Sakayu Shimizu

Kyoto Gakuen University, Kioto, Kyoto, Japan

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Publications (401)892.84 Total impact

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    ABSTRACT: The oil-producing zygomyceteMortierella alpina 1S-4 is known to accumulate beneficial polyunsaturated fatty acids. We identified the lig4 gene thatencodesfor a DNA ligase 4 homolog, which functions to repair double strand breaks by non-homologous end joining.We disrupted the lig4 gene to improvethe genetargeting efficiency in M. alpina. The M. alpina 1S-4 Δlig4strains showed no defect in vegetative growth, formation of spores, and fatty acid production, but exhibited high sensitivity to methyl methansulfonate, an agent that causes DNA double-strand breaks. Importantly, gene replacement of ura5 marker by CBXB marker occurred in 67% of Δlig4 strains and the genetargeting efficiency was 21-fold greater than that observed in disruption of the lig4 gene in the M. alpina 1S-4 host strain. Further metabolic engineering of the Δlig4 strains is expected to result in strains that produce higher levels of rare and beneficial polyunsaturated fatty acids and contribute tobasic research onthe zygomycete. Copyright © 2015. Published by Elsevier B.V.
    Journal of Biotechnology 06/2015; 208. DOI:10.1016/j.jbiotec.2015.05.020 · 2.87 Impact Factor
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    ABSTRACT: We investigated the omega-3 eicosatetraenoic acid (ETA) production by molecular breeding of the oleaginous fungus Mortierella alpina, which can slightly accumulate ETA only when cultivated at a low temperature. The endogenous ω3-desaturase gene or the heterologous Saprolegnia diclina Δ17 (sdd17m) desaturase gene were overexpressed in M. alpina S14, a Δ5-desaturation activity-defective mutant derived from M. alpina 1S-4. M. alpina S14 transformants introduced with the endogenous ω3-desaturase gene showed ETA at 42.1% content in the total lipids that was 84.2-fold and 3.2-fold higher than that of the wild-type strain 1S-4 and host strain S14, respectively, when cultivated at 12°C. No accumulation of ETA was observed at 28°C. In contrast, transformants with the heterologous sdd17m gene showed 24.9% of the content of total lipids at 28°C. These results indicated that these M. alpina S14 transformants are promising strains for the production of ETA, which is hard to obtain from natural sources. Copyright © 2015 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.
    Journal of Bioscience and Bioengineering 04/2015; 120(3). DOI:10.1016/j.jbiosc.2015.01.014 · 1.88 Impact Factor
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    ABSTRACT: To develop an efficient gene-targeting system in Mortierella alpina 1S-4, we identified the ku80 gene encoding the Ku80 protein, which is involved in the nonhomologous end-joining pathway in genomic double-strand break (DSB) repair, and constructed ku80 gene-disrupted strains via single-crossover homologous recombination. The Δku80 strain from M. alpina 1S-4 showed no negative effects on vegetative growth, formation of spores, and fatty acid productivity, and exhibited high sensitivity to methyl methanesulfonate, which causes DSBs. Dihomo-γ-linolenic acid (DGLA)-producing strains were constructed by disruption of the Δ5-desaturase gene, encoding a key enzyme of bioconversion of DGLA to ARA, using the Δku80 strain as a host strain. The significant improvement of gene-targeting efficiency was not observed by disruption of the ku80 gene, but the construction of DGLA-producing strain by disruption of the Δ5-desaturase gene was succeeded using the Δku80 strain as a host strain. This report describes the first study on the identification and disruption of the ku80 gene in zygomycetes and construction of a DGLA-producing transformant using a gene-targeting system in M. alpina 1S-4.
    Current Genetics 03/2015; 61(4). DOI:10.1007/s00294-015-0481-2 · 2.68 Impact Factor
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    ABSTRACT: The oleaginous fungus Mortierella alpina is known to accumulate eicosapentaenoic acid (EPA) only when cultivated at a low temperature (below 15°C). Here, we investigated EPA production at a ordinary temperature (28°C) by expressing the Saprolegnia diclina Δ17 desaturase gene (sdd17m) in M. alpina ST1358, an ω3-desaturation activity-defective mutant derived from M. alpina 1S-4. Expression of the exogenous gene was confirmed by EPA accumulation in transformants at both 28°C and 12°C. The EPA content in total lipids produced by transformants was over 20% at 28°C. Bench-scale fermentation with a 5-L jar fermentor showed that EPA content reached 26.4% of total fatty acids, and final EPA production reached 1.8 g/L. This is the first study to report the accumulation of EPA in M. alpina at a ordinary temperature, and provide a platform technology for the industrial production of EPA using M. alpina as a promising source for EPA.
