Atlas Journal of Biology 2 (2): 100–115, 2012 doi: 10.5147/ajb.2012.0092 A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) A Mutated Yeast Strain with Enhanced Ethanol Production Ef- ficiency and Stress Tolerance Naghmeh Hemmati1*, David A. Lightfoot1,2, and Ahmed Fakhoury3 1 Department of Plant Soil and Agricultural Systems, Southern Illinois University at Carbondale, Carbondale, IL 62901-4415, USA; 2 Department of Biochemistry and Molecular Biology, Southern Illinois University at Carbondale, Carbondale, IL 62901USA; 3 Department of Plant Soil and Agricultural Systems, Southern Illinois University at Carbondale, Carbondale, IL 62901 USA Received: July 15, 2011 / Accepted: May 6, 2012 __________________________________________________ * Corresponding author: naghmeh@siu.edu 100 Abstract One of the strategies to improve and optimize bio-ethanol production from new feed stocks is to develop new strains of Saccharomyces cerevisiae with tolerance to stresses. The main objectives here were to; generate S. cerevisiae mutants tolerant to high ethanol concentrations; test for their ability to ferment maize starch; and partially characterize the mu- tations responsible for the new phenotypes. A combination of mutagenesis, selection and cross-stress protection meth- ods were used. EMS (ethyl methanesulfonate) was used to mutagenize one S. cerevisiae strain. The mutagenized yeast strain was exposed to high concentrations of ethanol and tolerant mutants were isolated. Mutants showed improved ethanol yield (0.02-0.03 g/g of maize) and fermentation ef- ficiency (3-5%). Finally, AFLP (Amplified Fragment Length Polymorphism) was performed to identify polymorphisms in the mutants that might underlie the strains ethanol tolerance. The best performing mutant isolate had four altered gene transcripts encoding; an arginine uptake and canavanine re- sistance protein (CAN1); mitochondrial membrane proteins (SLS1); a putative membrane glycoprotein (VTH1); and cy- tochrome C oxidase (COX6; EC 1.9.3.1) among about 1,000 tested. It was concluded these mutations might underlie the improved ethanol production efficiency and stress tolerance. Keywords: AFLP, Mutagenesis, Polymorphism, SNPs, Ethanol tol- erance, CAN1, SLS1, VTH1, COX6, Ethanol yield, Fermentation Efficiency. Introduction Modern civilization has been greatly dependent on the oxi- dation of fossil fuel reserves over the past 150 years for energy production (Hambourger et al., 2009; Stephenson et al., 2011). Fossil fuel reserves are limited and their current oxidation rate is a major global and environmental concern, with complex and severe impacts on the climate of the planet (DECC, 2009; Ste- phenson et al., 2011). Several factors have contributed to a revival of interest in bio-fuels, both in the United States and worldwide (Bothast et al., 1999; Jeffries and Jin, 2004; Fargione et al., 2008). These include; a steep increase in the price of crude oil and crude oil- based products, an increase in the awareness of the detrimen- tal effects of burning fossil fuels, release of greenhouse gases; a renewed political awareness of the need for decreasing the reliance of the economy on foreign oil and the availability of land set aside from food crop production ideal for biofuel crops (Bothast et al., 1999; Jeffries and Jin, 2004). Saccharomyces cerevisiae has been broadly used for fuel ethanol production due to its ability to produce high concentra- tion of ethanol from simple sugars. Ethanol counts as a toxin for yeast cells and tolerance to it is closely related to ethanol productivity which is a major factor in industrial ethanol produc- tion (Jones, 1989). Improving and increasing understanding of the impact of ethanol toxicity on yeast cells will assist enhancing yeast ethanol tolerance and higher ethanol production (Demain, 2009; Stanley, 2010). Earlier studies showed that the acquired tolerance to former- ly lethal stress levels has been linked to the activation of specific A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) 101 stress response mechanisms during pre-exposure to the sub-le- thal stress (Plesset et al., 1982; Sanchez and Lindquist, 1990; Coote et al., 1991). The effects of pre-exposure to stress condi- tions have been studied in ethanol stress (Vriesekoop and