A knockout strain of CPR1 induced during fermentation of Saccharomyces cerevisiae KNU5377 is susceptible to various types of stress

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To investigate the tolerance factor of Saccharomyces cerevisiae KNU5377 against various types of environmental stress during fermentation, we identified the protein that is upregulated at high temperatures. The highly upregulated protein was high-score-matched as a cytoplasmic peptidyl-prolyl cis-trans isomerase, cyclophilin (Cpr1p), by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF). We constructed a CPR1-deleted KNU5377 strain (KNU5377Y cpr1Δ) to determine the roles of the protein under fermentative or stress condition. The growth of the S. cerevisiae KNU5377Y cpr1Δ strain was completely inhibited under the following conditions: heat (40°C), hydrogen peroxide (20–30 mM), menadione (0.3 mM), ethanol (16%), sulfuric acid (5 mm), and lactic acid (0.4–0.8%). However, the wild-type and cpr1Δ mutant of S. cerevisiae BY4741 as a positive control did not show differences in sensitivity to stress. It is interesting to note that the wild-type KNU5377Y and KNU5377Y cpr1Δ mutant showed high sensitivity against various stresses, particularly, acid stress such as in the presence of sulfuric and lactic acid. Although the alcohol fermentation rate of the KNU5377Y cpr1Δ mutant markedly decreased with an increase in temperature up to 40°C, we observed no decrease in that of the wild-type strain under the same conditions. These results suggest that CPR1 contributes to the stress tolerance of KNU5377 against various types of environmental stress caused during fermentation, thus leading to the physiological role of maintaining an alcohol fermentation yield, even at high temperatures such as 40°C.

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Strains and culture conditions

The yeast strains used in this study are listed in Table 1. KNU5377 was isolated from a sewage soil in Daegu, Korea. As a positive control, ATCC 24858 as an ethanol-tolerant strain from the American Type Culture Collection (ATCC) and W303 was purchased from the Korean Culture Center of Microorganisms (KCCM) were used, and the wild type and cpr1Δ mutant of BY4741 were purchased from the European S. cerevisiae Archives for Functional Analysis (EUROSCARF). Combined with the laboratory yeast

The identification of the Cpr1 protein induced during fermentation at 40°C

To gain insight into how KNU5377 can ferment at high temperatures, we first performed an expression analysis at the protein level by SDS–PAGE after fermentation at the indicated temperature (30°C, 37°C and 40°C). We focused on proteins of low molecular weight (MW) because proteins below 20 kDa and their relevant genes were highly upregulated in KNU5377 as determined by microarray analysis, two-dimensional (2D) gel electrophoresis, and SELDI-TOF MS under specific stresses such as heat, hydrogen

Discussion

S. cerevisiae KNU5377, originally named F38-1, was isolated and identified as a thermotolerant strain that can produce alcohol at high temperatures such as 40°C compared with the productivity of alcohol in laboratory control strains (5, 6). Moreover, the haploid KNU5377 strain (KNU5377Y) showed higher tolerance against many types of stress compared with the ATCC24858 and W303 strains.

In this study, an approximately 17-kDa protein, which was highly upregulated during fermentation at high

Acknowledgments

We thank Dr. Joseph Heitman for providing the Cpr1p antibody. This work was supported by a Korea Research Foundation Grant funded by the Korean Government (MOEHRD) (KRF-2005-202-C00289).

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