Methods for the positive selection of ethanol overproducing mutants from Saccharomyces cerevisiae

A method of making a S. cerevisiae strain with enhanced ethanol producing characteristics comprising,

• contacting a culture of a parent strain of S. cerevisiae with a sufficient amount of a first selection agent selected from the group consisting of oxythiamine, trehalose, bromopyruvate, glyoxylic acid and glucosamine for the selection agent to be toxic to the parent strain;
• growing the contacted culture on a plating medium containing a sugar as a carbon source;
• selecting candidate spontaneous mutant strains of the culture that grow on the plating medium;
• measuring an ethanol producing characteristic of the candidate spontaneous mutant strains to determine whether the candidate strain demonstrates enhanced ethanol production characteristics in comparison to the parent strain and if so, obtaining the S. cerevisiae strain with enhanced ethanol producing characteristics;
• contacting the obtained spontaneous mutant strain with enhanced ethanol production characteristics with an amount of a second selection agent selected from the group consisting of oxythiamine, trehalose, bromopyruvate, glyoxylic acid and glucosamine for the selection agent to be toxic to the strain, wherein the second selection agent differs from the first selection agent;
• and
• repeating the growing, selecting and measuring acts to obtain a second spontaneous mutant strain having enhanced ethanol production characteristics in comparison to the first obtained spontanerous mutant strain.