Mitsuyoshi Ueda

California Institute of Technology, Pasadena, California, United States

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Publications (206)432.54 Total impact

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    ABSTRACT: In Saccharomyces cerevisiae, we have demonstrated that organic solvent stress activated the pleiotropic drug resistance (PDR) pathway, which involves the transcription factors Pdr1p and Pdr3p. Pdr1p and Pdr3p are functionally homologous in multidrug resistance, although Pdr3p has been reported to have some distinct functions. Here, we analyzed the functions of Pdr1p and Pdr3p during the cellular response to isooctane, as a representative of organic solvents, and observed the differential functions of Pdr1p and Pdr3p. In response to organic solvent stress, only Pdr3p contributed to the regulation of downstream genes of the PDR pathway, while Pdr1p had a rather inhibitory role on transcriptional induction through competition with Pdr3p for binding to their recognition sequence, pleiotropic drug response element. Our results demonstrated that organic solvent stress was likely to damage mitochondria, causing generation of reactive oxygen species and mitochondrial fragmentation, and to activate retrograde signaling pathway via Pdr3p to upregulate PDR5 expression. Therefore, the unique function of Pdr3p in organic solvent stress distinguishes this pathway from the multidrug response.
    Current Genetics 12/2014; · 2.41 Impact Factor
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    ABSTRACT: Compared with ethanol, butanol has more advantageous physical properties as a fuel, and biobutanol is thus considered a promising biofuel material. Biobutanol has often been produced by Clostridium species; however, because they are strictly anaerobic microorganisms, these species are challenging to work with. We attempted to introduce the butanol production pathway into yeast Saccharomyces cerevisiae, which is a well-known microorganism that is tolerant to organic solvents. 1-Butanol was found to be produced at very low levels when the butanol production pathway of Clostridium acetobutylicum was simply introduced into S. cerevisiae. The elimination of glycerol production pathway in the yeast contributed to the enhancement of 1-butanol production. In addition, by the use of trans-enoyl-CoA reductase in the engineered pathway, 1-butanol production was markedly enhanced to yield 14.1 mg/L after 48 h of cultivation.
    Bioscience Biotechnology and Biochemistry 10/2014; · 1.27 Impact Factor
  • Jungu Bae, Kouichi Kuroda, Mitsuyoshi Ueda
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    ABSTRACT: Proximity effect is a form of synergistic effects exhibited when cellulases work within a short distance from each other, and this effect can be a key factor in enhancing saccharification efficiency. In this study, we evaluated the proximity effect between 3 cellulose-degrading enzymes displayed on the yeast-cell surface, that is, endoglucanase, cellobiohydrolase, and β-glucosidase. We constructed 2 kinds of arming yeasts through genome integration: ALL-yeast, which simultaneously displayed the 3 cellulases (thus, the different cellulases were near each other); and MIX-yeast, a mixture of 3 kinds of single-cellulase-displaying yeasts (the cellulases were far apart). The cellulases were tagged with a fluorescence protein or polypeptide to visualize and quantify their display. To evaluate the proximity effect, we compared the activities of ALL-yeast and MIX-yeast with respect to degrading phosphoric-acid-swollen cellulose after adjusting for the cellulase amounts. ALL-yeast exhibited 1.25 fold or 2.22 fold higher activity than MIX-yeast did at a yeast concentration of OD10 or OD0.1. At OD0.1, the distance between the 3 cellulases was greater than that at OD10 in MIX-yeast, but the distance remained the same in ALL-yeast; thus, the difference between the cellulose-degrading activities of ALL-yeast and MIX-yeast increased (to 2.22 fold) at OD0.1, which strongly supports the proximity effect between the displayed cellulases. Proximity effect was also observed for crystalline cellulose (Avicel). We expect the proximity effect to further increase when enzyme-display efficiency is enhanced, which would further increase cellulose-degrading activity. This arming-yeast technology can also be applied to examine proximity effects in other diverse fields.