    European Journal of Lipid Science and Technology 02/2015; DOI:10.1002/ejlt.201400657 · 1.81 Impact Factor
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    ABSTRACT: (4R,6R)-Actinol can be stereo-selectively synthesized from ketoisophorone by a two-step conversion using a mixture of two enzymes: Candida macedoniensis old yellow enzyme (CmOYE) and Corynebacterium aquaticum (6R)-levodione reductase. However, (4S)-phorenol, an intermediate, accumulates because of the limited substrate range of CmOYE. To address this issue, we solved crystal structures of CmOYE in the presence and absence of a substrate analogue p-HBA, and introduced point mutations into the substrate-recognition loop. The most effective mutant (P295G) showed two- and 12-fold higher catalytic activities toward ketoisophorone and (4S)-phorenol, respectively, than the wild-type, and improved the yield of the two-step conversion from 67.2 to 90.1 %. Our results demonstrate that the substrate range of an enzyme can be changed by introducing mutation(s) into a substrate-recognition loop. This method can be applied to the development of other favorable OYEs with different substrate preferences.
    ChemBioChem 01/2015; 16(3). DOI:10.1002/cbic.201402555 · 3.09 Impact Factor
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    ABSTRACT: AimTo find cis-11-eicosenoic acid (20:1ω9, EA)-producing microorganisms.Methods and ResultsWe found EA-producing fungi by screening about 300 fungal strains, and identified a major fatty acid accumulated in the Mortierella fungi as EA by means of GC-MS analysis. In particular, Mortierella chlamydospora CBS 529.75 produced a high amount of EA (36.3 mg g−1 of dried cells) on cultivation at 28°C for 4 days and then at 12°C for 3 days. In the result of lipid analysis, most of the EA was a component of triacylglycerols, not phospholipids.Conclusion We found that M. chlamydospora CBS 529.75 was the best producer for the microbial production of EA.Significance and Impact of the StudyEA is beneficial as a raw material for medical supplies and a moisturizing component of cosmetic creams. This is the first report of microbial production of EA.This article is protected by copyright. All rights reserved.
    Journal of Applied Microbiology 12/2014; 118(3). DOI:10.1111/jam.12725 · 2.48 Impact Factor
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    ABSTRACT: The front cover picture, provided by Makoto Hibi, Jun Ogawa et al., depicts Fe(II)/α-ketoglutarate (αKG)-dependent dioxygenases, which are hydroxylation biocatalysts catalyzing highly selective monooxygenation available to industrial applications. Recently, a great number of Fe(II)/αKG-dependent dioxygenases with a variety of functions has been found in the genome database, and collected into a biocatalyst library. One of these enzymes, N-succinyl L-amino acid β-hydroxylase, was applied to an enzymatic cascade system together with N-succinyl L-amino acid desuccinylase for the production of optically pure β-hydroxy α-amino acids. Details can be found in the full paper on pages 767–774 (M. Hibi, T. Kasahara, T. Kawashima, H. Yajima, S. Kozono, S. V. Smirnov, T. Kodera, M. Sugiyama, S. Shimizu, K. Yokozeki, and J. Ogawa, Adv. Synth. Catal. 2015, 357, 767–774; DOI: 10.1002/adsc.201400672).
    Advanced Synthesis & Catalysis 11/2014; 357(4). DOI:10.1002/adsc.201400672 · 5.66 Impact Factor
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    ABSTRACT: We found a new aldehyde oxidase (ALOD), which catalyzes the conversion of glycolaldehyde to glycolate, from Burkholderia sp. AIU 129. The enzyme further oxidized aliphatic aldehydes, an aromatic aldehyde, and glyoxal, but not glycolate or alcohols. The molecular mass of this enzyme was 130 kDa, and it was composed of three different subunits (αβγ structure), in which the α, β, and γ subunits were 76 kDa, 36 kDa, and 14 kDa, respectively. The N-terminal amino acid sequences of each subunit showed high similarity to those of putative subunits of xanthine dehydrogenase. Metals (copper, iron and molybdenum) and chelating reagents (α,α′-dipyridyl and 8-hydroxyquinoline) inhibited the ALOD activity. The ALOD showed highest activity at pH 6.0 and 50°C. Twenty mM glycolaldehyde was completely converted to glycolate by incubation at 30°C for 3 h, suggesting that the ALOD found in this study would be useful for enzymatic production of glycolate.