Pam- ment, 2005) and other stress conditions such as osmotic (Trollmo et al., 1988; Varela et al., 1992) and oxidative stresses (Davies et al., 1995). Stanley et al. (2010) proposed that pretreated yeast cultures showed a 70% reduction in the stress adaptation period when exposed to higher ethanol concentrations. There- fore, the isolation of stress resistant strains should include a pre- treatment phase. However, improving the efficiency of ethanol production in engineered strains is not trivial. Selection for small scale batch culture growth and the numbers of background mu- tations are the major issues that have been hard to overcome. The main objectives of the research reported here were to; gen- erate S. cerevisiae mutants with improved toleranance to high concentrations of ethanol; assess the ability of the mutants to produce bio-ethanol in larger scale cultures; and to partially characterize the mutations. Materials and Methods Strains The yeast strain (NCYC-1681 Brewing strain) that was devel- oped for fermentation was chosen for this experiment. The strain was; round-oval in colony shape; cream in color; had a shiny surface; and showed a smooth texture on agar. Cell viability was retained during fermentation to final ethanol concentrations of 15% (v/v). The strain was obtained from the National Collection of Yeast Cultures, Institute of Food Research, Norwich Research Park, Norwich, United Kingdom, NR4, 7UA. Media The complete growth medium for S. cerevisiae consisted of YPD liquid and solid medium (YPD Broth, Fisher). Yeast storage stock solutions were prepared by adding 15% (v/v) glycerol to complete growth medium of yeast after 24 hours and were stored at -80°C. Mutagenesis and Screening for Survival Rate A modified version of Burke et al. (2000) was used. Briefly, the cells were incubated at room temperature in 200 μl steril- ized distilled water containing 8% (w/v) EMS for 5-20 min with constant vortexing. The optimum lethal dose (LD) of each EMS concentration was calculated. The aim was to identify 70% to 80% lethality and was obtained by plating and incubating an appropriate dilution of cells at 30°C overnight. The growing colonies were isolated after 24 hours. Selection of Ethanol-Tolerant Mutants Previous researchers have demonstrated that pre-exposure of yeast to a sub-lethal amount of stressing agent such as etha- nol or heat can stimulate molecular responses resulting in resis- tance to higher levels of the same stress condition compared to control cells, cells without pre-exposure (Plesset et al., 1982; Sanchez and Lindquist, 1990; Coote et al., 1991). In the cur- rent research study, pre-treatment to stress condition (ethanol stress) and selection were performed after the mutagenesis step. Briefly, about 0.1 ml of the cell suspension (2 × 108 cell/ml) was added to 20 ml YPD liquid and cultivated at 30°C overnight. The cells were exposed to gradual additions of absolute ethanol (99.9% with 0.1% (v/v) methanol) to cultures. Concentrations were raised from 5% to 35%-40% (v/v) within 5 days (ad- ditions were 5% ethanol for the first day; 5% for the second day; 10% for the third day, 10% for the fourth day; and 5%- 10% for the fifth day). Positive controls (with 0% ethanol) were cultured under identical conditions. After the fifth day the cells were pelleted, washed and suspended in 2 ml sterilized distilled water. The resulting suspensions were plated on YPD-agar and cultured at 30°C overnight for mutant selection. This procedure was repeated more than 20 times and each time there were 3 replications. The growing colonies showed that the mutant strain was able to tolerate ethanol up to 40% (v/v). Testing Fermentation Abilities The mutants (two mutants) and the parent strain were sent to National Corn to Ethanol Research Center (NCERC) in Edwards- ville, IL to test for their fermentation ability (NCERC, 2007). The process was divided into three steps; preparation of yeast strains, liquefaction of starch substrate and fermentation. The ethanol yields and fermentation efficiencies for yeast strains (one control and two mutants) were calculated from the ethanol concentrations measured by HPLC after 64.5 hours of fermenta- tion. The statistical significance of the differences between sam- ples was evaluated using