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The yeast Pichia kudriavzevii N77-4 was isolated from the Korean traditional fermentation starter nuruk. In this study, fermentation performance and stress resistance ability of N77-4 was analyzed. N77-4 displayed superior thermotolerance (up to 44°C) in addition to enhanced acetic acid resistance compared to Saccharomyces cerevisiae. Moreover, N77-4 produced 7.4 g/L of ethanol with an overall production yield of 0.37 g/g glucose in 20 g/L glucose medium. However, in 250 g/L glucose medium the growth of N77-4 slowed down when the concentration of ethanol reached 14 g/L or more and ethanol production yield also decreased to 0.30 g/g glucose. An ethanol sensitivity test indicated that N77-4 was sensitive to the presence of 1% ethanol, which was not the case for S. cerevisiae. Furthermore, N77-4 displayed a severe growth defect in the presence of 6% ethanol. Because inositol biosynthesis is critical for ethanol resistance, expression levels of the PkINO1 encoding a key enzyme for inositol biosynthesis was analyzed under ethanol stress conditions. We found that ethanol stress clearly repressed PkINO1 expression in a dose-dependent manner and overexpression of PkINO1 improved the growth of N77-4 by 19% in the presence of 6% ethanol. Furthermore, inositol supplementation also enhanced the growth by 13% under 6% ethanol condition. These findings indicate that preventing downregulation in PkINO1 expression caused by ethanol stress improves ethanol resistance and enhances the utility of P. kudriavzevii N77-4 in brewing and fermentation biotechnology.

To achieve inorganic phosphate (Pi) homeostasis, cells must be able to sense intracellular and extracellular Pi concentrations. In the Pi signaling (PHO) pathway in Saccharomyces cerevisiae, high Pi represses genes involved in Pi uptake (e.g., PHO84) and Pi utilization (PHO5); conversely, the cyclin-dependent kinase inhibitor Pho81 inhibits the activity of the Pho80-Pho85 cyclin-cyclin dependent kinase complex in low-Pi conditions, leading to induction of these genes. However, how yeast senses Pi availability remains unresolved. To identify factors involved in Pi sensing upstream of the Pho81-Pho80-Pho85 complex, we generated and screened suppressor mutants of a Δpho84 strain that shows constitutive PHO5 expression. By a series of genetic tests, including dominance-recessiveness, complementation and tetrad analyses, three sef (suppressor of pho84 [pho eighty-four]) mutants (sef8, sef9 and sef10) were shown to contain a novel single mutation. The sef mutants suppressed the phenotype of constitutive PHO5 expression at the transcriptional level, but did not show restored Pi uptake capacity. An epistasis-hypostasis test revealed that the sef mutations were hypostatic to pho80 mutation, indicating that their gene products function upstream of the Pho81-Pho80-Pho85 complex in the PHO pathway. The sef mutations identified are associated with gene(s) that may be involved in the homeostasis of an intracellular Pi level-sensing mechanism in S. cerevisiae.

To achieve inorganic phosphate (Pi) homeostasis, cells must be able to sense intracellular and extracellular Pi concentrations. In the Pi signaling (PHO) pathway in Saccharomyces cerevisiae, high Pi represses genes involved in Pi uptake (e.g., PHO84) and Pi utilization (PHO5); conversely, the cyclin-dependent kinase inhibitor Pho81 inhibits the activity of the Pho80-Pho85 cyclin-cyclin dependent kinase complex in low-Pi conditions, leading to induction of these genes. However, how yeast senses Pi availability remains unresolved. To identify factors involved in Pi sensing upstream of the Pho81-Pho80-Pho85 complex, we generated and screened suppressor mutants of a Δpho84 strain that shows constitutive PHO5 expression. By a series of genetic tests, including dominance-recessiveness, complementation and tetrad analyses, three sef (suppressor of pho84 [pho eighty-four]) mutants (sef8, sef9 and sef10) were shown to contain a novel single mutation. The sef mutants suppressed the phenotype of constitutive PHO5 expression at the transcriptional level, but did not show restored Pi uptake capacity. An epistasis-hypostasis test revealed that the sef mutations were hypostatic to pho80 mutation, indicating that their gene products function upstream of the Pho81-Pho80-Pho85 complex in the PHO pathway. The sef mutations identified are associated with gene(s) that may be involved in the homeostasis of an intracellular Pi level-sensing mechanism in S. cerevisiae.

Exploiting bacterial DNA-acting enzymes and expanding their repertoire for genome engineering have been major technological and conceptual advances in molecular biology over the last decade. The CRISPR-Cas9 system offers many attractive superiorities, such as multiplexing, high precision, low cost, and simplicity compared to other strategies/systems/approaches known to date for gene editing. The efficient co-delivery of Cas9 and single guide RNA(s) into a desired cell and subsequent correct targeting of selected genomic fragment(s) are among the most critical and determining issues for CRISPR-Cas9-based genome engineering. CRISPR/Cas9 components can be transported into target cells via various delivery methods, including physical methods (such as electroporation and microinjection) as well as viral and non-viral methods. Physical and viral methods, with all their privileges, still suffer from disadvantages including induction of immune responses, cell damage, lack of high specificity, etc. We are witnessing a remarkable increase in the employment of nanomaterials as non-viral carriers for the delivery of the CRISPR/Cas9 system. Nanoparticles have so far presented numerous advantages such as ease of synthesis, high efficiency, low cost, size tunability, non-mutagenicity, non-immunogenicity, etc. with regard to the delivery of CRISPR/Cas9. Here, we will review the recent progress in the delivery of CRISPR/Cas9 system components via nanomaterials and outline future challenges.

Additional file 1. Addiitonal figures and tables.

A key mechanism of signal transduction in eukaryotes is reversible protein phosphorylation, mediated through protein kinases and protein phosphatases (PPases). Modulation of signal transduction by this means regulates many biological processes. Saccharomyces cerevisiae has 40 PPases, including seven protein phosphatase 2C (PP2C PPase) genes (PTC1–PTC7). However, their precise functions remain poorly understood. To elucidate their cellular functions and to identify those that are redundant, we constructed 127 strains with deletions of all possible combinations of the seven PP2C PPase genes. All 127 disruptants were viable under nutrient-rich conditions, demonstrating that none of the combinations induced synthetic lethality under these conditions. However, several combinations exhibited novel phenotypes, e.g. the Δptc5Δptc7 double disruptant and the Δptc2Δptc3Δptc5Δptc7 quadruple disruptant exhibited low (13°C) and high (37°C) temperature-sensitive growth, respectively. Interestingly, the septuple disruptant Δptc1Δptc2Δptc3Δptc4Δptc5Δptc6Δptc7 showed an essentially normal growth phenotype at 37°C. The Δptc2Δptc3Δptc5Δptc7 quadruple disruptant was sensitive to LiCl (0.4 m). Two double disruptants, Δptc1Δptc2 and Δptc1Δptc4, displayed slow growth and Δptc1Δptc2Δptc4 could not grow on medium containing 1.5 m NaCl. The Δptc1Δptc6 double disruptant showed increased sensitivity to caffeine, congo red and calcofluor white compared to each single deletion. Our observations indicate that S. cerevisiae PP2C PPases have a shared and important role in responses to environmental stresses. These disruptants also provide a means for exploring the molecular mechanisms of redundant PTC gene functions under defined conditions. Copyright © 2014 John Wiley & Sons, Ltd.

