RESEARCH PAPER New Biotechnology  Volume 29, Number 2  January 2012 CDC19 encoding pyruvate kinase is important for high-temperature tolerance in Saccharomyces cerevisiae Suthee Benjaphokee 1 , Preeyaporn Koedrith 2 , Choowong Auesukaree 3 , Thipa Asvarak 2 , Minetaka Sugiyama 1 , Yoshinobu Kaneko 1 , Chuenchit Boonchird 2 and Satoshi Harashima 1 1 Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita-shi, Osaka 565-0871, Japan 2 Department of Biotechnology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand 3 Department of Biology, Faculty of Science, Mahidol University, Rama VI Road, Bangkok 10400, Thailand Use of thermotolerant strains is a promising way to reduce the cost of maintaining optimum temperatures in the fermentation process. Here we investigated genetically a Saccharomyces cerevisiae strain showing a high-temperature (41 8C) growth (Htg + ) phenotype and the result suggested that the Htg + phenotype of this Htg + strain is dominant and under the control of most probably six genes, designated HTG1 to HTG6. As compared with a Htg  strain, the Htg + strain showed a higher survival rate after exposure to heat shock at 48 8C. Moreover, the Htg + strain exhibited a significantly high content of trehalose when cultured at high temperature and stronger resistance to Congo Red, an agent that interferes with cell wall construction. These results suggest that a strengthened cell wall in combination with increased trehalose accumulation can support growth at high temperature. The gene CDC19, encoding pyruvate kinase, was cloned as the HTG2 gene. The CDC19 allele from the Htg + strain possessed five base changes in its upstream region, and two base changes resulting in silent mutations in its coding region. Interestingly, the latter base changes are probably responsible for the increased pyruvate kinase activity of the Htg + strain. The possible mechanism leading to this increased activity and to the Htg + phenotype, which may lead to the activation of energy metabolism to maintain cellular homeostasis, is discussed. Introduction Bioethanol, a renewable eco-friendly fuel, is now considered to be an alternative to conventional gasoline. Saccharomyces cerevisiae are facultative anaerobes and under anaerobic conditions can ferment glucose to ethanol. Improvements in ethanol production by using genetically engineered yeast cells during the fermentation process may lead to a boost in the bioethanol production industry [1]. Among the desirable traits of strains required for efficient bioetha- nol production, tolerance to high temperature, acidity and ethanol is crucial for reducing cooling and ethanol recovery costs, and for minimizing the risk of contamination. Although considerable pro- gress has been made with respect to engineering yeast strains for stress tolerance [2], a full understanding of the molecular mechan- isms that confer tolerance to stress remains lacking. Thermotolerant yeast strains offer the advantage of conducting the bioethanol production process at elevated temperatures. Furthermore, isolation and selection of strains from nature is a promising way to obtain super strains exhibiting desirable phe- notypes such as ability to grow at high temperatures [3]. The optimum temperature for growth of S. cerevisiae ranges from 25 8C to 30 8C, and S. cerevisiae do not normally grow at tempera- tures higher than 40 8C. Recently, a high-temperature growth phenotype (Htg), which enables cells to grow at 41 8C, has been categorized as a quantitative trait that is controlled by multiple genes in S. cerevisiae [4]. In addition, MKT1, END3 and RHO2 were identified as Htg quantitative trait genes underlying the Htg Research Paper Corresponding author: Harashima, S. (harashima@bio.eng.osaka-u.ac.jp) 166 www.elsevier.com/locate/nbt 1871-6784/$ - see front matter ß 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.nbt.2011.03.007