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Mishima, Japan

Ikeda A.,Hiroshima University | Muneoka T.,Hiroshima University | Murakami S.,Hiroshima University | Hirota A.,Hiroshima University | And 10 more authors.
Journal of Cell Science | Year: 2015

In eukaryotic organisms, including mammals, nematodes and yeasts, the ends of chromosomes, telomeres are clustered at the nuclear periphery. Telomere clustering is assumed to be functionally important because proper organization of chromosomes is necessary for proper genome function and stability. However, the mechanisms and physiological roles of telomere clustering remain poorly understood. In this study,we demonstrate a role for sphingolipids in telomere clustering in the budding yeast Saccharomyces cerevisiae. Because abnormal sphingolipidmetabolismcauses downregulation of expression levels of genes involved in telomere organization, sphingolipids appear to control telomere clustering at the transcriptional level. In addition, the data presented here provide evidence that telomere clustering is required to protect chromosome ends from DNA-damage checkpoint signaling. As sphingolipids are found in all eukaryotes, we speculate that sphingolipid-based regulation of telomere clustering and the protective role of telomere clusters in maintaining genome stability might be conserved in eukaryotes. © 2015.

Ismail K.S.K.,Kobe University | Ismail K.S.K.,University Malaysia Perlis | Sakamoto T.,Kobe University | Hatanaka H.,Suntory Research Center | And 2 more authors.
Journal of Biotechnology | Year: 2013

Production of ethanol from xylose at high temperature would be an economical approach since it reduces risk of contamination and allows both the saccharification and fermentation steps in SSF to be running at elevated temperature. Eight recombinant xylose-utilizing Saccharomyces cerevisiae strains developed from industrial strains were constructed and subjected to high-temperature fermentation at 38 °C. The best performing strain was sun049T, which produced up to 15.2. g/L ethanol (63% of the theoretical production), followed by sun048T and sun588T, both with 14.1. g/L ethanol produced. Via transcriptomic analysis, expression profiling of the top three best ethanol producing strains compared to a negative control strain, sun473T, led to the discovery of genes in common that were regulated in the same direction. Identification of the 20 most highly up-regulated and the 20 most highly down-regulated genes indicated that the cells regulate their central metabolism and maintain the integrity of the cell walls in response to high temperature. We also speculate that cross-protection in the cells occurs, allowing them to maintain ethanol production at higher concentration under heat stress than the negative controls. This report provides further transcriptomics information in the interest of producing a robust microorganism for high-temperature ethanol production utilizing xylose. © 2012 Elsevier B.V.

Yamaguchi H.,Tohoku University | Hosoya M.,Tohoku University | Shimoyama T.,Tohoku University | Takahashi S.,Tohoku University | And 5 more authors.
Journal of Bioscience and Bioengineering | Year: 2012

Acetaldehyde (AA) accumulates in the oral cavity after alcohol intake and is responsible for an increased risk of alcohol-related upper aerodigestive tract (UDAT) cancer among aldehyde dehydrogenase 2-inactive heterozygotes in particular. Thus, the removal of AA from the saliva to a level below its mutagenic concentration (50 μM) after drinking is a potentially straightforward method for reducing the risk of alcohol-related UDAT cancer. Although microbial cells with AA-decomposing activity could potentially serve as a useful agent for the catalytic removal of AA from the saliva without the supplemental addition of cofactors, these cells generally exhibit strong AA-producing activity from ethanol, which is present in excess (50. mM) over AA (100 μM) in the saliva after drinking. In this study, we observed that Gluconobacter kondonii (GK) cells efficiently decomposed salivary AA (100-390 μM) without the supplemental addition of cofactors irrespective of the type of alcoholic beverages consumed, even in the presence of an excess of ethanol (63. mM). Hydrogen peroxide, which is carcinogenic in animal experiments, was not produced because of the AA removal. The GK cells incubated at 45°C and pH 3.5 for 15. h were killed, but they retained 80% of their original AA-decomposing activity. The treated cells were used as nonviable microcapsules that harbor a membrane-bound AA-decomposing activity. © 2012 The Society for Biotechnology, Japan.

