase activity (0.83 unit/ml). The agarase activity in the FY834/pRS-TG strain was about 2 folds higher than compared with activity of agarase expressed by inducible promoter (GAL10 promoter). Furthermore, the AGAG1 gene integrated by YIp system was stably expressed in 112 generations and activity of recombinant agarase was maintained.