Abstract
Corn-ethanol production requires enzymatic hydrolysis of the starch prior to yeast fermentation in a two-stage process. To create yeast capable of producing both endo- and exo-acting starch-degrading enzymes, recombinant constructs of Hordeum vulgare (barley) α-amylase and Lentinula edodes glucoamylase genes were integrated into the chromosomes of Saccharomyces cerevisiae. PCR amplification, dot blot hybridization, and Northern analysis confirmed that the genes were effectively integrated and transcribed. The expression of the enzymes purified from the yeast culture was analyzed by SDS-PAGE. 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 a-amylase and the glucoamylase gene constructs yielded recombinant yeast that expressed and secreted both enzymes in active forms. The recombinant yeast was able to hydrolyze starch with ∼77% conversion in laboratory cultures.