High-temperature ethanol fermentation has several benefits including a reduction in cooling cost, minimizing risk of bacterial contamination, and enabling simultaneous saccharification and fermentation. To achieve the efficient ethanol fermentation at high temperature, yeast strain that tolerates to not only high temperature but also the other stresses present during fermentation, e.g., ethanol, osmotic, and oxidative stresses, is indispensable. The C3253, C3751, and C4377 Saccharomyces cerevisiae strains, which have been previously isolated as thermotolerant yeasts, were found to be multiple stress-tolerant. In these strains, continuous expression of heat shock protein genes and intracellular trehalose accumulation were induced in response to stresses causing protein denaturation. Compared to the control strains, these multiple stress-tolerant strains displayed low intracellular reactive oxygen species levels and effective cell wall remodeling upon exposures to almost all stresses tested. In response to simultaneous multi-stress mimicking fermentation stress, cell wall remodeling and redox homeostasis seem to be the primary mechanisms required for protection against cell damage. Moreover, these strains showed better performances of ethanol production than the control strains at both optimal and high temperatures, suggesting their potential use in high-temperature ethanol fermentation.

Yeast (Saccharomyces cerevisiae) RNA is an important source of 5'-ribonucleotides that is used in both the food and pharmaceutical industries. Efficient transcription of rDNA is very important to construct yeast strains with high RNA content. The gene RRN10, which encodes, a component of the upstream activation factor, is essential to promote high-level transcription of rDNA. In our previous study, we isolated SupE strain as a dominant suppressor, which showed the ability to restore the severe growth defects and reduced RNA content caused by disruption of the RRN10 gene. SupE strain has multiple mutations which we designated collectively as SUPE. Further analysis on SUPE mutation indicated that RPL40A was responsible for suppression of defect caused by rrn10 disruption. However, there were no base changes in this gene as compared with the parental Δrrn10 strain, thus suggesting that an additional copy of RPL40A suppress the defects caused by Δrrn10 disruption, and that, in SupE strain, these defects are suppressed by increased transcription of RPL40A whose copy is doubled. When multiple copies of RPL40A were combined with SUPE mutation on an RRN10⁺ background, the resultant SupE strain had significantly higher RNA content than wild-type strain. In addition, increased transcription of RPL40B also showed significant effect to restore the growth defect and reduced RNA content caused by Δrrn10 disruption. We propose a model to explain how SUPE mutation increases the transcription of ribosomal protein genes such as RPL40A and RPL40B in SupE strain, resulting in an increase in RNA content.

Reversible phosphorylation is one of the key post-translational modifications for the regulation of many essential cellular processes. We have previously reported that the disruption of two protein phosphatase (PPase) genes, PTP2 and MSG5, causes calcium sensitivity indicating that functional redundancy exists between the two PPases in response to high extracellular calcium. In this paper, we found that the inactivation of calcineurin by the disruption of the calcineurin regulatory subunit, CNB1 or treatment with a calcineurin inhibitor, FK506, can suppress the calcium-sensitive phenotype of the ptp2Δmsg5Δ double disruptant. In the wake of a calcium-induced, calcineurin-driven signaling pathway activation, the calcium sensitivity of the ptp2Δmsg5Δ double disruptant can be suppressed by regulating the SLT2 pathway through the disruption of the major kinases in the SLT2 signal cascade that include BCK1, MKK1 and SLT2. Also, we show that PTP2 and MSG5 are key regulatory PPases that prevent over-activation of the calcium-induced signaling cascade under the parallel control of the SLT2 and calcineurin pathways.

The capability of multi-stress-tolerant Saccharomyces cerevisiae diploid strain TJ14 for the production of cellulosic bio-ethanol by semi-simultaneous saccharification and fermentation (SSSF) technology was evaluated under high-temperature conditions. At 39°C, the TJ14 produced 45 g/l ethanol by SSSF of 100 g (w/v)/l cellulose - a significantly higher concentration than reported in prevailing literature.

The simultaneous saccharification and fermentation process requires thermo-tolerant yeast to facilitate the enzymatic hydrolysis of cellulose. In this paper, we describe a Htg+ strain that exhibits confluent growth at high temperature (41 °C) and resistance to heat shock, ethanol, osmotic, oxidative and DNA damage stresses. HTG6, one of the six genes responsible for the thermotolerant phenotype was identified to be the gene RSP5 encoding a ubiquitin ligase. The RSP5 allele of the Htg+ strain, designated RSP5-C, possessed five, one and two base changes in the promoter, open reading frame and terminator region, respectively. The base changes in the promoter region of the RSP5-C allele were found to be responsible for the thermotolerant phenotype by strongly increasing transcription of the RSP5 gene and consequently causing a rise in the ubiquitination of cell proteins. Overexpression of the RSP5-BY allele present in the htg6 host strain (Htg-) conferred thermotolerance at 41°C, to this strain as in the case of RSP5-C allele. We also discovered that an Htg+ strain overexpressing the RSP5-C allele exhibits a more robust Htg+ phenotype against higher temperature (43 °C). The data presented here also suggest that overexpression of RSP5 could be applied to raise the upper limit of thermotolerance in S. cerevisiae strain used for industrial bioethanol production.

PCR-mediated chromosome splitting (PCS) was developed in the yeast Saccharomyces cerevisiae. It is based on homologous recombination and enables division of a chromosome at any point to form two derived and functional chromosomes. However, because of low homologous recombination activity, PCS is limited to a single site at a time, which makes the splitting of multiple loci laborious and time-consuming. Here we have developed a highly efficient and versatile chromosome engineering technology named CRISPR-PCS that integrates PCS with the novel genome editing CRISPR/Cas9 system. This integration allows PCS to utilize induced double strand breaks to activate homologous recombination. CRISPR-PCS enhances the efficiency of chromosome splitting approximately 200-fold and enables generation of simultaneous multiple chromosome splits. We propose that CRISPR-PCS will be a powerful tool for breeding novel yeast strains with desirable traits for specific industrial applications and for investigating genome function.