Duong C.T.,TU Berlin | Strack L.,TU Berlin | Futschik M.,Humboldt University of Berlin | Futschik M.,University of Algarve | And 6 more authors.
Metabolic Engineering | Year: 2011

Diacetyl causes an unwanted buttery off-flavor in lager beer. It is spontaneously generated from α-acetolactate, an intermediate of yeast's valine biosynthesis released during the main beer fermentation. Green lager beer has to undergo a maturation process lasting two to three weeks in order to reduce the diacetyl level below its taste-threshold. Therefore, a reduction of yeast's α-acetolactate/diacetyl formation without negatively affecting other brewing relevant traits has been a long-term demand of brewing industry. Previous attempts to reduce diacetyl production by either traditional approaches or rational genetic engineering had different shortcomings. Here, three lager yeast strains with marked differences in diacetyl production were studied with regard to gene copy numbers as well as mRNA abundances under conditions relevant to industrial brewing. Evaluation of data for the genes directly involved in the valine biosynthetic pathway revealed a low expression level of Sc. -ILV6 as a potential molecular determinant for low diacetyl formation. This hypothesis was verified by disrupting the two copies of Sc. -ILV6 in a commercially used lager brewers' yeast strain, which resulted in 65% reduction of diacetyl concentration in green beer. The Sc. -ILV6 deletions did not have any perceptible impact on beer taste. To our knowledge, this has been the first study exploiting natural diversity of lager brewers' yeast strains for strain optimization. © 2011 Elsevier Inc.

Hasunuma T.,Kobe University | Hori Y.,Kobe University | Sakamoto T.,Kobe University | Ochiai M.,Suntory Research Center | And 2 more authors.
Microbial cell factories | Year: 2014

BACKGROUND: Bioethanol produced by the yeast Saccharomyces cerevisiae is currently one of the most promising alternatives to conventional transport fuels. Lignocellulosic hemicelluloses obtained after hydrothermal pretreatment are important feedstock for bioethanol production. However, hemicellulosic materials cannot be directly fermented by yeast: xylan backbone of hemicelluloses must first be hydrolyzed by heterologous hemicellulases to release xylose, and the yeast must then ferment xylose in the presence of fermentation inhibitors generated during the pretreatment.RESULTS: A GIN11/FRT-based multiple-gene integration system was developed for introducing multiple functions into the recombinant S. cerevisiae strains engineered with the xylose metabolic pathway. Antibiotic markers were efficiently recycled by a novel counter selection strategy using galactose-induced expression of both FLP recombinase gene and GIN11 flanked by FLP recombinase recognition target (FRT) sequences. Nine genes were functionally expressed in an industrial diploid strain of S. cerevisiae: endoxylanase gene from Trichoderma reesei, xylosidase gene from Aspergillus oryzae, β-glucosidase gene from Aspergillus aculeatus, xylose reductase and xylitol dehydrogenase genes from Scheffersomyces stipitis, and XKS1, TAL1, FDH1 and ADH1 variant from S. cerevisiae. The genes were introduced using the homozygous integration system and afforded hemicellulolytic, xylose-assimilating and inhibitor-tolerant abilities to the strain. The engineered yeast strain demonstrated 2.7-fold higher ethanol titer from hemicellulosic material than a xylose-assimilating yeast strain. Furthermore, hemicellulolytic enzymes displayed on the yeast cell surface hydrolyzed hemicelluloses that were not hydrolyzed by a commercial enzyme, leading to increased sugar utilization for improved ethanol production.CONCLUSIONS: The multifunctional yeast strain, developed using a GIN11/FRT-based marker recycling system, achieved direct conversion of hemicellulosic biomass to ethanol without the addition of exogenous hemicellulolytic enzymes. No detoxification processes were required. The multiple-gene integration technique is a powerful approach for introducing and improving the biomass fermentation ability of industrial diploid S. cerevisiae strains.

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