    Applied and Environmental Microbiology 10/2014; · 3.95 Impact Factor
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    ABSTRACT: Chitosan oligosaccharides longer than a hexamer have higher bioactivity than polymer or shorter oligosaccharides, such as the monomer or dimer. In our previous work, we generated Paenibacillus fukuinensis chitosanase-displaying yeast using yeast cell surface displaying system and demonstrated the catalytic base. Here we investigated the specific function of putative four amino acid residues Trp159, Trp228, Tyr311, and Phe406 engaged in substrate binding. Using this system, we generated chitosanase mutants in which the four amino acid residues were substituted with Ala and the chitosanase activity assay and HPLC analysis were performed. Based on these results, we demonstrated that Trp159 and Phe406 were critical for hydrolyzing both polymer and oligosaccharide, and Trp228 and Tyr311 were especially important for binding to oligosaccharide, such as the chitosan-hexamer, not to the chitosan polymer. From the results, we suggested the possibility of the effective strategy for designing useful mutants that produce chitosan oligosaccharides holding higher bioactivity.
    Bioscience Biotechnology and Biochemistry 07/2014; 78(7):1177-1182. · 1.27 Impact Factor
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    ABSTRACT: Phenotypic and genomic heterogeneity among single cells in a cell population leads to inaccuracy and obscuration in research about mammalian cell differentiation. In order to address the problems regarding bulk analysis on heterogeneous cell populations, it is necessary to accurately regulate and analyze changes in differentiating cells at the single-cell level. To investigate the single-cell changes in PC12 neuronal differentiation that occur when inhibited by U0126, an inhibitor of mitogen-activated protein kinase kinase (MEK), we directly injected the chemical into individual target cells and analyzed the outcomes (neurite outgrowth) at the single-cell level. As a result, we could accurately regulate the quantity of U0126 being introduced into each target cell, which was previously not possible using the common method of simply adding the inhibitor to the culture medium. It was possible analyze the inhibitive effect of U0126 even when the injected quantity was lower than the lower limit for inhibition when added to culture medium (0.1 μM, identical to 1.2 × 108 molecules per each cell on dish). In particular, injection of 1.5 × 107 molecules into each cell resulted in a 59% decrease of the mean total neurite length. Time-course analysis of neurite outgrowth at the single-cell level using fluorescence staining method showed that the changes in neurite length of differentiating PC12 cells were not homogeneous, but were largely variable across individual target cells.
    Cell Biology International 05/2014; · 1.64 Impact Factor
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    ABSTRACT: We have engineered a system that holds potential for use as a safety switch in genetically modified yeasts. Human apoptotic factor BAX (no homolog in yeast), under the control of the FBP1 (gluconeogenesis enzyme) promoter, was conditionally expressed to induce yeast cell apoptosis after glucose depletion. Such systems might prove useful for the safe use of genetically modified organisms.
    Bioscience Biotechnology and Biochemistry 02/2014; 78(2):358-62. · 1.27 Impact Factor
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    ABSTRACT: The viability of employing brown macroalgae as a future renewable energy and chemical resource has so far proven to be challenging because of the low efficiency of the energy extraction process from alginate, which forms the principle, difficult-to-degrade component. In contrast to currently employed alginate-metabolizing microbial approaches, we here extract energy from macroalgae, for the first time, through a fuel cell system that exploits the electrochemical oxidation of alginate by gold nanoparticle-decorated functionalized carbon nanotubes without any external input of energy. The analyses suggest that the electrochemical oxidation process induces partial oxidation of alginate and, in addition to bioenergy, also yields valuable chemicals, which paves the way for the future production of energy and feedstock materials from inedible biomass.
    ChemCatChem 01/2014; 6(1). · 5.18 Impact Factor
  • Kouichi Kuroda, Mitsuyoshi Ueda
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    ABSTRACT: The cell surface display system in yeast enables the innovative strategy for improving cellular functions in a wide range of applications such as biofuel production, bioremediation, synthesis of valuable chemicals, recovery of rare metal ions, development of biosensors, and high-throughput screening of proteins/peptides library. Display of enzymes for polysaccharide degradation enables the construction of metabolically engineered whole-cell biocatalyst owing to the accessibility of the displayed enzymes to high-molecular-weight polysaccharides. In addition, along with fluorescence-based activity evaluation, fluorescence-activated cell sorting (FACS), and yeast cell chip, the cell surface display system is an effective molecular tool for high-throughput screening of mutated proteins/peptides library. In this article, we describe the methods for cell surface display of proteins/peptides of interest on yeast, evaluation of display efficiency, and harvesting of the displayed proteins/peptides from cell surface.