Yeast RNA is a good source of nucleotide seasoning, and more than half of yeast RNA consists of ribosomal RNA (rRNA). Previously, we reported the development of a Saccharomyces cerevisiae strain displaying a 1.4- to 2.3-times higher RNA content than the wild-type strain through the isolation of dominant suppressors (designated SupA to SupG strains) from a Δrrn10 disruptant showing decreased rRNA transcription. In the present study, the cloning of one of the genes responsible for the suppression was attempted using a genomic library from the SupD strain. NOP15, a gene involved in ribosome biogenesis, was found to be responsible for suppressing the growth defect of the Δrrn10 disruptant. The isolated NOP15 allele (designated NOP15(T-279C)) possessed a single T to C substitution at nucleotide position-279 of NOP15. The transcription level of NOP15(T-279C) in the originally isolated SupD strain was 2-fold higher than that in the Δrrn10 disruptant. Furthermore, a dose-dependent relationship between the transcription level of NOP15 and total amount of RNA in the Δrrn10 disruptant was observed: the enhanced transcription due to the NOP15(T-279C) allele is involved in the suppression mechanisms in the SupD strain. Introduction of the NOP15(T-279C) allele into the wild-type strain increased the total RNA content by 1.4-fold. These results indicate that the transcription level of NOP15 is an important determinant of the productivity of RNA and that its increased transcription provides an effective approach to obtain higher RNA yields in yeast.

To gain more insight into adaptation response to lactic-acid stress in yeast, a genome-wide screening for genes whose disruption caused hypersensitivity to 4.0% l-lactic acid (pH 2.8) was performed using the gene deletion collection of Saccharomyces cerevisiae. We identified 107 genes that contributed significantly to the ability of yeast cells to adapt lactic-acid stress. More than 30% of the genes identified in this screening were newly identified to be involved in mechanisms for adaptation response to lactic acid. We found that protein urmylation by Uba4 and N-terminal acetylation by Nat3 were involved in lactic acid adaptation mechanisms. Functional categorization of the genes followed by microscopic analysis revealed that a variety of cellular functions were involved in adaptation response to lactic acid and function associated with vacuolar transport played important roles in adaptation response to lactic acid. We also found that vacuole fragmented immediately upon exposure to lactic- and hydrochloric-acid stress. In addition, our analysis revealed that lactic-acid stress significantly reduced the amount of intracellular amino acids. Amino acid supplementation recovered the adaptation deficiency to lactic acid, suggesting that intracellular amino-acid homeostasis plays important roles in adaptation response to lactic-acid stress. These data suggest that enhancing vacuolar integrity, as well as maintaining intracellular amino-acid homeostasis may be an efficient approach to confer resistance to lactic-acid stress.

Use of super strains exhibiting tolerance to high temperature, acidity and ethanol is a promising way to make ethanol production economically feasible. We describe here the breeding and performance of such a multiple-tolerant strain of Saccharomyces cerevisiae generated by a spore-to-cell hybridization technique without recombinant DNA technology. A heterothallic strain showing a high-temperature (41°C) tolerant (Htg(+)) phenotype, a derivative from a strain isolated from nature, was crossed with a homothallic strain displaying high-ethanol productivity (Hep(+)), a stock culture at the Thailand Institute of Scientific and Technological Research. The resultant hybrid TJ14 displayed ability to rapidly utilize glucose, and produced ethanol (46.6g/l) from 10% glucose fermentation medium at high temperature (41°C). Not only ethanol productivity at 41°C but also acid tolerance (Acd(+)) was improved in TJ14 as compared with its parental strains, enabling TJ14 to grow in liquid medium even at pH 3. TJ14 maintained high ethanol productivity (46.0g/l) from 10% glucose when fermentation was done under multiple-stress conditions (41°C and pH 3.5). Furthermore, when TJ14 was subjected to a repeated-batch fermentation scheme, the growth and ethanol production of TJ14 were maintained at excellent levels over ten cycles of fermentation. Thus, the multiple-stress (Htg(+) Hep(+) Acd(+)) resistant strain TJ14 should be useful for cost-effective bioethanol production under high-temperature and acidic conditions.

Polylactic acid plastics are receiving increasing attention for the control of atmospheric CO2 emissions. Lactic acid, the building block for polylactic acid, is produced by fermentation technology from renewable carbon sources. The yeast Saccharomyces cerevisiae, harboring the lactate dehydrogenases gene (LDH), produces lactic acid at a large scale due to its strong acid resistance, to its simple nutritional requirements and to its ease of genetic engineering. Since improvement of lactic acid resistance is correlated with an increase of lactic acid production under non-neutralizing condition, we isolated a novel gene that enhances lactic acid resistance using a multi-copy yeast genomic DNA library. In this study, we identified the ESBP6 gene, which increases lactic acid resistance when overexpressed and which encodes a protein with similarity to monocarboxylate permeases. Although ESBP6 was not induced in response to lactic acid stress, it caused weak but reproducible sensitivity to lactic acid when disrupted. Furthermore, intracellular pH in the ESBP6 overexpressing strain was higher than that in the wild-type strain under lactic acid stressed condition, suggesting that Esbp6 plays some roles in lactic acid adaptation response. The ESBP6 overexpressing strain carrying the LDH gene induced 20% increase in lactic acid production compared with the wild-type strain carrying the LDH gene under non-neutralizing conditions. These results indicate that overexpression of ESBP6 provides a novel and useful tool to improve lactic acid resistance and lactic acid production in yeast.

The Saccharomyces cerevisiae Yvh1, a dual-specificity protein phosphatase involved in glycogen accumulation and sporulation, is required for normal vegetative growth. To further elucidate the role of Yvh1, we generated dominant mutants suppressing the slow growth caused by YVH1 disruption. One of the mutant alleles, designated as SVH1-1 (suppressor of Δyvh1 deletion), was identical to MRT4 (mRNA turnover) that contained a single-base substitution causing an amino acid change from Gly(68) to Asp. Mrt4(G68D) restored the deficiencies in growth and rRNA biogenesis that occurs in absence of Yvh1. Here, we report that the interaction between Mrt4 and Yvh1 is also essential for normal glycogen accumulation and mRNA decay as well as the induction of sporulation genes IME2, SPO13 and HOP1. The Mrt4(G68D) could restore the plethora of phenotypes we observed in absence of Yvh1. We found that Yvh1 is not essential for wild-type induction of the transcriptional regulator of these genes, IME1, suggesting that either translation or post-translational modification to activate Ime1 has been compromised. Since a defect in ribosome biogenesis in general can be related to other various defects, the ribosome biogenesis defect caused by absence of Yvh1 might be an indirect cause of observed phenotypes.

