Overproduction of glycolytic enzymes in yeast

Eight different enzymes for glycolysis and alcoholic fermentation were overproduced in a common Saccharomyces cerevisiae strain by placing their genes on multicopy vectors. The specific enzyme activities were increased between 3.7- and 13.9-fold above the wild-type level. The overproduction of the different glycolytic enzymes had no effect on the rate of ethanol formation, even with those enzymes that catalyse irreversible steps: hexokinase, phosphofructokinase and pyruvate kinase. Also the simultaneous increase in the activities of pairs of enzymes such as pyruvate kinase and phosphofructokinase or pyruvate decarboxylase and alcohol dehydrogenase, did not increase the rate of ethanol production. The levels of key glycolytic metabolites were also normal, compared to the reference strain.