SACCHAROMYCES CEREVISIAE STRAINS

摘要

The present invention relates to a method of preparing a strain of sugar fermenting Saccharomyces cerevisiae with capability to ferment xylose, wherein said method comprises different procedural steps. The method comprises mating a first sporulated Saccharomyces cerevisiae strain with a second Saccharomyces cerevisiae haploid strain. Thereafter, screening for mated cells is performed, growing such mated cells, and verifying that mated cells exhibit basic morphology by microscopic inspection. Thereafter, creation of a mixture of the mated cells is performed, subjecting the mixture to continuous chemostat lignocellulose cultivation and obtaining the sugar fermenting Saccharomyces cerevisiae cells with capability to ferment xylose is performed. The invention also comprises strains obtained by said method.

主权项

1. 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 an amino acid sequence of SEQ ID NO 1 having xylose reductase activity or encoding a fragment of said amino acid sequence having xylose reductase activity, wherein said strain is obtained by the following steps:
a) sporulating a first strain of Saccharomyces cerevisiae for providing at least 20 tetrads of said strain, b) introducing DNA, encoding for xylose reductase and xylitol dehydrogenase obtained from Scheffersomyces stipitis and xylulokinase obtained from Saccharomyces cerevisiae, into a second strain of Saccharomyces cerevisiae, c) mating the first sporulated Saccharomyces cerevisiae strain with the second Saccharomyces cerevisiae strain evolved on xylose and in a haploid state by mixing cells of said Saccharomyces cerevisiae haploid strain with each tetrad obtained in step a) to provide mated cells on an YPD agar plate, d) screening for mated cells on xylose and geneticin agar plates, e) growing 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 microscopic inspection and selecting such mated cells with basic morphological features, g) creation of a mixture of the mated cells with basic morphology features in equal amounts from step f), h) subjecting the mixture to continuous chemostat cultivation firstly in a microaerobic environment and thereafter in a anaerobic environment using feeding strategy with defined xylose medium feed for at least 0.08 h−1 in cyclus of feed and disrupted feed in a cyclus time range of a few hours, and thereafter with constant feed for at least 0.13 h−1, i) subjecting the mixture to continuous chemostat cultivation in an anaerobic environment with cells from step h) using lignocellulose feeding with xylose medium for at least 0.08 h−1 in cyclus of feed and disrupted feed in a cyclus time range of a few hours, and with constant feed rate for at least 0.13 h−1, j) obtaining the sugar fermenting Saccharomyces cerevisiae cells with capability to ferment xylose by collecting said cells from the chemostat reactor, k) diluting the cells from chemostat into water, then plating cells onto YPD agar plate to obtain single colonies, and incubating at 30° C. for at least 4 days.