analysis of variance (ANOVA). DNA Extraction Genomic DNA was obtained following Chung, (1996). DNA quality was assessed by spectrophotometer (Spectronic, Madi- son, WI) by calculating the A260/A280 nm ratios and the A260 nm values were used to determine DNA concentrations. The DNA was stored at -20°C for the subsequent experiments. AFLP (Amplified Fragment Length Polymorphism) and Transforma- tion AFLP analysis was carried out using the AFLP Microorganism Primer Kit (Invitrogen, Carlsbad, CA). Polyacrylamide gel elec- trophoresis (PAGE) was divided to five major steps; preparation of glass plates; assembling and pouring the gel; electrophoresis for 2 h at 200V; fixing and silver staining gels; and extracting the bands with altered intensities in mutants or control strains from the gel. PCR was performed using band DNA as a template with the appropriate selective AFLP primers. Amplified bands were sep- arated and purified by agarose gel electrophoresis. QIAquick Gel Extraction Kit (Qiagen, Hilden, Germany) was used to ex- tract the bands from the agarose gel. pGEM®-T or pGEM®-T A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) 102 Easy Vectors Kit from Promega (Madison, WI) was used for li- gation. Transformations were made into E.coli as follows. The tubes containing the ligation reactions were centrifuged to col- lect contents at the bottom of the tubes. Just 1μl of the ligation reaction was added to 10 μl of thawed JM109 High Efficiency Competent CellsTM. The reaction was mixed gently. The tubes were placed on ice for 20 minutes. The cells were placed on a heat plate for 45–50 seconds at exactly 42°C and immediately after that tubes were returned to ice for 2 minutes. About 900 μl of LB medium was added to each tube and was incubated at 37°C for 1.5 hours. About 100 μl of each transformation reaction was plated on LB agar (0.625% (w/v)) with 100 μg/ ml ampicillin, 100 μg/ml X-gal and IPTG (80 μg/ml). The plates were incubated at 37°C overnight. White and blue colonies were identified after incubation. Plasmid DNA Purification and Sequencing Two or three white colonies from each sample were cultured in separate tubes containing 5 ml LB medium and incubated in a shaker and incubator (150 rpm) at 37 °C overnight. Wizard® Plus SV Minipreps DNA Purification System Kit (Promega) was used for isolation and purification of plasmid DNA. A PCR reac- tion was performed on the plasmid DNA and DNA quality was assessed and the size of each fragment was estimated after agarose gel electrophoresis. DNA sequencing was carried out at the Iowa State University DNA Facility. The DNA sequences were analyzed by VecScreen (vector screen), BLAST (basic local alignment search tool) at NCBI (Na- tional Center for Biotechnology Information) and SGD (Saccha- romyces Genome Database). SNPs (single nucleotide polymor- phisms) in the sequences were recognized and recorded. Results and Discussion Fermentation Two mutants were isolated that could grow when ethanol con- centrations were 35-40% (v/v) in media. In contrast the par- ent strain did not grow at concentrations above 15 %. Medium scale fermentation data indicated mutants had better ethanol yields (Figure 1) and fermentation efficiency than the parent strain (Figure 2). Polymorphic AFLP band isolation Overall, 10 bands were identified polymorphic from the primer pairs tested (Figure 3). The primer pairs generated about 10 discernible bands. Of those, 8 were dominant bands in the mutant. They were labeled as M1 to M8. Only two domi- nant polymorphic bands were found in control and absent from the mutant. They were labeled as C1-C2. The estimated size of the DNA fragments that showed polymorphism varied from 250 to 495 bp. From the 10 DNA fragments identified, 8 (C2, M2, M3, M4, M5, M6, M7 and M8) were successfully isolated. After transformation, minipreps and PCR steps, 6 DNA fragments were successfully Figure 1. Ethanol yield for all yeast strains that were tested, the paren- tal control and the two mutants. The error bars represent one standard deviation of three independent replicate fermentations. The ethanol yields were calculated for each yeast strain from the ethanol concen- trations measured by HPLC after 64.5 hours of fermentation. The sta- tistical significance of the differences