During ethanol fermentation, yeast cells are exposed to various stresses that have negative effects on cell growth, cell survival, and fermentation ability. This study, therefore, aims to develop Kluyveromyces marxianus-adapted strains that are multi-stress tolerant and to increase ethanol production at high temperatures through a novel evolutionary adaptation procedure. K. marxianus DMKU 3-1042 was subjected to repetitive long-term cultivation with gradual increases in temperature (RLCGT), which exposed cells to various stresses, including high temperatures. In each cultivation step, 1% of the previous culture was inoculated into a medium containing 1% yeast extract, 2% peptone, and 2% glucose, and cultivation was performed under a shaking condition. Four adapted strains showed increased tolerance to ethanol, furfural, hydroxymethylfurfural, and vanillin, and they also showed higher production of ethanol in a medium containing 16% glucose at high temperatures. One showed stronger ethanol tolerance. Others had similar phenotypes, including acetic acid tolerance, though genome analysis revealed that they had different mutations. Based on genome and transcriptome analyses, we discuss possible mechanisms of stress tolerance in adapted strains. All adapted strains gained a useful capacity for ethanol fermentation at high temperatures and improved tolerance to multi-stress. This suggests that RLCGT is a simple and efficient procedure for the development of robust strains.

The intrinsic mechanism of the thermotolerance of Kluyveromyces marxianus was investigated by comparison of its physiological and metabolic properties at high and low temperatures. After glucose consumption, the conversion of ethanol to acetic acid became gradually prominent only at high temperature (45°C) and eventually caused a decline in viability, which was prevented by exogenous glutathione. Distinct levels of reactive oxygen species (ROS), glutathione, and NADPH suggest greater accumulation of ROS and enhanced ROS-scavenging activity at a high temperature. Fusion and fission forms of mitochondria were dominantly observed at 30°C and 45°C, respectively. Consistent results were obtained by temperature up-shift experiments including transcriptomic and enzymatic analyses, suggesting a change of metabolic flow from glycolysis to the pentose phosphate pathway. Results of this study suggest that K. marxianus survives at a high temperature by scavenging ROS via metabolic change for a period until a critical concentration of acetate is reached. IMPORTANCE Kluyveromyces marxianus , a thermotolerant yeast, can grow well at temperatures over 45°C, unlike Kluyveromyces lactis , which belongs to the same genus, or Saccharomyces cerevisiae , which is a closely related yeast. K. marxianus may thus bear an intrinsic mechanism to survive at high temperatures. This study revealed the thermotolerant mechanism of the yeast, including ROS scavenging with NADPH, which is generated by changes in metabolic flow.