    Methods in molecular biology (Clifton, N.J.) 01/2014; 1152:137-55. · 1.29 Impact Factor
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    ABSTRACT: Candida albicans malate dehydrogenase (Mdh1p) has been screened by previous proteome studies as a candidate for a vaccine against candidiasis. In this study, recombinant Mdh1 protein with a His-tag was produced in Escherichia coli and evaluated as an immunogenic protein against candidiasis. Mdh1p was administrated to mice by two methods subcutaneous injection and intranasal administration before challenging them with a lethal dose of C. albicans. After vaccination of Mdh1p, antibody responses were observed. To evaluate the vaccination effect of Mdh1p, survival tests were performed after 35 d. Although all control mice died within 24 d or 25 d, 100% and 80% of mice survived with subcutaneous and intranasal administration, respectively. Therefore, our results indicate that, among C. albicans antigens examined thus far, Mdh1p is currently the most effective antigen for use as a vaccine for C. albicans.
    Biocontrol science. 01/2014; 19(1):51-5.
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    ABSTRACT: Uranium is one of the most important metal resources, and the technology for the recovery of uranyl ions (UO22+) from aqueous solutions is required to ensure a semi-permanent supply of uranium. The NikR protein is a Ni2+-dependent transcriptional repressor of the nickel-ion uptake system in Escherichia coli, but its mutant protein (NikRm) is able to selectively bind uranyl ions in the interface of the two monomers. In this study, NikRm protein with ability to adsorb uranyl ions was displayed on the cell surface of Saccharomyces cerevisiae. To perform the binding of metal ions in the interface of the two monomers, two metal-binding domains (MBDs) of NikRm were tandemly fused via linker peptides and displayed on the yeast cell surface by fusion with the cell wall-anchoring domain of yeast α-agglutinin. The NikRm-MBD-displaying yeast cells with particular linker lengths showed the enhanced adsorption of uranyl ions in comparison to the control strain. By treating cells with citrate buffer (pH 4.3), the uranyl ions adsorbed on the cell surface were recovered. Our results indicate that the adsorption system by yeast cells displaying tandemly fused MBDs of NikRm is effective for simple and concentrated recovery of uranyl ions, as well as adsorption of uranyl ions.
    Biomolecules. 01/2014; 4(2):390-401.
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    ABSTRACT: Falsirhodobacter sp. alg1 is an alginate-degrading bacterium, the second species from the nonphototrophic bacterial genus Falsirhodobacter. We report the first draft genome of a bacterium from this genus and point out possible important features related to alginate assimilation and its evolutionary aspects.
    Genome announcements. 01/2014; 2(4).
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    ABSTRACT: Organic solvents are toxic to living cells. In eukaryotes, cells with organic solvent tolerance have only been found in Saccharomyces cerevisiae. Although several factors contributing to organic solvent tolerance have been identified in previous studies, the mechanism of how yeast cells naturally respond to organic solvent stress is not known. We demonstrated that the pleiotropic drug resistance (PDR) pathway contributed to response to organic solvent stress. Activation of the PDR pathway by mutations in the transcription factors Pdr1p and Pdr3p led to organic solvent tolerance. Exposure to organic solvents also induced transcription levels of PDR5, which encodes a major drug efflux pump. Overproduction of Pdr5p improved organic solvent tolerance, presumably by exporting organic solvents out of the cell. In addition, we showed that the cell wall integrity (CWI) pathway was induced in response to organic solvents to upregulate genes encoding the cell wall-related proteins Wsc3p and Ynl190wp. WSC3 and YNL190W were upregulated independently of the PDR pathway. Among the components of the CWI pathway, the cell surface sensors (Wsc3p and Mid2p) and the transcription factors (Swi4p and Swi6p) appeared to be particularly involved in the response to organic solvents. Our findings indicate that S. cerevisiae activates two different signaling pathways, the PDR pathway and the CWI pathway, to cope with stresses from organic solvents.
