Cloning and expression of a Saccharomyces diastaticus glucoamylase gene in Saccharomyces cerevisiae and Schizosaccharomyces pombe

Abstract

A recombinant plasmid pool of the Saccharomyces diastatcus genome was constructed in plasmid YEp13 and used to transform a strain of Saccharomyces cerevistae. Six transformants were obtained which expressed amylolytic activity. The plasmids each contained a 3.9-kilobase (kb) BamHI fragment, and all of these fragments were cloned in the same orientations and had ideittical restriction maps, which differed from the map of the STAI gene (I. Yamashita and S. Fukui, Agric. Biol. Chemi. 47:2689-2692. The glucoamylase activity exhibited by aU S. cerevisiae transformants was approximately 100 times less than that of the donor strain. An even lower level of activity was obtained when the recombinant plasmid was introduced into Schizosaccharomyces pombe. No expression was observed in Escherichia coli. The 3.9-kb BamHI fragment hybridized to two sequences (4.4 and 3.9 kb) in BamHI-digested S. diastaticus DNA, regardless of which DEX (STA) gene S. diastaticus contained, and one sequence (3.9 kb) in BamHI-digested S. cerevisiae DNA. Tetrad analysis of crosses involving untransformed S. cerevisiae and S. diastaticus indicated that the 4.4-kb homologous sequence cosegregated with the glucoamylase activity, whereas the 3.9-kb fragment was present in each of the meiotic products. Poly(A)+ RNA fractions from vegetative and sporulating diploid cultures of S. cerevisiae and S. diasticus were probed with the 3.9-kb BamHI fragment. Two RNA species, measuring 2.1 and 1.5 kb, were found in both the vegetative and sporulating cultures of S. diastaticus, whereas one 1.5-kb species was present only in the RNA from sporulating cultures of S. cerevisiae.Saccharomyces diastaticus is closely related to Saccharomyces cerevisiae, except that S. diastaticus has the ability to produce and secrete glucoamylase (1). The genetics and biochemistry of glucoamylase production have been well studied, most recently by Erratt and Stewart (11-13), Tamaki (26), and Yamashita and Fukui (27-30). The independent investigations of Erratt and Stewart (11) and Tamaki (26) have led to two nomenclatures for designating the genes for starch fermentation. Tamaki (26) demonstrated the existence of three polymeric genes for starch fermentation: STAI, STA2, and STA3. Erratt and Stewart (11-13) used DEX to designate the gene for dextrin (starch) fermentation; they also found three polymeric genes for dextrin fermentation, one of which was shown to be allelic to STA3. Recently, it was deternined that DEXI is allelic to STA2 and DEX2 is allelic to STAI (lOa).As a continuation of our earlier work on glucoamylase from S. diastaticus, this yeast was used as a donor for the cloning of a glucoamylase gene into S. cerevisiae by functional complementation, using plasmid YEp13 (4) as the cloning vehicle. Functional complementation has been used successfully for cloning other genes in S. cerevisiae (3,16) and, more recently, for cloning the STAJ, DEXI (STA2), and STA3 genes from S. diastaticus (19,28,31 related to the sporulation-specific glucoamylase gene from S. cerevisiae (8) be...

Glucoamylases: Microbial Sources, Industrial Applications and Molecular Biology ? A Review

“…This acquired ability is of great interest for the production of ethanol from starch derivatives (Mercier and Colonna 1988). Erratt and Nasim (1986) cloned the glucoamylase gene from S. cerevisiae var. diastaticus in Saccharomyces cerevisiae and Schizosaccharomyces pombe.…”

Expression and regulation of glucoamylase from the yeast Schwanniomyces castellii

“…However, heat shock promotes expression of heat shock proteins and only gradually affects posttranslational levels of cell-associated and extracellular glucoamylase, suggesting that proteolysis is the other level of glucose action which is involved. These results suggest that glucoamylase expression is tightly regulated in a manner similar to that of the enzymes responsible for galactose and maltose metabolism in Saccharomyces yeasts (7,9,26).…”

The S1, S2 and SGA1 ancestral genes for the STA glucoamylase genes all map to chromosome IX in Saccharomyces cerevisiae

“…These genes are absent in S. cerevi.siue, but a related gene SGAI (previously SAG, SGA and Asfa), encoding an intracellular, sporulation-specific glucoamylase, is present *Corresponding author CCC 0749~503X195/080783-05 '1' 1995 by John Wiley & Sons Ltd (Clancy et al, 1982;Yamashita and Fukui, 1985). The restriction maps of STAI, STA2 and STA3 are identical (Pretorius et ul., 1986b(Pretorius et ul., , 1991Yamashita et al, 1985a), whereas SGAI (Erratt and Nasim 1986b) is homologous to the middle and 3' regions of the STA genes. Two additional sequences, SI and S2, present in various S. cerevisiae strains exhibit homology to the 5' regions of the STA genes (Pretorius et al, 1986b;Yamashita et al, 1985a).…”