between hybrids was evaluated using analysis of variance (ANOVA). Statistically significant differences were detected among the ethanol yields for the yeast strains that were tested (P = 1.79 x 10-5), where P is the probability that the ethanol yield for all yeast strains is the same. Strains with same letter were not statistically significantly different from each other. Figure 2. Fermentation efficiency for all yeast strains that were tested, the parental control and the two mutants. The error bars represent one standard deviation of three independent replicate fermentations. The fermentation efficiency was calculated for each yeast strain from the ethanol concentrations measured by HPLC after 64.5 hours of fermen- tation. The statistical significance of the differences between hybrids was evaluated using analysis of variance (ANOVA). Statistically signifi- cant differences were detected among the ethanol yields for the yeast strains that were tested (P = 1.79 x 10-5), where P is the probability that the ethanol yield for all yeast strains is the same. Strains with same letter were not statistically significantly different from each other. isolated and DNA sequence analyzed (C2 464 bp; M2 468 bp; M3 450 bp; M4 446 bp; M5 457 bp and M7 327 bp). Gene identification BLAST searches indicated that three fragments, C2, M4, and M5, showed high similarity (C2 98%; M4 98%; M5 99%) to the CAN1 gene,YEL063C (Supplementary Figure 1, Section A). CAN1 gene family members encode membrane proteins with about 590 amino acids that function as arginine permeases (SGD # CAN1/YEL063C ). The CAN1 gene is required for ar- ginine uptake by yeast cells and canavanine resistance by selec- tive exclusion (Rak et al., 2007). Two SNPs were found between the mutant and the wild type allele in the alignment between C2 and CAN1 gene (Table 1; Supplementary Figure 1, section C). Both SNPs, were in coding regions (Supplementary Figure 1, section B). One of the SNPs changed the amino acid sequence and produced nonsynony- mous amino acid but the other one did not change the amino acid sequence and produced synonymous change in amino acid sequence (Annotation Table). One SNP was found between the mutant and the wild type allele in the alignment between M4 insert and the expected CAN1 gene sequence (Supplementary Figure 1, section E). The only SNP was in coding regions (Supplementary Figure 1, sec- tion D). This SNP did not change the amino acid sequence and produced synonymous amino acid (Annotation Table). Two SNPs were found between the mutant and the wild type allele in the alignment between M5 insert and the expected CAN1 gene sequence (Supplementary Figure 1, section G). Both SNPs, were in coding regions (Supplementary Figure 1, section F). One of the SNPs changed the amino acid sequence and produced nonsynonymous amino acid but the other one did not change the amino acid sequence and produced synonymous change in amino acid sequence (Annotation Table). The transportation systems for many amino acids are pro- ton symports that an amino acid molecule enters the cell along with a proton (H+). The H+ ion entry to the cell reduces cellular pH. The cell preventive reaction from acidification of cytoplasm, maintaining cell pH and secondary transport mechanisms, is exporting the proton into the outside medium by a membrane bound ATPase that acts as a transporter which actively pumps hydrogen ions. During the fermentation for ethanol production, amino acid transport in yeast cells was strongly inhibited by the significant amounts of ethanol. The reason that yeast cells prevent the uptake of amino acids along with protons would be related to stopping too many protons from entering the cell (Dharmadhikari, 2007, Aguilera et al., 2006, Monteiro and Sa´- Correia, 1998; Ogawa et al., 2000; Rosa and Sa´-Correia, 1991). Many research studies presented that membrane structure and function are the predominant target of ethanol stress. Expo- sure of yeast to ethanol results in increased membrane fluidity and consequential decrease in membrane integrity (Mishra and Prasad, 1989). Teixeira et al. (2009) demonstrated a detailed physiological and molecular study that illustrates changes in plasma membrane organization and function of yeast cells as a response to ethanol