[Cloning and expression of Trichoderma reesei endoglucanase III (EG III) gene in Saccharomyces cerevasiae]

Abstract

Using Congo red-staining method, one positive clone with CMCase activity was isolated from the Trichoderma ressei cDNA gene bank constructed in Saccharomyces cerevasiae. Sequencing result showed that the 1.5 kb-length DNA fragment inserted in the recombinant plasmid encoded EG III gene from T. reesei. Enzymatic characterization of the EG III produced by recombinant S. cerevasiae was analyzed. The experimental results indicated that the optimum pH and temperature for EG III are 5.0 and 60 degrees C, respectively. The effects of secretory system components SSO 2 and SEB1 of S. cerevisiae on EG III secretion were examined. The results indicated that the amount of EG III secreted by the strain with SSO 2-overexpression was highest among the different recombinant S. cerevisiae strains, showed that SSO 2 is a rate-limiting component of the secretory machinery in the process of EG III secretion. Furthermore, the EG III expression level was increased 5.3 times by deletion. Furthermore, the EG III expression level was increased 5.3 times by deletion of the 98 bp in 5' untranslated region of eg3 mRNA sequence. This result suggested that the regulation region could exist in the 5' untranslated region of EG III mRNA, which is recognized by the gene expression related factors of S. cerevasiae.