Development of a screening method for the identification of a novel Saccharomyces cerevisiae mutant over-expressing Trichoderma reesei cellobiohydrolase II

Gundllapalli S.B. ; Otero R.R.C. ; Pretorius I.S. (2006)


In a previous study we showed that the fusion of the cellulose-binding domain (CBD2) from Trichoderma reesei cellobiohydrolase II to a β-glucosidase (BGL1) enzyme from Saccharomycopsis fibuligera significantly hindered its expression and secretion in Saccharomyces cerevisiae. This suggests that the possible low secretion of heterologous cellulolytic enzymes in S. cerevisiae could be attributed to the presence of a cellulose-binding domain (CBD) in these enzymes. The aim of this study was to increase the extracellular production of the chimeric CBD2-BGL1 enzyme (designated CBGL1) in S. cerevisiae. To achieve this, CBGL1 was used as a reporter enzyme for screening mutagenised S. cerevisiae strains with increased ability to secrete CBD-associated enzymes such as cellulolytic enzymes. A mutant strain of S. cerevisiae, WM91-CBGL1, which exhibited up to 200 U L-1 of total activity, was isolated. Such activity was approximately threefold more than that of the parental host strain. Seventy-five per cent of the activity was detected in the extracellular medium. The mutant strain transformed with the T. reesei CBH2 gene produced up to threefold more cellobiohydrolase enzyme than the parental strain, but with 50% of the total activity retained intracellularly. The cellobiohydrolase enzymes from the parent and mutant strains were partially purified and the characteristic properties analysed.

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