stress. The results agreed with the previous researches (Meaden et al., 1999; Rosa and Sa´-Correia, 1991; Salgueiro et al., 1988). The desired phenotype, tolerance to high concentration of ethanol, might be related to mutations that have been observed in CAN1 gene. The polymorphism observed in the CAN1 gene may have resulted in molecular modifications (via EMS mutagen- esis and pre-treatment to stress condition) that improved the up- take of arginine (amino acid) in the presence of high concentra- tion of ethanol. The higher tolerance to ethanol in mutants might be related to improved amino acid up-take during fermentation. M2 fragment showed high similarity (99%) to the SLS1 gene. Two SNPs were found between the mutant and the wild type allele in the alignment between the M2 insert and SLS1 gene sequence (Table 1; Supplementary Figure 2, section C). Both SNPs were in coding regions (Supplementary Figure 2, section B). Both SNPs did not change amino acid sequence and resulted in synonymous amino acid (Annotation Table). The SLS1 gene is located on Crick strand of chromosome XII and encoded for mi- tochondrial membrane proteins with 643 amino acids, (Supple- mentary Figure 2, Section A). SLS1 gene is one of the required factors for assembly of respiratory-chain enzyme complexes. It also coordinates in expression of mitochondria-encoded genes and delivery of mRNA to membrane-bound translation machin- ery (SGD, 2008). The finding here is further evidence that the major targets of ethanol are membrane structure and function as described by earlier researches (Mishra and Prasad, 1989; Teixeira et al., 2009). Benitez and Codon (2003) also indicat- ed that the destructive effect sites of ethanol activity in yeast are the plasma membrane, hydrophobic proteins of the cell, mitochondrial membranes, nuclear membrane, vacuolar mem- brane, endoplasmic reticulum, and hydrophobic proteins in the cytoplasm. Previous research showed Respiratory Deficient (RD) mutants had lower growth rate, fermentation rate and respira- tion rate than aerobically growing wild strain of yeast. RD mu- tants were anaerobically grown cells which did not have active mitochondria and oxidative metabolism. The results illustrated the functional mitochondria was necessary for a greater toler- ance to ethanol in yeast cells. It was suggested that the fac- 103 A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) Table 1. List of geneic non-synonymous mutations detected by DNA sequence of bands showing polymorphisms by AFLP of ge- nomic DNA (Figure 3). Gene Name Gene ID Segment SNP Amino Acid CAN1 856646 C2 C453T A117A CAN1 856646 C2 T627G S176A CAN1 856646 M4 , M5 G566T V155V CAN1 856646 M5 T637A I178N SLS1 850830 M2 G645A K98K SLS1 850830 M2 G675C L108L VTH1 854634 M3 Insertion361ATA A102D,T VTH1 854634 M3 T369A N104K VTH1 854634 M3 T372C Y105Y VTH1 854634 M3 T374A F106Y VTH1 854634 M3 A445G N130D COX6 856448 M7 T732C A94A tor responsible for the improvement in ethanol tolerance was not respiratory metabolism itself but majorly was more about the differences in cellular components, membrane lipids and the physiology derived the ability of yeast cell to respire (Aguilera and Benitez, 1985; Van Uden, 1989). Teixeira et al (2009) indicated that the 30 common mitochon- drial genes were also yeast resistant genes to high concentra- tions of ethanol, suggesting mitochondrial functions were essen- tial for ethanol tolerance even in the presence of glucose. These results were also reconfirmed the previous findings that ethanol tolerance depends on the stability of the mitochondrial genome (Jimenez and Benitez, 1988). The desired phenotype, tolerance to high concentration of ethanol, might be related to mutations that have been observed in SLS1 gene. The polymorphism observed in the SLS1 gene may have resulted in molecular modifications (via EMS mutagenesis and pre-treatment to stress condition) that improved cellular components, membrane lipid compositions and modified mem- brane fluidity which are required for higher ethanol tolerance during anaerobic fermentation (Aguilera and Benitez, 1985; Van Uden, 1989; Teixeira et al., 2009). The M3 Sequence Showed High Similarity (96%) to the VTH1 Gene, YIL173W. Four mutations and indel, three nucleotide in- sertions, were detected