Abstract
An industrial strain of Saccharomyces cerevisiae, NRRL Y-132, was genetically engineered to stably secrete barley α-amylase by introducing an expression cassette containing the α-amylase cDNA and a dominant selectable marker into the ribosomal DNA loci of the yeast chromosome. Batch fermentation studies with this strain showed that the integrated expression cassette was mitotically stable for over 100 generations of continuous culture. In addition, mutation of cysteine 95 to alanine in barley α-amylase was found to increase the recombinant strain's starch hydrolyzing ability. The integrated strain showed higher starch hydrolyzing ability and ethanol production on both soluble and raw starch than the strain transformed with an episomal-based yeast expression plasmid. The results indicate that integration of the gene cassette into multiple copy loci should be given consideration when designing amylolytic yeast strains.
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