    Current Genetics 12/2013; · 2.41 Impact Factor
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    ABSTRACT: A display system for adding new protein functions to the cell surfaces of microorganisms has been developed, and applications of the system to various fields have been proposed. With the aim of constructing a cell surface environment suitable for protein display in Saccharomyces cerevisiae, the cell surface structures of cell wall mutants were investigated. Four cell wall mutant strains were selected by analyses using a GFP display system via a GPI anchor. β-Glucosidase and endoglucanase II were displayed on the cell surface in the four mutants, and their activities were evaluated. mnn2 deletion strain exhibited the highest activity for both the enzymes. In particular, endoglucanase II activity using carboxymethylcellulose as a substrate in the mutant strain was 1.9-fold higher than that of the wild-type strain. In addition, the activity of endoglucanase II released from the mnn2 deletion strain by Zymolyase 20 T treatment was higher than that from the wild-type strain. The results of GFP and endoglucanase displays suggest that the amounts of enzyme displayed on the cell surface were increased by the mnn2 deletion. The enzyme activity of the mnn2 deletion strain was compared with that of the wild-type strain. The level of endoglucanase II activity using carboxymethylcellulose as a substrate was higher than that of β-glucosidase activity using p-nitrophenyl-β-glucopyranoside as a substrate, suggesting that the cell surface environment of the mnn2 deletion strain facilitates the binding of high-molecular-weight substrates to the active sites of the displayed enzymes. This article is protected by copyright. All rights reserved.
    Yeast 12/2013; · 1.96 Impact Factor
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    ABSTRACT: Clostridium cellulovorans has been one of promising microorganisms to use biomass efficiently; however the basic metabolic pathways have been completely unknown. We carried out 13C-isotopomer-based target metabolome analysis, or carbohydrate conversion process analysis, for more profound understanding of metabolic pathways of the bacterium. Our findings that pyruvate + oxaloacetate, fumarate, and malate inside and outside cells exhibited 13C incorporation suggest that C. cellulovorans exactly fixed CO2 and partly operated the TCA cycle in a reductive manner. Accompanied with CO2 fixation, the microorganism was also found to produce and secrete lactate. Overall, our study demonstrates that a part of C. cellulovorans metabolic pathways related to glycolysis and the TCA cycle are involved in CO2 fixation.
    AMB Express. 10/2013; 3(1):61.
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    ABSTRACT: A thermotolerant Saccharomyces cerevisiae yeast strain, YK60-1, was bred from a parental strain, MT8-1, via stepwise adaptation. YK60-1 grew at 40°C, a temperature at which MT8-1 could not grow at all. YK60-1 exhibited faster growth than MT8-1 at 30°C. To investigate the mechanisms how MT8-1 acquired thermotolerance, DNA microarray analysis was performed. The analysis revealed the induction of stress-responsive genes such as those encoding heat shock proteins and trehalose biosynthetic enzymes in YK60-1. Furthermore, nontargeting metabolome analysis showed that YK60-1 accumulated more trehalose, a metabolite that contributes to stress tolerance in yeast, than MT8-1. In conclusion, S. cerevisiae MT8-1 acquired thermotolerance by induction of specific stress-responsive genes and enhanced intracellular trehalose levels.
    Biotechnology Progress 09/2013; · 1.85 Impact Factor
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    ABSTRACT: The cellulosome is a complex of cellulosomal proteins bound to scaffolding proteins. This complex is considered the most efficient system for cellulose degradation. Clostridium cellulovorans, known to produce cellulosomes, changes the composition of its cellulosomes depending on the growth substrate. However, studies have only investigated cellulosomal proteins; changes in non-cellulosomal proteins have rarely been examined. In this study, we performed a quantitative proteome analysis of the whole exoproteome of C. cellulovorans, including cellulosomal and non-cellulosomal proteins, to illustrate how various substrates are efficiently degraded. C. cellulovorans was cultured with cellobiose, xylan, pectin, or phosphoric acid swollen cellulose (PASC) as the sole carbon source. PASC was used as a cellulose substrate for more accurate quantitative analysis. Using an isobaric tag method and a liquid chromatography mass spectrometer equipped with a long monolithic silica column, 639 proteins were identified and quantified in all 4 samples. Among these, 79 proteins were involved in saccharification, including 35 cellulosomal and 44 non-cellulosomal proteins. We compared protein abundance by spectral count and found that cellulosomal proteins were more abundant than non-cellulosomal proteins. Next, we focused on the fold change of the proteins depending on the growth substrate. Drastic changes were mainly observed among the non-cellulosomal proteins. These results indicate that cellulosomal proteins were primarily produced to efficiently degrade any substrate, and that non-cellulosomal proteins were specifically produced to optimize the degradation of a particular substrate. This study highlights the importance of non-cellulosomal proteins as well as cellulosomes for the efficient degradation of various substrates.
