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United States Department of Agriculture

Agricultural Research Service

Research Project: EVOLUTIONARY ENZYMES AND SEPARATION PROCESSES FOR IMPROVED BIOREFINING OF CROPS AND RESIDUES Title: Chromosomal integration of recombinant alpha-amylase and glucoamylase genes in saccharomyces cerevisiae for starch conversion

Authors
item Wong, Dominic
item Batt-Throne, Sarah
item Robertson, George
item Lee, Charles
item Wagschal, Kurt

Submitted to: Industrial Biotechnology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: December 17, 2009
Publication Date: June 16, 2010
Citation: Wong, D., Batt Throne, S.B., Robertson, G.H., Lee, C.C., Wagschal, K.C. 2010. Chromosomal integration of recombinant alpha-amylase and glucoamylase genes in saccharomyces cerevisiae for starch conversion. Industrial Biotechnology. 6:112-118.

Interpretive Summary: The production of fuel ethanol from cornstarch involves the breakdown of starch to simple sugars by enzymatic reactions, followed by fermentation of the sugar to ethanol by the yeast Saccharomyces cerevisiae. The enzyme usage, estimated at 1 g per gallon of ethanol, contributes considerable to the cost and requires an elaborate process infrastructure. Development of engineered yeast to provide a cell-based system that is capable of both enzyme hydrolysis and fermentation with high yields could have significant technical and economic impact on the current process. It would eliminate added enzymes and result in a simple unit operation and fermentation. In order to enable the yeast to use starch for fermentation, we have engineered yeast strains to produce starch-degrading enzymes, '-amylase and glucoamylase, in active forms. The genes were co-integrated into the yeast chromosome, and co-expression of the genes was confirmed and characterized. The resulting yeast acquired the capacity of hydrolyzing starch with a yield of about 77% conversion.

Technical Abstract: Recombinant constructs of barley '-amylase and Lentinula edodes glucoamylase genes were integrated into the chromosomes of Saccharomyces cerevisiae. The insertion was confirmed by PCR amplification of the gene sequence in the chromosomes. The expression was analyzed by SDS-PAGE of the enzymes purified from the yeast culture. Secretion of the active enzymes was individually monitored during growth of the yeast using assays specific for exo- and endo-amylolytic activities. Co-integration of the '-amylase and the glucoamylase gene constructs yielded recombinant yeast that expressed and secreted both enzymes. This recombinant yeast was able to hydrolyze starch with a yield of ~77% conversion.

Last Modified: 11/22/2014
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