To save cost and input energy for bioethanol production, a consolidated continuous solid-state fermentation system composed of a rotating drum reactor, a humidifier, and a condenser was developed. Biomass, saccharifying enzymes, yeast, and a minimum amount of water are introduced into the system. Ethanol produced by simultaneous saccharification and fermentation is continuously recovered as vapor from the headspace of the reactor, while the humidifier compensates for the water loss. From raw corn starch as a biomass model, 95 +/- 3, 226 +/- 9, 458 +/- 26, and 509 +/- 64 g l(-1) of ethanol solutions were recovered continuously when the ethanol content in reactor was controlled at 10-20, 30-50, 50-70 and 75-85 g kg-mixture(-1), respectively. The residue showed a lesser volume and higher solid content than that obtained by conventional liquid fermentation. The cost and energy for intensive waste water treatment are decreased, and the continuous fermentation enabled the sustainability of enzyme activity and yeast in the system.

We describe the cloning and functional characterization of the fatty acid elongase gene HpELO2, a homologue of the HpELO1 gene required for the production of C24:0 in the yeast Hansenula polymorpha. The open reading frame (ORF) of HpELO2 consists of 1,035 bp, encoding 344 amino acids, sharing about 65% identity with that of Saccharomyces cerevisiae Elo2. Expression of HpELO2 rescued the lethality of the S. cerevisiae elo2Delta elo3Delta double disruptant. An accumulation of C18:0 and a significant increase and decrease in the levels of C24:0 and C26:0, respectively, were observed in the Hpelo2Delta disruptant. These results supported an idea that HpELO2 encodes a fatty acid elongase involved in the elongation of C18:0 to very long-chain fatty acids. The Hpelo1Delta Hpelo2Delta double disruption was nonviable, suggesting that HpELO1 and HpELO2 are the only two genes necessary for the biosynthesis in H. polymorpha. Interestingly, transcription of HpELO2 and HpELO1 were found to be transiently up-regulated by exogenous long-chain fatty acids; however, this up-regulation was not observed with HpELO1 and HpELO2 genes driven by the constitutively expressed promoter of the HpACT gene, suggesting that exogenous fatty acids specifically trigger the transcriptional induction of HpELO1 and HpELO2 through their promoter regions.

Saccharomyces cerevisiae, for centuries the yeast that has been the workhorse for the fermentative production of ethanol, is now also a model system for biological research. The recent development of chromosome-splitting techniques has enabled the manipulation of the yeast genome on a large scale, and this has allowed us to explore questions with both biological and industrial relevance, the number of genes required for growth and the genome organization responsible for the ethanol production. To approach these questions, we successively deleted portions of the yeast genome and constructed a mutant that had lost about 5% of the genome and that gave an increased yield of ethanol and glycerol while showing levels of resistance to various stresses nearly equivalent to those of the parental strain. Further systematic deletion could lead to the formation of a eukaryotic cell with a minimum set of genes exhibiting appropriately altered regulation for enhanced metabolite production.

Segmental aneuploidy can play an important role in environmental adaptation. However, study of segmental aneuploids is severely hampered by the difficulty of creating them in a designed fashion. Here, we describe a PCR-mediated chromosome duplication (PCDup) technology that enables the generation of segmental aneuploidy at any desired chromosomal region in Saccharomyces cerevisiae. We constructed multiple strains harboring 100 kb to 200 kb segmental duplications covering the whole of the S. cerevisiae genome. Interestingly, some segmental aneuploidies confer stress tolerance, such as to high temperature, ethanol and strong acids, while others induce cell lethality and stress sensitivity, presumably as result of the simultaneous increases in dosages of multiple genes. We suggest that our PCDup technology will accelerate studies into the phenotypic changes resulting from alteration of gene dosage balance of multiple genes and will provide new insights into the adaptive molecular mechanisms in the genome in segmental aneuploidy-derived human diseases.

Saccharomyces cerevisiae strains from industrial and natural geographical environments are reported to show great variation in copy number of chromosomal regions. Such variation contributes to the mechanisms underlying adaptation to different environments. Here, we created and phenotypically analyzed segmentally haploidized strains, each harboring a deletion of one copy of approximately 100-300 kb of the left or right terminal region of 16 chromosomes in a diploid strain by using a PCR-mediated chromosomal deletion method. No haploidized strain of the 158-kb deleted right terminal region of chromosome III or the 172-kb deleted right terminal region of chromosome VI was produced; however, segmentally haploidized strains of the remaining 30 terminal regions were obtained. Among these 30 strains, two exhibited higher lactic acid resistance and two displayed higher thermo-tolerance at 41°C versus the host diploid strain. By contrast, four and two segmentally haploidized strains showed sensitivity to 6% lactic acid and low temperature at 13°C, respectively. The effect of the decreased copy number of the chromosomal terminal regions on ethanol production was analyzed. As compared with the host diploid strain, a 3.8% and 4.3% improvement in ethanol production in 10% glucose medium was observed for two strains in which one of two copies of the 197-kb left terminal region of chromosome V and one of two copies of the 195-kb left terminal region of chromosome X was deleted, respectively. These results indicate that artificial segmental haploidization might contribute to improvement of industrially important phenotypes and provide a new approach to breeding superior yeast strains.

The budding yeast Saccharomyces cerevisiae has great potential to tolerate weak organic acid stress. This trait is becoming important for refining bioproduction systems for chemicals such as lactic acid. In this chapter, we summarize mechanisms of S. cerevisiae adaptation to weak organic acids. For example, yeast cells counteract lipophilic organic acids such as benzoic or sorbic acid by enhancing the production of plasma membrane transporters, including Pdr12, which pump the acid anions out of cells. For acetic acid stress, a less lipophilic organic acid, yeast cells increase their resistance through inactivation of the plasma membrane channel Fps1, which facilitates diffusional entry of acetic acid. On the other hand, protons generated by dissociation of weak organic acids are actively pumped out of the cell by the plasma membrane proton transporter Pma1. During the period of adaptation, the transcription activators War1 and Haa1 are important in stimulating transcription-mediated adaptation responses. Haa1 is also essential to activate the adaptation response to lactic acid stress. Our recent analysis suggested that one of the adaptation responses to lactic acid stress is mediated by nuclear localization of Haa1. This nuclear localization is thought to be linked to the extent of Haa1 phosphorylation, which seems to be important for Msn5-mediated nuclear export of Haa1. Studies for improving lactic acid resistance are also discussed from the point of view of efficient lactic acid production by S. cerevisiae.

IRE1 and HAC1 are essential for the unfolded protein response in the endoplasmic reticulum (ER). IRE1- and HAC1-disruptants require high concentrations of inositol for its normal growth. The ALG6, ALG8, and ALG10 genes encode the glucosyltransferases necessary for the completion of the synthesis of the lipid-linked oligosaccharide used for the asparagine-linked glycosylation of proteins in that order. Here we show that, given a combination of the hac1 defect with a disruption of ALG6, ALG8, and ALG10, no strains grow on inositol-free medium. However, the growth defect of the hac1-alg10 double disrupted was partially, but significantly, suppressed by the addition of inositol to the medium. These results indicate that inositol, according to the numbers of glucose residues in the oligosaccharide, plays an important role in the stress response and quality control of glycoproteins in the ER.

