Improved Stress Tolerance of Saccharomyces cerevisiae by CRISPR-Cas-Mediated Genome Evolution
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In bioprocesses, a microorganism with high tolerance to various stresses would be advantageous for efficient bio-based chemical production. Yeast Saccharomyces cerevisiae has long been used in the food industry because of its safety and convenience, and genetically engineered S. cerevisiae strains have been constructed and used for the production of various bio-based chemicals. In this study, we developed a novel genome shuffling method for S. cerevisiae using CRISPR-Cas. By using this, the thermotolerant mutant strain T8-292, which can grow well at 39 °C, was successfully created. The strain also showed higher cell viability in low pH and high ethanol concentration. In addition, the differences in genome structure between mutant and parent strains were suggested by random amplified polymorphic DNA PCR method. Our genome shuffling method could be a promising strategy for improvement of various stress tolerance in S. cerevisiae.
KeywordsCRISPR-Cas δ sequence DNA repair Genome shuffling Saccharomyces cerevisiae Stress tolerance
This work was partly supported by the Japan Society for the Promotion of Science KAKENHI (grant number JP18K14069) and KAKENHI Specific Support Operation of Osaka Prefecture University to RY.
- 3.Abdel-Banat, B. M. A., Hoshida, H., Ano, A., Nonklang, S., & Akada, R. (2010). High-temperature fermentation: how can processes for ethanol production at high temperatures become superior to the traditional process using mesophilic yeast? Applied Microbiology and Biotechnology, 85(4), 861–867.PubMedGoogle Scholar
- 8.Abe, H., Fujita, Y., Takaoka, Y., Kurita, E., Yano, S., Tanaka, N., & Nakayama, K. (2009). Ethanol-tolerant Saccharomyces cerevisiae strains isolated under selective conditions by over-expression of a proofreading-deficient DNA polymerase δ. Journal of Bioscience and Bioengineering, 108(3), 199–204.PubMedGoogle Scholar
- 10.Shi, D., Wang, C., & Wang, K. (2009). Genome shuffling to improve thermotolerance, ethanol tolerance and ethanol productivity of Saccharomyces cerevisiae. Journal of Industrial Microbiology & Biotechnology, 36(1), 139–147.Google Scholar
- 17.Couto, M. M. B., van der Vossen, J. M. B. M., Hofstra, H., & Huis in’t Veld, J. H. J. (1994). RAPD analysis: a rapid technique for differentiation of spoilage yeasts. International Journal of Food Microbiology, 24(1-2), 249–−260.Google Scholar
- 18.Rasband, W. S. ImageJ, U. S. National Institutes of Health, Bethesda, Maryland, USA, https://imagej.nih.gov/ij/, 1997–2018. Accessed in 2016.
- 29.Kitichantaropas, Y., Boonchird, C., Sugiyama, M., Kaneko, Y., Harashima, S., & Auesukaree, C. (2016). Cellular mechanisms contributing to multiple stress tolerance in Saccharomyces cerevisiae strains with potential use in high-temperature ethanol fermentation. AMB Express, 6(1), 107.PubMedPubMedCentralGoogle Scholar
- 32.Cenis, J. L. (1993). Identification of Four MajorMeloidogynespp. by Random Amplified Polymorphic DNA (RAPD-PCR). American Physical Society, 83(1), 76–80.Google Scholar