Insertion of transposon in the vicinity of SSK2 confers enhanced tolerance to furfural in Saccharomyces cerevisiae.

Furfural is one of the major inhibitors generated during sugar production from cellulosic materials and, as an aldehyde, inhibits various cellular activities of microorganisms used, leading to prolonged lag time during ethanologenic fermentation. Since Saccharomyces cerevisiae strains tolerant to furfural are of great economic benefit in producing bioethanol, much effort to obtain more efficient strains continues to be made. In this study, we examined the furfural tolerance of transposon mutant strains (Tn 1-5) with enhanced ethanol tolerance and found that one of them (Tn 2), in which SSK2 is downregulated at the transcriptional level, displayed improved furfural tolerance. Such phenotype was abolished by complementation of the entire open reading frame of SSK2, which encodes a mitogen-activated protein (MAP) kinase kinase kinase of the high osmolarity glycerol (HOG) signaling pathway, suggesting an inhibitory effect of SSK2 in coping with furfural stress. Tn 2 showed a significant decrease in the intracellular level of reactive oxygen species (ROS) and early and high activation of Hog1p, a MAP kinase integral to the HOG pathway in response to furfural. The transcriptional levels of CTT1 and GLR1, two of known Hog1p downstream target genes whose protein products are involved in reducing ROS, were increased by 43 % and 56 % respectively compared with a control strain, probably resulting in the ROS decrease. Tn 2 also showed a shortened lag time during fermentation in the presence of furfural, resulting from efficient conversion of furfural to non-toxic (or less toxic) furfuryl alcohol. Taken together, the enhanced furfural tolerance of Tn 2 is suggested to be conferred by the combined effect of an early event of less ROS accumulation and a late event of efficient detoxification of furfural.