Enhancement of β-1,3-Glucanase Activity by Sequential δ-Sequence Mediated Integration in Saccharomyces cerevisiae

Beta-1,3-glucanase is widely used in various biotechnological and industrial processes, with over- production required to enable versatile utilization. We examined the overexpression of β-1,3-glucanase (EXGA) from Aspergillus oryzae using δ-sequence-mediated integration. We constructed pRSδ-exgA and pRSδK-exgA plasmids for integration of the EXGA gene into various chromosomes of Saccharomyces cerevisiae. These plasmids contain the ADH1 promoter for constitutive expression, a signal sequence (exoinulinase signal sequence (INU1 s.s)) for secretory production, and a δ-sequence for integration of β-1,3-glucanase. The pRSδ-exgA plasmid was transformed into the S. cerevisiae BY4742△exg1 strain, and β-1.3-glucanase was stably overexpressed and secreted. Another plasmid, pRSδK-exgA, was in- troduced into the S. cerevisiae BY4742△exg1 (YKY082) strain, and overexpression of β-1,3-glucanase was examined by inducible integration under geneticin selection. The activity of β-1,3-glucanase in- creased in accordance with a rise in the geneticin concentration, with 0.8 mg/ml of geneticin suitable for overexpression of β-1,3-glucanase. Subsequently, pRSδK-exgA was repeatedly transformed for se- quential δ-integration. The activity of β -1,3-glucanase reached about 0.063 unit/ml/OD 600, 0.095 unit/ml/OD600, 0.131 unit/ml/OD600 and 0.165 unit/ml/OD 600 by the first, second, third, and fourth round of integration, respective ly. According to the increase in the activity of β-1,3-glucanase by se- quential δ-integration, the copy number (integration rate) of the EXGA gene also increased in various chromosomes. These results suggest that recombinant β-1,3- glucanase activity can be sequentially in- creased by repeated δ-sequence integration.