Efficient one-step starch utilization by industrial strains of Saccharomyces cerevisiae expressing the glucoamylase and α-amylase genes from Debaryomyces occidentalis

Abstract

Amylolytic industrial polyploid strains of Saccharomyces cerevisiae (ATCC 4126, ATCC 9763 and ATCC 24858) expressing a glucoamylase gene (GAM1) or an alpha-amylase gene (AMY) from Debaryomyces occidentalis were developed. The glucoamylase activity of S. cerevisiae ATCC 9763 expressing the GAM1 gene was 3.7-times higher than that of D. occidentalis. On the other hand, alpha-amylase activity in the corresponding strain expressing the D. occidentalis AMY gene increased 10-times relative to D. occidentalis. These two recombinant yeast strains expressing the GAM1 gene and AMY gene, respectively were cultured simultaneously to produce both glucoamylase and alpha-amylase for efficient one-step utilization of starch. Growth, substrate utilization and enzyme activity of these strains are described.

Construction of a direct starch-fermenting industrial strain of Saccharomyces cerevisiae producing glucoamylase, α-amylase and debranching enzyme

“…An industrial polyploid strain of S. cerevisiae ATCC 9763 distillery yeast (Ness et al 1993) was used as the host for the yeast transformation experiment. Plasmid YIpAURG1 and YIpdAURSGd (Ghang et al 2005(Ghang et al , 2007 were used to clone the GA1 gene and GAM1 gene, respectively. YIpdAURDpSAd and YIpGB2 (Choi et al 2002;Lim et al 2008) served as the backbones of the d-integrative system and the 18S rDNA-integrative system, respectively.…”

Yeast glucoamylases: molecular-genetic and structural characterization

“…All these yeasts synthesize "the true glucoamylases" belonging in the sequence-based classification of carbohydrate-active enzymes (Cantarel et al 2009) to the family 15 of glycoside hydrolases. They share a common mechanism of action involving inversion of the anomeric configuration (Sauer et al 2000) in contrast to another group of exo-acting starch-hydrolyzing yeast enzymes from Candida albicans (Sturtevant et al 1999), Debaryomyces occidentalis (Ghang et al 2007) and Schwanniomyces occidentalis (Sato et al 2005), previously named as glucoamylases. These enzymes are, however, typical α-glucosidases and belong to the glycoside hydrolase family 31.…”

Construction of Amylolytic Industrial Strains of Saccharomyces cerevisiae for Improved Ethanol Production from Raw Starch

“…Manipulation of S. cerevisiae to expresses high RSDE activities, via δ-integration and rDNA-integration of the RSDE genes, can be useful for the improvement of ethanol productivity from raw starch (Kim et al, 2011;Yamada et al, 2012). We recently constructed δ -integrative and rDNA-integrative vectors to allow for the multiple integration of amylase genes into the chromosomes of industrial strains (Ghang et al, 2007;Kim et al, 2010). In this study, we constructed 18S rDNA-integrative vectors containing RSDE genes, such as Bacillus amyloliquefaciens α-amylase (Amy) and Aspergillus awamori glucoamylase genes (GA1), and integrated them into the RSDE-producing parental strain with δ-integrated GA1 and Debaryomyces occidentalis α -amylase (AMY) genes (ATCC 9763/YIpδAGSAδ) in an effort to develop industrial strains of S. cerevisiae with higher RSDE activities.…”

“…ATCC 9763/YIpδAGSAδ was constructed from the industrial strain of S. cerevisiae ATCC 9763 (Kim et al, 2011) and was used as the host for the yeast transformation experiment. Plasmids YIpδAURAGδ and YIpδAURSAδ (Ghang et al, 2007) were used as sources of GA1 and AMY genes, respectively. Plasmid YIpSG2rD (Kim et al, 2010) was employed as the backbone of the rDNA-integrative system.…”

“…1). The glucoamylase and α-amylase activities were quantified at pH 6.0 and 40℃ using the PGO/ODAD assay (Sigma) and dinitrosalicylic acid method, respectively (Ghang et al, 2007). All assays were repeated in triplicate and the mean values were calculated.…”