Chromosome engineering techniques that can manipulate a large segment of chromosomal DNA are useful not only for studying the organization of eukaryotic genomes but also for the improvement of industrially important strains. Toward the development of techniques that can efficiently manipulate a large segment of chromosome, we have previously reported a one-step chromosome splitting technique in a haploid Saccharomyces cerevisiae cell, with which we could successfully split yeast chromosome 11, XIII, or XI into two halves to create a haploid strain having 17 chromosomes. We have now constructed chromosome splitting vectors bearing ADE2, HIS3, LEU2, or TRP1 marker, and by using these vectors, we could successively split yeast chromosomes to create a novel yeast haploid strain having up to 21 chromosomes. The specific growth rates of yeast strains carrying more than 16 chromosomes up to 21 did not differ significantly, suggesting that yeast cells can harbor more chromosomes than they do in their natural state, that is, 16 chromosomes, without serious effects on their growth.

The Saccharomyces cerevisiae Siw14, a tyrosine phosphatase involved in the response to caffeine, participates in regulation of the phosphorylation and intracellular localization of Gln3, a GATA transcriptional activator of nitrogen catabolite repression-sensitive genes. In Δsiw14 cells, the phosphorylation level of Gln3 is decreased and the nuclear localization of Gln3 is stimulated by caffeine. However, the mechanism by which Siw14 controls the localization and function of Gln3 remains unclear, although the nuclear localization of Gln3 is known to be induced by activation of the type 2A phosphatases (PP2As) Pph21 and Pph22, and the type 2A-related phosphatase Sit4. In this study, we show that the increased nuclear localization of Gln3 in response to caffeine caused by disruption of the SIW14 gene is dependent on the Sit4 and PP2A phosphatases. We also show that decreased phosphorylation of Gln3 caused by disruption of the SIW14 gene is completely suppressed by deletion of both PPH21 and PPH22, but only partially suppressed by deletion of SIT4. Taking these results together, we conclude that Siw14 functions upstream of Pph21 and Pph22 as an inhibitor of the phosphorylation and localization of Gln3, and that Sit4 acts independently of Siw14.

The phosphorylation status of cellular proteins results from an equilibrium between the activities of protein kinases and protein phosphatases (PPases). Reversible protein phosphorylation is an important aspect of signal transduction that regulate many biological processes in eukaryotic cells. The Saccharomyces cerevisiae genome encodes 40 PPases, including seven members of the protein phosphatase 2C subfamily (PTC1 to PTC7). In contrast to other PPases, the cellular roles of PTCs have not been investigated in detail. Here, we sought to determine the cellular role of PTC6 in S. cerevisiae with disruption of PTC genes. We found that cells with Δptc6 disruption were tolerant to the cell wall-damaging agents Congo red (CR) and calcofluor white (CFW); however, cells with simultaneous disruption of PTC1 and PTC6 were very sensitive to these agents. Thus, simultaneous disruption of PTC1 and PTC6 gave a synergistic response to cell wall damaging agents. The level of phosphorylated Slt2 increased significantly after CR treatment in Δptc1 cells and more so in Δptc1Δptc6 cells; therefore, deletion of PTC6 enhanced Slt2 phosphorylation in the Δptc1 disruptant. The level of transcription of KDX1 upon exposure to CR increased to a greater extent in the Δptc1Δptc6 double disruptant than the Δptc1 single disruptant. The Δptc1Δptc6 double disruptant cells showed normal vacuole formation under standard growth conditions, but fragmented vacuoles were present in the presence of CR or CFW. Our analyses indicate that S. cerevisiae PTC6 participates in the negative regulation of Slt2 phosphorylation and vacuole morphogenesis under cell wall stress conditions.

Chromosome engineering enables large-scale genome manipulation and can be used as a novel technology for breeding of yeasts. PCR-mediated chromosome splitting (PCS) offers a powerful tool for chromosome engineering by enabling a yeast chromosome to be split at any desired site. By applying PCS, a huge variety of chromosome combinations can be created and the best strain under specific conditions can be selected-a technology that we have called genome reorganization. Once the optimal strain is obtained, chromosome constitutions need to be maintained stably; however, mini-chromosomes of less than 50 kb are at relatively high frequency lost during cultivation. To overcome this problem, in this study we screened for multicopy suppressors of the high loss of mini-chromosomes by using a multicopy genomic library of Saccharomyces cerevisiae. We identified a novel gene, YCR041W, that stabilizes mini-chromosomes. The translational product of YCR041W was suggested to play an important role in increasing stability for mini-chromosome maintenance, probably by decreasing the rate of loss during mitotic cell division. The stabilization of mini-chromosomes conferred by YCR041W overexpression was completely dependent on the silencing protein Sir4, suggesting that a process related to telomere function might be involved in mini-chromosome stabilization. Overexpression of YCR041W stabilized not only a yeast artificial chromosome vector, but also a mini-chromosome derived from a natural chromosome. Taking these results together, we propose that YCR041W overexpression can be used as a novel chromosome engineering tool for controlling mini-chromosome maintenance and loss.

Improvement of the lactic acid resistance of the yeast Saccharomyces cerevisiae is important for the application of the yeast in industrial production of lactic acid from renewable resources. However, we still do not know the precise mechanisms of the lactic acid adaptation response in yeast and, consequently, lack effective approaches for improving its lactic acid tolerance. To enhance our understanding of the adaptation response, we screened for S. cerevisiae genes that confer enhanced lactic acid resistance when present in multiple copies and identified the transcriptional factor Haa1 as conferring resistance to toxic levels of lactic acid when overexpressed. The enhanced tolerance probably results from increased expression of its target genes. When cells that expressed Haa1 only from the endogenous promoter were exposed to lactic acid stress, the main subcellular localization of Haa1 changed from the cytoplasm to the nucleus within 5 min. This nuclear accumulation induced upregulation of the Haa1 target genes YGP1 , GPG1 , and SPI1 , while the degree of Haa1 phosphorylation observed under lactic acid-free conditions decreased. Disruption of the exportin gene MSN5 led to accumulation of Haa1 in the nucleus even when no lactic acid was present. Since Msn5 was reported to interact with Haa1 and preferentially exports phosphorylated cargo proteins, our results suggest that regulation of the subcellular localization of Haa1, together with alteration of its phosphorylation status, mediates the adaptation to lactic acid stress in yeast.

