Hae-Sun Yun

Construction of hsf1 Knockout-mutant of a Thermotolerant Yeast Strain Saccharomyces cerevisiae KNU5377

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.

Cyclophilin A Cpr1 Protein Modulates the Response of Antioxidant Molecules to Menadione- induced Oxidative Stress in Saccharomyces cerevisiae KNU5377Y

Objectives The cellular function of cyclophilin A (CypA) differs between organisms, even though CypA is conserved in both prokaryotes and eukaryotes. The purpose of this study was to elucidate the role of activated CypA isoform CPR1 in the antioxidative mechanisms of Saccharomyces cerevisiae under menadione (MD)-induced oxidative stress. Methods Four S. cerevisiae strains, KNU5377Y (kwt) and BY4741 (bwt), and their isogenic cpr1⊿ mutant strains (kc1 and bc1), were treated with MD, at a concentration ranging between 0.25 and 0.4 mM. Cpr1-mediated antioxidative effects were analyzed by measuring the levels of cellular glutathione (GSH) and ascorbate (AsA)-like molecules in yeast. Results GSH and AsA-like reductant molecule concentrations were more reduced in the presence of MD in the kc1 strain than in the kwt strain; whereas, there was no significant difference between the bwt and bc1 strains under the same conditions. In kc1 strain samples, we observed a reduction in the expression of proteins related both to GSH synthesis and the recycling system, and simultaneously, downregulation of GSH synthetase and GSH reductase activities were also evident. Oxidative stress in the kc1 strain was alleviated by the application of the GSH and AsA analog. Conclusion These results indicate that activated Cpr1 modulates the response of antioxidant molecules involved in cellular redox homeostasis of KNU5377Y during oxidative stress induced by MD.