title: 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.
authors.affiliation: Guinness Brewing Worldwide Research Centre, Dublin, Ireland.
authors.forename: B T
authors.initials: BT
authors.lastname: Kinsella
authors.forename: B A
authors.initials: BA
authors.lastname: Cantwell
mesh_terms: D001483:Base Sequence; D002175:Candida / Q000201:enzymology / Q000235:genetics*; D003001:Cloning, Molecular; D003470:Culture Media; D004271:DNA, Fungal / Q000737:chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D015966:Gene Expression Regulation, Fungal; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids; D011401:Promoter Regions, Genetic; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics*; D014170:Transformation, Genetic; D000520:alpha-Glucosidases / Q000096:biosynthesis / Q000235:genetics*
chemical_list: D003470:Culture Media; D004271:DNA, Fungal; D000520:alpha-Glucosidases