Method of preparing a strain of Saccharomyces cerevisiae and a method of fermentation to produce ethanol

A method of preparing a strain of Saccharomyces cerevisiae comprising at least one native XKS1 gene in its genome encoding xylulokinase, at least one native XDH1 gene in its genome encoding xylitol dehydrogenase, and at least one modGre3 gene in its genome, said modGre3 gene encoding the protein of SEQ ID NO:

• 1 having xylose reductase activity, wherein said method comprises the following steps:
• a) sporulating a first strain of Saccharomyces cerevisiae to provide at least 20 tetrads of said strain,
• b) introducing DNA encoding a xylose reductase and a xylitol dehydrogenase obtained from Scheffersomyces stipitis and a xylulokinase obtained from Saccharomyces cerevisiae, into a second strain of Saccharomyces cerevisiae which is a haploid strain that has the β-lactamase gene removed and has been evolved to utilize xylose, wherein said second strain of Saccharomyces cerevisiae is a haploid strain obtained from (i) removing the β-lactamase gene in the deposited yeast strain Taurus01 with deposit number CBS102679, Taurus03 with deposit number CBS128138, Taurus04 with deposit number CBS128139, Taurus07 with deposit number CBS128140, or Taurus10 with deposit number CBS128141, and (ii) evolving the strains of (i) to utilize xylose,
• c) mating the first sporulated Saccharomyces cerevisiae strain with the Saccharomyces cerevisiae haploid strain obtained in step b) by mixing the Saccharomyces cerevisiae haploid strain obtained in step b) with each tetrad obtain in step a) to provide mated cells on a YPD agar plate,
• d) screening for mated cells on xylose and geneticin agar plates,
• e) growing the mated cells from step d) in minimal defined xylose liquid medium,
• f) verifying that the mated cells exhibit basic morphology features of budding yeast by microscope inspection and selecting such mated cells with basic morphological features,
• g) creating a mixture of the mated cells with basic morphological features by including in equal amounts each of the selected mated cells exhibiting basic morphological features of budding yeast from step f),
• h) subjecting the mixture to continuous chemostat cultivation in a chemostat reactor firstly in a microaerobic environment and thereafter in an anaerobic environment using a feeding strategy wherein a defined xylose medium is fed in cycles of feeding and disrupted feeding at a dilution rate of at least 0.08 h−1 in a cycle time range of a few hours, followed by a constant feed at a dilution rate of at least 0.13 h−1,
• i) subjecting the mixture to a continuous chemostat cultivation in an anaerobic environment using a feeding strategy wherein lignocellulose and a xylose medium are fed in cycles of feeding and disrupted feeding at a dilution rate of at least 0.08 h−1 in a cycle time range of a few hours, followed by a constant feed at a dilution rate of at least 0.13 h−1,
• j) obtaining sugar fermenting Saccharomyces cerevisiae cells with capability to ferment xylose by collecting said cells subjected to the continuous chemostat cultivation of step i) which are capable of fermenting xylose from the chemostat reactor, and
• k) diluting the cells capable of fermenting xylose of step j) with water, plating the cells onto a YPD agar plate, and incubating at 30° C. for at least 4 days to obtain single colonies.