between the mutant and the wild type allele in the alignment between the M3 fragment and the VTH- 1gene sequence (Table 1; Supplementary Figure 3, section C). All four SNPs and indel, three nucleotide insertions, were in cod- ing region (Supplementary Figure 3, section B). Three of four SNPs changed the amino acid sequence and produced nonsyn- onymous amino acid. One of them did not change the amino acid sequence and produces a synonymous amino acid. The three nucleotide insertions, indel, changed the amino sequence and produced two nonsynonymous amino acids (Annotation Table ). The VTH1 gene is located on Watson strand of chro- mosome IX (Supplementary Figure 3, Section A). It encodes for putative membrane glycoprotein with 1,549 amino acids. And it may function in vacuolar protein sorting (SGD, 2008). Ethanol can disrupt the vacuolar membrane and release the proteases into cytoplasm which results in inactivation of intracel- lular enzymes (Van Uden, 1989). Teixeira et al. (2009) dem- onstrated that many of the genes required for ethanol toler- ance in yeast are related to intracellular trafficking, including vacuolar protein targeting, endosome transport, and transport mediated by the endosomal sorting complexes. They described that the target point to overcome stress imposed by lipophilic agents such as ethanol, are the membrane transporters. Ethanol stress alters vacuole morphology from segregated structures to a single, large organelle (Meaden et al., 1999). Chandler et al. (2004) found that gene expression profiles of ethanol stressed cells are different in the later stages of ethanol stress. And the major induced genes (YRO2, ALD4, ARG4, LAP4, PCL5, SSU1, YGL117W) at this stage are associated with energy utilization, general stress response and vacuole function. Fujita et al. (2006) found hundred and thirty-seven mutants as ethanol sensitive mu- tant with a considerable number of vacuole function-related genes being necessary for growth in the presence of ethanol stress. The outcomes of different research studies of genome- 104 wide screens for ethanol tolerance commonly agree on genes associated with vacuole function and amino acid biosynthesis as important factors for ethanol tolerance (Stanley et al., 2010). The desired phenotype, tolerance to high concentration of ethanol, might be related to mutations that have been observed in VTH1 gene. The polymorphism observed in the VTH1 gene may have resulted in molecular modification (via EMS mutagen- esis and pre-treatment to stress condition) that had positive ef- fects on vacuole morphology, alteration in vacuole-based func- tions such as maintaining intracellular pH and ion homoeostasis, vacuole protein sorting and transportation in the cell (Stanley et al. 2010). The M7 sequence showed high similarity (99%) to COX6 gene (YHR051W). One mutation was observed in COX6 gene. The SNP was found between the mutant gene and the wild type al- lele in the alignment between the M7 fragment and the COX6 gene sequence (Supplementary Figure 4, section C). SNP was in coding region (Table 1; Supplementary Figure 4, section B). The SNP did not change the amino acid sequence and produced synonymous amino acid (Annotation Table). The COX6 gene is located on Watson strand of chromosome VIII and encodes for subunit IV of cytochrome C oxidase (EC 1.9.3.1; Supplementary Figure 4 Section A). The protein contained 148 amino acids and is the terminal member of the mitochondrial inner membrane electron transport chain and its expression is regulated by oxy- gen concentrations (SGD, 2008). Mitochondrion, the organelle that is found in all eukaryotic cells is required for cellular res- A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) Figure 3. AFLP fingerprints of genomic DNA between mutant 1 (lane 1) and the wild type strain (lane2). Amplification products are shown from primers EcoRI/AC and MseI/G. There was 20 μg DNA per lane. The 100 bp Promega DNA ladder was used as a size standard (lane 3). Arrowed are 10 polymorphic bands detected and eluted from this gel for DNA sequencing. piration. The additional mitochondria rolls include participation in the biosynthesis of organic acids, amino acids, and phospho- lipids, the degradation of fatty acids and the storage of metal ions (Scheffler, 