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KMID : 0613820190290080916
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Journal of Life Science
2019 Volume.29 No. 8 p.916 ~ p.921
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Isolation of a Mutant with Thermotolerance and Ethanol Tolerance Using Proofreading-deficient DNA Polymerases in Saccharomyces cerevisiae
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Kim Yeon-Hee
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Abstract
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In this study, we constructed a biological system that exhibited thermotolerance, ethanol tolerance, and increased ethanol productivity using a random mutagenesis method. We attempted to isolate a thermotolerant mutant using proofreading-deficient DNA polymerase δ and ε encoded by the pol3 and pol2 genes, respectively, in Saccharomyces cerevisiae. To obtain mutants that could grow at high temperatures (38℃and 40℃), random mutagenesis of AMY410 (pol2-4) and AMY126 (pol3-01) strains was induced. The parental strains (AMY410 and AMY126) grew poorly at temperatures higher than 38℃. By stepwise elevation of the incubation temperature, AMY410-Ht (heat tolerance) and AMY126- Ht strains that proliferated at 40℃ were obtained. These strains were further incubated in medium containing 6% and 8% ethanol and then AMY410-HEt (heat and ethanol tolerance) and AMY126-HEt strain with ethanol tolerance at an 8% ethanol concentration was obtained. The AMY126-HEt strain grew even at an ethanol concentration of 10%. Furthermore, following the addition of high concentrations of glucose (5% and 10%), an AMY126-HEt3 strain with increased ethanol productivity was isolated. This strain produced 24.7 g/l of ethanol (95% theoretical conversion yield) from 50 g/l of glucose. The findings demonstrate that a new biological system (yeast strain) showing various phenotypes can be easily and efficiently bred by random mutagenesis of a proofreading- deficient mutant.
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KEYWORD
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Ethanol tolerance, proofreading-deficient mutant, random mutagenesis, Saccharomyces cerevisiae, thermotolerance
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