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

Abstract

To develop a strain of Saccharomyces cerevisiae that produces ethanol directly from starch, two integrative vectors were constructed to allow the simultaneous multiple integration of the Aspergillus awamori glucoamylase gene (GA1) and the Debaryomyces occidentalis alpha-amylase gene (AMY) and glucoamylase with debranching activity gene (GAM1) into the chromosomes of an industrial strain of S. cerevisiae. The GA1 and AMY genes were constitutively expressed under the ADC1 promoter in S. cerevisiae using the double delta-integration system. The GAM1 gene was constitutively expressed under the corresponding promoter using the double 18S rDNA-integration system. The recombinant industrial strain secreting biologically active alpha-amylase, glucoamylase and debranching enzyme was able to ferment starch to ethanol in a single step. The new strain produced 8% (v/v) ethanol (62.8 g l(-1)) from 20% (w/v) soluble starch after 2 days, fermentation.

Raw starch fermentation to ethanol by an industrial distiller’s yeast strain of Saccharomyces cerevisiae expressing glucoamylase and α-amylase genes

“…A genetically-manipulated S. cerevisiae that could synthesize and secrete amylolytic enzymes might prove useful for the economically important processes mentioned above (de Moraes et al 1995;Marin et al 2001;Shigechi et al 2004). Many genes of filamentous fungi and yeasts that encode for amylolytic enzymes have been expressed in S. cerevisiae (Lin et al 1998;Kim et al 2010). For efficient ethanol production, recombinant S. cerevisiae that co-expresses glucoamylase and a-amylase require both high amylolytic activity and stable expression during direct ethanol fermentation from starch.…”

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

“…This result suggests that the secretion of raw starch degrading α-amylase from B. amyloliquefaciens under its own signal sequences occurred at relatively low levels (Southgate et al, 1993). The activities of α-amylase and glucoamylase improved with increasing copy numbers via rDNA-integration (Kim et al, 2010), reaching 6.95 U/ml and 1.08 U/ml, respectively in ATCC 9763/YIpδAGSAδ/YIpSA2rD and ATCC 9763/YIpδ AGSAδ/YIpAG2rD. These values were 1.2-times and 1.9-times higher than the respective activities of the parental strain (Kim et al, 2011).…”

“…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.…”

“…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. The YPD medium [1% (w/v) yeast extract, 2% (w/v) bacto-peptone, and 2% (w/v) glucose] was employed for the propagation of S. cerevisiae.…”

“…1). Recently, Kim et al (2010) reported δ-integrated and rDNA-integrated copy numbers of 46 and 12 for the α-amylase and glucoamylase genes, respectively in the recombinant yeast strain exhibiting high α-amylase and glucoamylase activity, as determined by real-time PCR analysis. Therefore, the accumulation of sequential δ-integration and rDNA-integration of RSDE genes can result in increasing the copy number of the corresponding genes and improving RSDE activities (Choi et al, 2002).…”