1999). Majority of the yeast genes encoding mi- tochondrial proteins are subject to glucose repression (DeRisi et al., 1997), and repression under hypoxic conditions (Ter Linde and Yde, 2002) which fermentation of alcoholic beverages is a good example because the concentration of glucose is high while the concentration of oxygen is low. Several studies suggested the presence of mitochondria during the fermentation of alcoholic beverages. For example, data from a genome-wide studies indi- cated that genes encoding mitochondrial proteins were at higher rate of expression during the brewing of sake (Japanese rice wine), one of the alcoholic beverages (Wu et al., 2006; Kitagaki and Shirnoi, 2007). Teixeira et al. (2009) indicated a high pro- portion of the mitochondrion- related genes were also required for ethanol stress resistance. They were mostly involved in mito- chondrial protein synthesis, respiration, and mitochondrial DNA maintenance. Scheffler, (1999) and Kitagaki and Shirnoi, (2007) pointed out that it is biologically reasonable for the presence of mitochondria during alcohol fermentation. The reason is that yeast cells require to rapidly transfer from anaerobic to aero- bic metabolism even in anaerobic conditions. For example when glucose is depleted and oxygen becomes available yeast cells use mitochondria to utilize the produced alcohol. Various studies commonly agree that the first obstacle that yeast cells under ethanol stress initially struggle with is maintain- ing energy production which leads to an increment in expression of genes associated with energy-generating activities such as glycolysis and mitochondrial function and decrease in expres- sion rates of many genes associated with energy demanding processes, such as growth (Stanley et al., 2010). The desired phenotype, tolerance to high concentration of ethanol, might be related to mutations that have been observed in COX6 gene. The polymorphism observed in the COX6 gene may have resulted in molecular modifications (via EMS muta- genesis and pre-treatment to stress condition) that improved the energy-generating activities, protein synthesis, respiration, and mitochondrial DNA maintenance in mutants (Stanley et al., 2010). Conclusions From the molecular point of view, the polymorphisms that resulted from EMS mutagenesis might have stimulated the ac- tivation of specific molecular stress response mechanisms in mentioned genes which resulted in higher levels of resistance to stress conditions, high concentration of ethanol. Interestingly mutant showed better growth than the wild type under the lower available carbon stress conditions caused by media derived from cellulosic biomass (Hemmati, Lightfoot and Anderson, unpublished). In future genome sequencing followed by gene knockouts and/or complementing the mutation(s) might identify the significant polymorphisms underlying the altered growth during ethanol stress. From the fermentation point of view and future investiga- tion of kinetics of ethanol production, it would be very valuable to evaluate the mutants’ activity under the enhanced enzymatic fermentation provided by Thomas (2009). It might provide more insight to stress tolerance among mutants. 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Wu B, X Zheng, Y Araki, B Sahara, B Takagi, and B Shimoi (2006) Global gene expression analysis of yeast cells during sake brewing. Appl. Environ Microb 72: 7353-7358. A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) 106 Hemmati et al., 2012 - Supplementary Data Supplementary Figure 1. Section A: Ideogram of the CAN1 gene on chromosome V and a snapshot of predicted structural informa- tion about the protein/cDNA sequence. A legend on the right-hand side of the image indicates what data that section of the image is displaying. SS: Secondary Structure, D: Disordered Regions, TM: Transmembrane Regions, CC: Coiled Coil Regions and SP: Signal Peptide. Cited from Saccharomyces Genome Database, SGD, and Yeast Resource Center, YRC. A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) 107 Supplementary Figure 1. Section B: CAN1 gene coding region is marked by start codon ATG and stop codon TAG in larger and bold fonts. The region that C2 segment matches with CAN1 gene is highlighted in gray. Section C: The