A key mechanism of signal transduction in eukaryotes is reversible protein phosphorylation, mediated through protein kinases and protein phosphatases (PPases). Modulation of signal transduction by this means regulates many biological processes. Saccharomyces cerevisiae has 40 PPases, including seven protein phosphatase 2C (PP2C PPase) genes (PTC1-PTC7). However, their precise functions remain poorly understood. To elucidate their cellular functions and to identify those that are redundant, we constructed 127 strains with deletions of all possible combinations of the seven PP2C PPase genes. All 127 disruptants were viable under nutrient-rich conditions, demonstrating that none of the combinations induced synthetic lethality under these conditions. However, several combinations exhibited novel phenotypes, e.g. the Δptc5Δptc7 double disruptant and the Δptc2Δptc3Δptc5Δptc7 quadruple disruptant exhibited low (13°C) and high (37°C) temperature-sensitive growth, respectively. Interestingly, the septuple disruptant Δptc1Δptc2Δptc3Δptc4Δptc5Δptc6Δptc7 showed an essentially normal growth phenotype at 37°C. The Δptc2Δptc3Δptc5Δptc7 quadruple disruptant was sensitive to LiCl (0.4 m). Two double disruptants, Δptc1Δptc2 and Δptc1Δptc4, displayed slow growth and Δptc1Δptc2Δptc4 could not grow on medium containing 1.5 m NaCl. The Δptc1Δptc6 double disruptant showed increased sensitivity to caffeine, congo red and calcofluor white compared to each single deletion. Our observations indicate that S. cerevisiae PP2C PPases have a shared and important role in responses to environmental stresses. These disruptants also provide a means for exploring the molecular mechanisms of redundant PTC gene functions under defined conditions.

Various genome engineering technologies have been developed in the yeast Saccharomyces cerevisiae. One such key technology is PCR-mediated chromosome-splitting technology, designated PCS. The aim of PCS is to cut a chromosome at any chosen site into two smaller pieces and to make these “newly generated chromosomes” behave as functional chromosomes. PCS splits a chromosome very efficiently (more than 70 %) and allows repeated splitting because the built-in Cre/loxP site-specific recombination system facilitates the use of marker recycling. Subsequently, PCR-mediated chromosome deletion (PCD) technology was developed as a derivative technology of PCS. PCD facilitates the deletion of any chromosomal region, irrespective of an internal or terminal location. Genome reorganization (GReO) technology was also developed on the basis of PCS. In GReO, a huge variety of genomic constitutions are generated from a master strain harboring a few dozen mini-chromosomes constructed by PCS when it undergoes combinatorial mini-chromosome loss during mitosis. PCD and GReO technology have been exploited in the breeding of yeast. Several strains with a large-scale deletion ranging from 400 to 500 kb were constructed by PCD. Some of these strains produced twofold more ethanol and glycerol than the parental strain. The gene expression profiles revealed that the physiological adjustment from fermentative to oxidative metabolism, including stimulation of mitochondrial function, does not occur in these strains. GReO technology has been successfully used to create ethanol-tolerant strains showing a more than tenfold-higher specific growth rate in the presence of 11 % ethanol as compared with the parental strain. Thus, these genome engineering technologies provide a new tool for breeding novel yeasts useful for industrial applications.

It is well known that the accumulation of an unfolded protein in the endoplasmic reticulum (ER) initiates the unfolded protein response (UPR). The UPR induces the transcriptional upregulation of multiple ER resident proteins involved in protein folding (for reviews, see references 20, 23, and 44). BiP/GRP78 is an abundant protein residing in the ER and essential for protein folding and protein sorting as a molecular chaperone. The structure of BiP is highly conserved from higher eukaryotes to yeast. In the yeast Saccharomyces cerevisiae, the BiP protein is encoded by KAR2. As in mammalian cells, the expression of KAR2 in yeast cells is induced by a variety of treatments, such as the addition of tunicamycin, which causes the accumulation of the unfolded protein in the ER. IRE1 encodes a bifunctional protein with transmembrane kinase and endoribonuclease activities that transmits the stress signal from the ER to the nucleus. The accumulation of the unfolded protein triggers Ire1p oligomerization, thereby inducing autophosphorylation, resulting in subsequent elicitation of the kinase and RNase activities. Activated Ire1p, together with the tRNA ligase encoded by RLG1 and Ada5p, causes unconventional splicing of HAC1 mRNA. HAC1 mRNA splicing allows efficient translation of Hac1p, which has a basic leucine zipper domain and functions as a transcriptional factor for genes regulated by the UPR, such as KAR2, PDI1, and FKB2. Since Hac1p is necessary for IRE1-mediated KAR2 induction as a positive transcription factor, mutants having a defect in ire1 or hac1 are unable to induce the transcription of KAR2, resulting in an inability of yeast cells to grow under stress conditions such as with the addition of tunicamycin. The IRE1 gene was first identified as the gene for inositol prototrophy (Ino+) of S. cerevisiae (29), and the HAC1 gene was isolated as a multicopy suppressor gene for the ire1 mutation (26). Mutants having a defect in ire1 or hac1 show inositol auxotrophy (Ino−) due to an inability to fully induce the expression of the INO1 gene, which encodes a rate-limiting enzyme for inositol synthesis (4, 24, 26). In S. cerevisiae, inositol is synthesized de novo in cells through the conversion of glucose 6-phosphate to inositol 1-phosphate, followed by dephosphorylation (5). The former reaction is mediated by inositol 1-phosphate synthase encoded by INO1 (6). Inositol is also taken up into cells in a carrier-mediated manner. S. cerevisiae possesses two distinct inositol transport systems. The major transport system is encoded by ITR1, and the minor one is encoded by ITR2 (30, 31). It is known that the expression of INO1 and ITR1, as well as a number of genes for enzymes involved in the synthesis of phospholipids in S. cerevisiae, is repressed in cells grown in the presence of inositol and derepressed in cells grown in the absence of inositol (32). INO1 and other coregulated genes of phospholipid biosynthesis contain one or two stretches of a conserved cis-acting promoter element, termed the inositol-choline-responsive element (ICRE). The INO2 and INO4 genes encode basic helix-loop-helix proteins that form a heterodimer and function as a transcriptional factor through binding to the ICRE (1, 41). Mutants having a defect in not only ino1 but also ino2 or ino4 exhibit the Ino− phenotype (10, 12). Several other mutants also show the Ino− phenotype. For example, mutations in the large subunit of RNA polymerase II (40) and the TATA binding protein (2, 43) lead to the Ino− phenotype due to an inability to express the INO1 gene. Depletion of the general transcription factor TFIIA also impairs INO1 activation (21). Cells having defects in the SWI1, SWI2, and SWI3 genes, which encode components of the SWI-SNF chromatin-remodeling complex, exhibit a derepression defect of INO1 (33–35). Furthermore, deletion of the INO80 gene, which is an SNF2-SWI2 paralogue and encodes a component of the INO80 chromatin-remodeling complex, prevents the efficient expression of INO1 (7, 42). On the other hand, mutations in the SIN3 and UME6 genes lead to high-level INO1 expression (15, 16). The SIN3 and UME6 gene products are components of a large complex that contains the RPD3 gene product, a histone deacetylase (17, 18, 39). Deletion of the RPD3 gene also leads to high-level INO1 expression. Additionally, a mutation in the OPI1 gene that encodes a protein containing leucine zipper and polyglutamine stretch motifs leads to an inositol overproduction phenotype (47). Little is known about the mechanism by which defects of the IRE1 or HAC1 gene lead to a decrease in INO1 expression or about the mechanism by which inositol regulates INO1 expression. In this study, we attempted to isolate and characterize the yeast gene that can suppress the Ino− phenotype of the Δhac1 strain when present in multiple copies. Here, we show that multiple copies of truncated ITC1 can suppress the Ino− phenotype of the Δire1 and Δhac1 strains and that the Isw2p-Itc1p complex usually represses INO1 expression.