    Applied and Environmental Microbiology 08/2013; · 3.95 Impact Factor
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    ABSTRACT: Candida albicans is an opportunistic pathogen that causes fatal disease if the host immunity is compromised. The mortality rate of systemic candidiasis is very high; hence, there is a ceaseless demand for novel pharmaceuticals. In this study, quantitative time-course proteomics of C. albicans during adaptation to fetal bovine serum (FBS) is described. Survival in blood is essential for virulence of C. albicans, and a detailed analysis is required. We cultivated C. albicans in FBS for 0-180min, and determined quantitative time-course variations of 1024 proteins in the cultured cells by using an LC-MS/MS system with a long monolithic silica capillary column. Clustering analysis identified FBS-induced proteins associated with detoxification of oxidative species, high-affinity glucose transport, citrate cycle, oxidative phosphorylation, and iron acquisition. Furthermore, we identified possible virulence factors such as orf19.4914.1 (named Blood-induced peptide 1, Blp1). Heterologous expression of BLP1 in Saccharomyces cerevisiae shortened the lag phase and resulted in a pleiotropic stress-tolerance phenotype, indicating a possible role for quick adaptation to a stressful environment. While further experiments are necessary to prove virulence of the identified factors, systematic identification of candidate virulence proteins in this study will lead to further understanding of virulence of C. albicans. Biological Significance This paper describes time-course proteomics of C. albicans during adaptation to serum, which is an essential process for fatal systemic candidiasis. Using a LC-MS/MS system with a monolithic silica capillary column, we have successfully characterized time-course variations of 1024 proteins. Among them, orf19.4914.1 (Blp1) was identified as a novel pleiotropic stress-tolerance peptide, which could have an important role for virulence of C. albicans.
    Journal of proteomics 08/2013; · 5.07 Impact Factor
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    ABSTRACT: The development of simple, portable, inexpensive, and rapid analytical methods for detecting and monitoring toxic heavy metals are important for the safety and security of humans and their environment. Herein we describe the application of phytochelatin (PC) synthase, which plays a critical role in heavy metal responses in higher plants and green algae, in a novel fluorescent sensing platform for cadmium (Cd). We first created surface-engineered yeast cells on which the PC synthase from Arabidopsis (AtPCS1) was displayed with retention of enzymatic activity. The general concept for the sensor is based on the Cd level-dependent synthesis of PC2 from glutathiones by AtPCS1-displaying yeast cells, followed by simple discriminative detection of PC2 via sensing of excimer fluorescence of thiol-labeling pyrene probes. The intensity of excimer fluorescence increased in the presence of Cd up to 1.0 μM in an approximately dose-dependent manner. This novel biosensor achieved a detection limit of as low as 0.2 μM (22.5 μg/L) for Cd. Although its use may be limited by the fact that Cu and Pb can induce cross-reaction, the proposed simple biosensor holds promise as a method useful for cost-effective screening of Cd contamination in environmental and food samples. The AtPCS1-displaying yeast cells also might be attractive tools for dissection of the catalytic mechanisms of PCS. © 2013 American Institute of Chemical Engineers Biotechnol. Prog., 2013.
