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KMID : 0613820060160030454
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Journal of Life Science
2006 Volume.16 No. 3 p.454 ~ p.458
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Construction of hsf1 Knockout-mutant of a Thermotolerant Yeast Strain Saccharomyces cerevisiae KNU5377
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Kim Il-Sup
Yun Hae-Sun
Choi Hye-Jin
Sohn Ho-Yong
Yu Choon-Bal
Kim Jong-Guk
Jin Ingn-Yol
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Abstract
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HSF1 is the heat shock transcription factor in Saccharomyces cerevisiae. S. cerevisiae KNU5377 can ferment at high temperature such as 40℃. We have been the subjects of intense study because Hsf1p mediates gene expression not only to heat shock, but to a variety of cellular and environmental stress challenges. Basing these facts, we firstly tried to construct the hsf1 gene-deleted mutant. PCR-method for fast production of gene disruption cassette was introduced in a thermotolerant yeast S. cerevisiae KNU5377, which allowed the addition of short flanking homology region as short as 45 bp suffice to mediate homologous recombination to kanMX module. Such a cassette is composed of linking genomic DNA of target gene to the selectable marker kanMX4 that confers geneticin (G418) resistance in yeast. That module is extensively used for PCR-based gene replacement of target gene in the laboratory strains. We describe here the generation of hsf1 gene disruption construction using PCR product of selectable marker with primers that provide homology to the hsf1 gene following separation of haploid strain in wild type yeast S. cerevisiae KNU5377. Yeast deletion overview containing replace cassette module, deletion mutant construction and strain confirmation in this study used Saccharomyces Genome Deletion Project (http:://www-sequence.standard.edu/group/yeast_deletion_project). This mutant by genetic manipulation of wild type yeast KNU5377 strain will provide a good system for analyzing the research of the molecular biology underlying their physiology and metabolic process under fermentation and improvement of their fermentative properties.
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KEYWORD
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Gene disruption, hsf1 gene, Saccharomyces cerevisiae KNU5377
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