gray region from section A is aligned with C2 segment and polymorphisms are marked by larger and bold fonts. A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) 108 Supplementary Figure 1. Section D: CAN1 gene coding region is marked by start codon ATG and stop codon TAG in larger and bold fonts. The region that M4 segment matches with CAN1 is highlighted in gray. Section E: The gray region from section A is aligned with M4 segment and polymorphisms are marked by larger and bold fonts. Supplementary Figure 1, Section C: The gray region from section A is aligned with C2 segment and polymorphisms are marked by larger and bold fonts. A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) 109 Supplementary Figure 1. Section E: The gray region from section A is aligned with M4 segment and polymorphisms are marked by larger and bold fonts. Supplementary Figure 1. Section F: CAN1 gene coding region is marked by start codon ATG and stop codon TAG in larger and bold font. The region that M5 segment matches with CAN1 is highlighted in gray. Supplementary Figure 1. Section G: The gray region from section A is aligned with M5 segment and polymorphisms are marked by larger and bold font. Supplementary Figure 2, Section A: Ideogram of the SLS1 gene on chromosome XII and a snapshot of predicted structural informa- tion about the protein/cDNA sequence. A legend on the right-hand side of the image indicates what data that section of the image is displaying. SS: Secondary Structure, D: Disordered Regions, TM: Transmembrane Regions, CC: Coiled Coil Regions and SP: Signal Peptide. Cited from Saccharomyces Genome Database, SGD, and Yeast Resource Center, YRC. A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) 110 Supplementary Figure 2. Section C: The gray region from section A is aligned with M2 segment and polymorphisms are marked by larger and bold fonts. A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) 111 Supplementary Figure 2, Section B: SLS1 gene coding region is marked by start codon ATG and stop codon TAA in larger and bold fonts. The region that M2 segment matches with SLS1 is highlighted in gray. Supplementary Figure 3, Section A: Ideogram of the VTH1 gene on chromosome IX and a snapshot of predicted structural informa- tion about the protein/cDNA sequence. A legend on the right-hand side of the image indicates what data that section of the image is displaying. SS: Secondary Structure, D: Disordered Regions, TM: Transmembrane Regions, CC: Coiled Coil Regions and SP: Signal Peptide. Cited from Saccharomyces Genome Database, SGD, and Yeast Resource Center, YRC. A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) 112 A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) 113 Supplementary Figure 3. Section B: VTH1 gene coding region is marked by start codon ATG in larger and bold fonts. The region that M3 segment matches with VTH1 is highlighted in gray. Supplementary Figure 3. Section C: The gray region from section A is aligned with M3 segment and polymorphisms are marked by larger and bold fonts. Supplementary Figure 4. Section A: Ideogram of the COX6 gene on chromosome VIII and a snapshot of predicted structural in- formation about the protein/cDNA sequence. The approximate locations of point mutations, base substitutions, are marked by pink arrows. A legend on the right-hand side of the image indicates what data that section of the image is displaying. SS: Secondary Structure, D: Disordered Regions, TM: Transmembrane Regions, CC: Coiled Coil Regions and SP: Signal Peptide. Cited from Saccha- romyces Genome Database, SGD, and Yeast Resource Center, YRC. A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) 114 Supplementary Figure 4. Section B: COX6 gene coding region is marked by start codon ATG and TAA in larger and bold fonts. The region that M7 segment matches with COX6 is highlighted in gray. Section C: The gray region from section A is aligned with M7 segment and polymorphisms are marked by larger and bold fonts. A tla s Jo ur na l o f Bi ol og y - IS SN 2 15 8- 91 51 . P ub lis he d By A tla s Pu bl ish in g, L P (w w w .a tla s- pu bl ish in g. or g) 115 Supplementary Figure 4. Section C: The gray region from section A is aligned with M7 segment and polymorphisms are marked by larger and bold fonts.