    Biotechnology Progress 08/2013; · 1.85 Impact Factor
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    ABSTRACT: Secreted aspartic peptidases (Saps) are a group of ten acidic hydrolases considered as key virulence factors of Candida albicans. These enzymes supply the fungus with nutrient amino acids as well as are able to degrade the selected host's proteins involved in the immune defense. Our previous studies showed that the human menstrual discharge is exceptionally rich in bactericidal hemoglobin (Hb) fragments - hemocidins. However, to date, the genesis of such peptides is unclear. The presented study demonstrates that the action of C. albicans isozymes Sap1-Sap6, Sap8 and Sap9, but not Sap7 and Sap10, toward human hemoglobin leads to limited proteolysis of this protein and generates a variety of antimicrobial hemocidins. We have identified these peptides and checked their activity against selected microorganisms representative for human vagina. We have also demonstrated that the process of Hb hydrolysis is most effective at pH 4.0, characteristic for vagina, and the liberated peptides showed pronounced killing activity toward Lactobacillus acidophilus, and to a lower degree, Escherichia coli. However, only a very weak activity toward Staphylococcus aureus and Candida albicans was noticed. These findings provide interesting new insights into pathophysiology of human vaginal candidiasis and suggest that C. albicans may be able to compete with the other microorganisms of the same physiological niche using the microbicidal peptides generated from the host protein.
    Peptides 08/2013; · 2.52 Impact Factor
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    ABSTRACT: Hypoxia has critical effects on the physiology of organisms. In the yeast Saccharomyces cerevisiae, glycolytic enzymes, including enolase (Eno2p), formed cellular foci under hypoxia. Here, we investigated the regulation and biological functions of these foci. Focus formation by Eno2p was inhibited temperature independently by the addition of cycloheximide or rapamycin or by the single substitution of alanine for the Val22 residue. Using mitochondrial inhibitors and an antioxidant, mitochondrial reactive oxygen species (ROS) production was shown to participate in focus formation. Focus formation was also inhibited temperature dependently by an SNF1 knockout mutation. Interestingly, the foci were observed in the cell even after reoxygenation. The metabolic turnover analysis revealed that [U-13C]glucose conversion to pyruvate and oxaloacetate was accelerated in focus-forming cells. These results suggest that under hypoxia, S. cerevisiae cells sense mitochondrial ROS and, by the involvement of SNF1/AMPK, spatially reorganize metabolic enzymes in the cytosol via de novo protein synthesis, which subsequently increases carbon metabolism. The mechanism may be important for yeast cells under hypoxia, to quickly provide both energy and substrates for the biosynthesis of lipids and proteins independently of the tricarboxylic acid (TCA) cycle and also to fit changing environments.
    Eukaryotic Cell 08/2013; 12(8). · 3.59 Impact Factor

Publication Stats

2k Citations
432.54 Total Impact Points

Institutions

  • 2013
    • California Institute of Technology
      Pasadena, California, United States
    • Chubu University
      Касугай, Aichi, Japan
  • 2010–2013
    • Japan Society for the Promotion of Science
      Edo, Tōkyō, Japan
  • 2008–2013
    • Hyogo University of Health Sciences
      Kōbe, Hyōgo, Japan
  • 1983–2013
    • Kyoto University
      • • Division of Applied Life Sciences
      • • Department of Synthetic Chemistry and Biological Chemistry
      Kyoto, Kyoto-fu, Japan
  • 2011
    • Fukui National College Of Technology
      Hukui, Fukui, Japan
  • 2006–2011
    • Mie University
      • • Department of Life Sciences
      • • Graduate School of Bioresources
      Tu, Mie, Japan
  • 2006–2010
    • University of Fukui
      • Department of Applied Chemistry and Biotechnology
      Fukui-shi, Fukui-ken, Japan
  • 2002–2009
    • Kobe University
      • Department of Chemical Science and Engineering
      Kōbe-shi, Hyogo-ken, Japan
  • 2006–2007
    • Osaka University
      • Department of Biotechnology
      Suita, Osaka-fu, Japan
  • 1998
    • Kinki University
      Ōsaka, Ōsaka, Japan
    • Universität Stuttgart
      Stuttgart, Baden-Württemberg, Germany
  • 1983–1992
    • Japan Women's University
      • Department of Chemical and Biological Sciences
      Tokyo, Tokyo-to, Japan