Since elevated ethanol is a major stress during ethanol fermentation, yeast strains tolerant to ethanol are highly desirable for the industrial scale ethanol production. A technology called global transcriptional machinery engineering (gTME), which exploits a mutant library of SPT15 encoding the TATA-binding protein of Saccharomyces cerevisiae (Alper et al., 2006; Science 314: 1565 Science 314: -1568, seems to a powerful tool for creating ethanol-tolerant strains. However, the ability of created strains to tolerate high ethanol on rich media remains unproven. In this study, a similar strategy was used to obtain five strains with enhanced ethanol tolerance (ETS1-5) of S. cerevisiae. Comparing global transcriptional profiles of two selected strains ETS2 and ETS3 with that of the control identified 42 genes that were commonly regulated with twofold change. Out of 34 deletion mutants available from a gene knockout library, 18 were ethanol sensitive, suggesting that these genes were closely associated with ethanol tolerance. Eight of them were novel with most being functionally unknown. To establish a basis for future industrial applications, strains iETS2 and iETS3 were created by integrating the SPT15 mutant alleles of ETS2 and ETS3 into the chromosomes, which also exhibited enhanced ethanol tolerance and survival upon ethanol shock on a rich medium. Fermentation with 20% glucose for 24 h in a bioreactor revealed that iETS2 and iETS3 grew better and produced approximately 25% more ethanol than a control strain. The ethanol yield and productivity were also substantially enhanced: 0.31 g/g and 2.6 g/L/h, respectively, for control and 0.39 g/g and 3.2 g/L/h, respectively, for iETS2 and iETS3. Thus, our study demonstrates the utility of gTME in generating strains with enhanced ethanol tolerance that resulted in increase of ethanol production. Strains with enhanced tolerance to other stresses such as heat, fermentation inhibitors, osmotic pressure, and so on, may be further created by using gTME.

dAggregation of misfolded protein in the endoplasmic reticulum (ER) induces a cellular protective response to ER stress, the unfolded protein response (UPR), which is mediated by a basic leucine zipper (bZIP) transcription factor, Hac1p/Xbp1. In this study, we identified and studied the molecular functions of a HAC1 homolog from the thermotolerant yeast Hansenula polymorpha (HpHAC1). We found that the HpHAC1 mRNA contains a nonconventional intron of 177 bp whose interaction with the 5= untranslated region is responsible for the translational inhibition of the HpHAC1 mRNA. The H. polymorpha hac1-null (Hphac1⌬) mutant strain grew slowly, even under normal growth conditions, and was less thermotolerant than the wild-type (WT) strain. The mutant strain was also more sensitive to cell wall-perturbing agents and to the UPR-inducing agents dithiothreitol (DTT) and tunicamycin (TM). Using comparative transcriptome analysis of the WT and Hphac1⌬ strains treated with DTT and TM, we identified HpHAC1-dependent core UPR targets, which included genes involved in protein secretion and processing, particularly those required for N-linked protein glycosylation. Notably, different glycosylation and processing patterns of the vacuolar glycoprotein carboxypeptidase Y were observed in the WT and Hphac1⌬ strains. Moreover, overexpression of active HpHac1p significantly increased the N-linked glycosylation efficiency and TM resistance. Collectively, our results suggest that the function of HpHac1p is important not only for UPR induction but also for efficient glycosylation in H. polymorpha. The endoplasmic reticulum (ER) represents the central organelle of the eukaryotic cell in which the crucial steps of protein folding, modification, and selection for transport to other compartments take place. Perturbation of ER homeostasis through, for example, the accumulation of misfolded proteins or defects in the ER membrane leads to the activation of intracellular signaling pathways referred to as the unfolded protein response (UPR), ERassociated protein degradation (ERAD), and the calnexin cycle (CNX) for glycoprotein quality control (1). UPR induces multiple protective cellular events required for proper protein folding and misfolded protein degradation. The underlying regulatory mechanism and modes of action of UPR have been intensively studied in the traditional yeast Saccharomyces cerevisiae (2). UPR is mediated by a master transcription factor, Hac1p, in S. cerevisiae (ScHac1p) (3), and the synthesis of functionally active Hac1p is triggered via nonconventional splicing of the HAC1 mRNA, which is mediated by the endonuclease activity of the ER stress sensor, Ire1p (4). This unusual splicing of HAC1 mRNA induces the translation of HAC1 mRNA by eliminating base pairing between the 5= untranslated region (UTR) and the splicing site (5). The newly synthesized Hac1p is then shuttled into the nucleus and functions as an active basic leucine zipper (bZIP) transcription factor during ER stress. Hac1p binds to a UPR element (UPRE) within the pr...