Analysis of the expression and secretion of the Candida tsukubaensis alpha-glucosidase gene in the yeast Saccharomyces cerevisiae

Abstract

The alpha-glucosidase gene of Candida tsukubaensis is contained within a 3.47 kb BamH1-Mlul fragment which, when introduced into Saccharomyces cerevisiae AH22 on a yeast-Escherichia coli shuttle vector, allows the transformants to utilize maltose as sole carbon source. Thus, the cloned gene confers a dominant selectable phenotype on transformed strains of S. cerevisiae which are otherwise unable to grow in nutrient media containing maltose, dextrin or other alpha-1.4-linked alpha-D-glucopyranosides, specifically hydrolysed by the alpha-glucosidase. The cloned enzyme expressed in yeast is secreted into the extracellular medium in a glycosylated form which accounts for up to 60% of the secreted protein and has a molecular size of 70-80 kilodalton (kDa). Deglycosylation of the alpha-glucosidase showed that the enzyme is composed of two distinct polypeptides with subunit molecular weights of 63-65 kDa (peptide 1) and 50-52 kDa (peptide 2). An increase in the level of expression of the alpha-glucosidase by yeast transformants in selective minimal medium was obtained by using a vector with increased copy number containing the leu2-d gene as selectable marker. The alpha-glucosidase gene promoter functions more effectively than the Gall-10 promoter in directing alpha-glucosidase expression in S. cerevisiae. It also directs the expression of high levels of beta-galactosidase activity in yeast when fused to a promoterless E. coli lacZ gene. Expression of the alpha-glucosidase gene under the control of its own promoter is constitutive, orientation dependent and not subject to catabolite repression.