Extractive lactic acid fermentation has recently been paid a great deal of attention. The problem with such a process is, however, that only undissociated lactate can be extracted. Therefore, lactic acid fermentation at low pH values is desirable. In the present study, we modified the metabolism of yeast (not lactic acid producing bacteria often cultivated at pH of 6–7) by expressing the lactate dehydrogenase (LDH) gene for the production of lactate at low pH values. For this purpose, the plasmid pADNS which contains the ADH1 promoter was used as a host vector, and a heterologous gene region, cDNA- LDH-A (encoding bovine lactate dehydrogenase) digested from plasmid pLDH12 was digested and ligated into the aforementioned two host vectors. The resultant plasmids were then transformed into Saccharomyces cerevisiae DS37. Using this recombinant S. cerevisiae strain, several batch and fed-batch fermentations at aerobic, microaerobic, and anaerobic conditions were conducted at several pH values (4.5-3.5). Since the recombinant S. cerevisiae produced a considerable amount of ethanol as well as lactate (about 10 g/ l ), we disrupted several pyruvate decarboxylase (PDC) genes to suppress the ethanol formation. Among the PDC genes, PDC1, PDC5 and PDC6, PDC1 had the greatest effect on the cell growth and ethanol production. The plasmid which containing the LDH-A structure gene was then transformed into the mutant strain lacking the PDC1 gene. Cultivation of this strain improved the lactate yield from glucose (from 0.155 to 0.20) while suppressing ethanol formation (from 0.35 to 0.20).

Yeast fatty acid synthase (Fas) comprises two subunits, α6 and β6, encoded by FAS2 and FAS1, respectively. To determine features of yeast Fas that control fatty acyl chain length, chimeric genes were constructed by combining FAS sequences from Saccharomyces cerevisiae (ScFAS) and Hansenula polymorpha (HpFAS), which mostly produces C16 and C18 fatty acids, respectively. The C16/C18 ratios decreased from 2.2 ± 0.1 in wild-type S. cerevisiae to 1.0 ± 0.1, 0.5 ± 0.2 and 0.8 ± 0.1 by replacement of ScFAS1, ScFAS2 and ScFAS1 ScFAS2 with HpFAS1, HpFAS2 and HpFAS1 HpFAS2, respectively, suggesting that the α, but not β subunits play a major role in determining fatty acyl chain length. Replacement of phosphopantetheinyl transferase (PPT) domain with the equivalent region from HpFAS2 did not affect C16/C18 ratio. Chimeric Fas2 containing half N-terminal ScFas2 and half C-terminal HpFas2 carrying H. polymorpha ketoacyl synthase (KS) and PPT gave a remarkable decrease in C16/C18 ratio (0.6 ± 0.1), indicating that KS plays a major role in determining chain length.

Two fatty acid desaturase genes have been cloned: HpFAD2 and HpFAD3 encode Hansenula polymorpha Δ12-fatty acid desaturase (HpFad2) and Δ15-fatty acid desaturase (HpFad3), which are responsible for the production of linoleic acid (LA, C18:2, Δ9, Δ12) and α-linolenic acid (ALA, αC18:3, Δ9, Δ12, Δ15), respectively. The open reading frame of the HpFAD2 and HpFAD3 genes is 1215bp and 1239bp, encoding 405 and 413 amino acids, respectively. The putative amino acid sequences of HpFad2 and HpFad3 share more than 60% similarity and three conserved histidine-box motifs with other known yeast Fad homologs. Hpfad2Δ disruptant cannot produce C18:2 and αC18:3, while the deletion of HpFAD3 only causes the absence of αC18:3. Heterologous expression of either the HpFAD2 or the HpFAD3 gene in Saccharomyces cerevisiae resulted in the presence of C18:2 and αC18:3 when the C18:2 precursor was added. Taken together, these observations indicate that HpFAD2 and HpFAD3 indeed encode Δ12- and Δ15-fatty acid desaturases that function as the only ones responsible for desaturation of oleic acid (C18:1) and linoleic acid (C18:2), respectively, in H. polymorpha. Because a Fatty Acid Regulated (FAR) region and a Low Oxygen Response Element (LORE), which are responsible for regulation of a Δ9-fatty acid desaturase gene (ScOLE1) in S. cerevisiae, are present in the upstream regions of both genes, we investigated whether the transcriptional levels of HpFAD2 and HpFAD3 are affected by supplementation with nutrient unsaturated fatty acids or by low oxygen conditions. Whereas both genes were up-regulated under low oxygen conditions, only HpFAD3 transcription was repressed by an excess of C18:1, C18:2 and C18:3, while the HpFAD2 transcript level did not significantly change. These observations indicate that HpFAD2 expression is not controlled at the transcriptional level by fatty acids even though it contains a FAR-like region. This study indicates that HpFAD2 may be regulated by post-transcriptional mechanisms, whereas HpFAD3 may be mainly controlled at a transcriptional level.

Breeding of yeast strains with higher RNA content is important because yeast RNA is a significant source of 5′-ribonucleotides, which have considerable use in both the food and pharmaceutical industries. Ribosomal RNA (rRNA) is an important source of yeast RNA as it accounts for about 80 % of total RNA content. We previously reported a dominant suppressor mutant of an rrn10 disruptant named SupE, which displays the ability not only to restore diminished RNA content caused by rrn10 disruption but also to increase the transcription level of ribosomal protein (RP) genes on an ∆rrn10 background in Saccharomyces cerevisiae. Here, to construct an S. cerevisiae strain with higher RNA content, we investigated the effect of increasing the copy number of the rDNA gene on a ∆rrn10 SUPE background. We successfully constructed a SupE strain with two copies of the rDNA cluster (ca. 300 rDNA genes) by using chromosome-splitting technology. The RNA content of this strain was 61 % higher than that of the SupE strain with a single copy of the rDNA cluster (ca. 150 rDNA genes), and 40 % higher than that of the wild-type strain with two copies of the rDNA cluster. A further increase in RNA content of 47 % was achieved by multicopy expression of the RPL40A gene in the SupE strain with two copies of the rDNA cluster. These observations suggest that we have constructed an S. cerevisiae strain with two copies of the rDNA cluster, which has achieved a considerably higher RNA content. Furthermore, the strategy taken in this study provides an effective approach to constructing S. cerevisiae strains with high potential for yeast food biotechnology.

In Saccharomyces cerevisiae, disruption of both protein phosphatase genes, PTP2 and MSG5, causes calcium sensitivity while additional disruption of protein kinase genes BCK1, MKK1, SLT2, MCK1, YAK1 and SSK2 confers calcium tolerance. Although the roles of BCK1, MKK1 and SLT2 have been characterized recently, the mechanism of suppression of the calcium sensitivity by SSK2 disruption is poorly understood. In this study, genetic analysis revealed a novel, high osmolarity glycerol (HOG)-independent suppressor function of Ssk2 in relation to the Ptp2 and Msg5-mediated calcium signaling. Through microarray analysis, we identified 19 genes with distinct pattern of expression that is likely involved in the calcium sensitive phenotype of the ptp2Δmsg5Δ double disruptant. Furthermore, we found msn2Δ and bcy1Δ as suppressors of the calcium sensitive phenotype. Our results suggest the interrelationship of a HOG-independent function of Ssk2, transcription factor Msn2, protein kinase A-related protein Bcy1 and 19 rise and fall genes as responsible for the suppression mechanism of the ptp2Δmsg5Δ double disruptant by ssk2Δ disruption.

To create strains that have high productivity of lactic acid without neutralization, a genome-wide screening for strains showing hyper-resistance to 6% l-lactic acid (pH 2.6) was performed using the gene deletion collection of Saccharomyces cerevisiae. We identified 94 genes whose disruption led to resistance to 6% lactic acid in rich medium. We also found that multiple combinations of Δdse2, Δscw11, Δeaf3, and/or Δsed1 disruption led to enhanced resistance to lactic acid depending upon their combinations. In particular, the quadruple disruptant Δdse2Δscw11Δeaf3Δsed1 grew well in 6% lactic acid with the shortest lag phase. We then introduced an exogenous lactate dehydrogenase gene (LDH) into those single and multiple disruptants to evaluate their productivity of lactic acid. It was found that the quadruple disruptant displaying highest lactic-acid resistance showed a 27% increase of lactic-acid productivity as compared with the LDH-harboring wild-type strain. These observations suggest that disruption of multiple genes whose deletion leads to lactic-acid resistance is an effective way to enhance resistance to lactic acid, leading to high lactic-acid productivity without neutralization.

We sought to breed an industrially useful yeast strain, specifically an ethanol-tolerant yeast strain that would be optimal for ethanol production, using a novel breeding method, called genome reconstruction, based on chromosome splitting technology. To induce genome reconstruction, Saccharomyces cerevisiae strain SH6310, which contains 31 chromosomes including 12 artificial mini-chromosomes, was continuously cultivated in YPD medium containing 6% to 10% ethanol for 33 days. The 12 mini-chromosomes can be randomly or specifically lost because they do not contain any genes that are essential under high-level ethanol conditions. The strains selected by inducing genome reconstruction grew about ten times more than SH6310 in 8% ethanol. To determine the effect of minichromosome loss on the ethanol tolerance phenotype, PCR and Southern hybridization were performed to detect the remaining mini-chromosomes. These analyses revealed the loss of mini-chromosomes no. 11 and no. 12. Mini-chromosome no. 11 contains ten genes (YKL225W, PAU16, YKL223W, YKL222C, MCH2, FRE2, COS9, SRY1, JEN1, URA1) and no. 12 contains fifteen genes (YHL050C, YKL050W-A, YHL049C, YHL048C-A, COS8, YHLComega1, ARN2, YHL046W-A, PAU13, YHL045W, YHL044W, ECM34, YHL042W, YHL041W, ARN1). We assumed that the loss of these genes resulted in the ethanol-tolerant phenotype and expect that this genome reconstruction method will be a feasible new alternative for strain improvement.

For industrial applications, fermentation of ethanol at high temperature offers advantages such as reduction in cooling costs, reduced risk of microbial contamination and higher efficiency of fermentation processes including saccharification and continuous ethanol stripping. Three thermotolerant Saccharomyces cerevisiae isolates (C3723, C3751 and C3867) from Thai fruits were capable of growing and producing 38 g/L ethanol up to 41°C. Based on genetic analyses, these isolates were prototrophic and homothallic, with dominant homothallic and thermotolerant phenotypes. After short-term (30 min) and long-term (12 h) exposure at 37°C, expression levels increased for the heat stress-response genes HSP26, SSA4, HSP82, and HSP104 encoding the heat shock proteins small HSP, HSP70, HSP90 and the HSP100 family, respectively. In isolates C3723 and C3867, expression was significantly higher than that in reference isolates W303 and TISTR5606 for TPS1 encoding trehalose-6-phosphate synthase, NTH1 encoding neutral trehalase and GSY1 encoding glycogen synthase. The results suggested that continuous high expression of heat stress-response genes was important for the long-term, heat stress tolerance of these thermotolerant isolates.

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°C) 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°C. 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.

A highly efficient technique, termed PCR-mediated chromosome splitting (PCS), was used to create cells containing a variety of genomic constitutions in a haploid strain of Saccharomyces cerevisiae. Using PCS, we constructed two haploid strains, ZN92 and SH6484, that carry multiple mini-chromosomes. In strain ZN92, chromosomes IV and XI were split into 16 derivative chromosomes, seven of which had no known essential genes. Strain SH6484 was constructed to have 14 mini-chromosomes carrying only non-essential genes by splitting chromosomes I, II, III, VIII, XI, XIII, XIV, XV, and XVI. Both strains were cultured under defined nutrient conditions and analyzed for combinatorial loss of mini-chromosomes. During culture, cells with various combinations of mini-chromosomes arose, indicating that genomic reorganization could be achieved by splitting chromosomes to generate mini-chromosomes followed by their combinatorial loss. We found that although non-essential mini-chromosomes were lost in various combinations in ZN92, one mini-chromosome (18kb) that harbored 12 genes was not lost. This finding suggests that the loss of some combination of these 12 non-essential genes might result in synthetic lethality. We also found examples of genome-wide amplifications induced by mini-chromosome loss. In SH6484, the mitochondrial genome, as well as the copy number of genomic regions not contained in the mini-chromosomes, was specifically amplified. We conclude that PCS allows for genomic reorganization, in terms of both combinations of mini-chromosomes and gene dosage, and we suggest that PCS could be useful for the efficient production of desired compounds by generating yeast strains with optimized genomic constitutions.