    Journal of Bioscience and Bioengineering 10/2014; 119(4). DOI:10.1016/j.jbiosc.2014.09.005 · 1.88 Impact Factor
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    ABSTRACT: The polyunsaturated fatty acids (PUFAs) include many functional lipids. The microbial metabolism of C18 PUFAs is known to produce their bioactive isomers, such as conjugated fatty acids and hydroxy fatty acids, but there is little information on that of C20 PUFAs. In this study, we aimed to obtain anaerobic bacteria for the ability to produce novel PUFA from C20 PUFAs. Through the screening of about 100 strains of anaerobic bacteria, Clostridium bifermentans JCM 1386 was selected as a strain with the ability to saturate PUFAs during anaerobic cultivation. This strain converted arachidonic acid (cis-5,cis-8,cis-11,cis-14-eicosatetraenoic acid) and eicosapentaenoic acid (cis-5,cis-8,cis-11,cis-14,cis-17-eicosapentaenoic acid) into cis-5,cis-8,trans-13-eicosatrienoic acid and cis-5,cis-8,trans-13,cis-17-eicosatetraenoic acid, giving yields of 57% and 67% against the added PUFAs, respectively. This is the first report of the isolation of the bacterium transforming C20 PUFAs into corresponding non-methylen-interrupted fatty acids. We further investigated the substrate specificity of the biohydrogenation by this strain and revealed that it can convert two cis double bonds at ω6 and ω9 positions in various C18 and C20 PUFAs into a trans double bond at ω7 position. This study should serve to open up the development of novel potentially bioactive PUFAs.
    The Journal of Lipid Research 07/2014; 55(9). DOI:10.1194/jlr.M045450 · 4.42 Impact Factor
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    ABSTRACT: We revealed that Ochrobactrum sp. AIU 033, which accumulated a high concentration of glyoxylate from glycolate, produced an enzyme catalyzing oxidation of glycolate to glyoxylate. The enzyme further oxidized lactate and primary alcohols (C2-C10), but did not oxidize glyoxylate, ethylene glycol, glycerol, or methanol. The K-m, value for glycolate (167 mM) was higher than that for primary alcohols. The glycolate oxidase activity was optimum at pH 5.5, and more than 80% of the enzyme activity remained in the pH range from 5.5 to 6.5 and at below 35 degrees C. The enzyme had a molecular mass of 130 kDa and was composed of an alpha(2)beta(2) structure, in which the la subunit was 52 kDa and the beta subunit was 14 kDa. The enzyme was a flavoprotein and contained two iron atoms. The N-terminal sequences of the 52 kDa subunit and 14 kDa subunit had high similarity to those of putative glucose-methanol-choline oxidoreductases and putative 2-keto-gluconate dehydrogenase. These findings implied that the enzyme was a novel type of alcohol oxidase exhibiting glycolate oxidase activity. The enzyme accumulated glyoxylate with time, but oxalate, which is the oxidation product of glyoxylate, was not detected. This result also indicated that the enzyme catalyzed the formation of glyoxylate in the resting cell-reaction and thus could be useful in the enzymatic production of glyoxylate.
    Journal of Molecular Catalysis B Enzymatic 07/2014; 105. DOI:10.1016/j.molcatb.2014.03.022 · 2.13 Impact Factor
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    ABSTRACT: L-allo-Threonine aldolase (LATA), a pyridoxal-5'-phosphate-dependent enzyme from Aeromonas jandaei DK-39, stereospecifically catalyzes the reversible interconversion of L-allo-threonine to glycine and acetaldehyde. Here, the crystal structures of LATA and its mutant LATA_H128Y/S292R were determined at 2.59 and 2.50 Å resolution, respectively. Their structures implied that conformational changes in the loop consisting of residues Ala123-Pro131, where His128 moved 4.2 Å outwards from the active site on mutation to a tyrosine residue, regulate the substrate specificity for L-allo-threonine versus L-threonine. Saturation mutagenesis of His128 led to diverse stereoselectivity towards L-allo-threonine and L-threonine. Moreover, the H128Y mutant showed the highest activity towards the two substrates, with an 8.4-fold increase towards L-threonine and a 2.0-fold increase towards L-allo-threonine compared with the wild-type enzyme. The crystal structures of LATA and its mutant LATA_H128Y/S292R reported here will provide further insights into the regulation of the stereoselectivity of threonine aldolases targeted for the catalysis of L-allo-threonine/L-threonine synthesis.
    Acta Crystallographica Section D Biological Crystallography 06/2014; 70(Pt 6):1695-1703. DOI:10.1107/S1399004714007664 · 2.67 Impact Factor
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    ABSTRACT: (R)-3-Quinuclidinol, a useful compound for the synthesis of various pharmaceuticals, can be enantioselectively produced from 3-quinuclidinone by 3-quinuclidinone reductase. Recently, a novel NADH-dependent 3-quinuclidionone reductase (AtQR) was isolated from Agrobacterium tumefaciens, and showed much higher substrate-binding affinity (> 100 fold) than the reported 3-quinuclidionone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of AtQR at 1.72 Å. Three NADH-bound protomers and one NADH-free protomer form a tetrameric structure in an asymmetric unit of crystals. NADH not only acts as a proton donor, but also contributes to the stability of the α7 helix. This helix is a unique and functionally significant part of AtQR and is related to form a deep catalytic cavity. AtQR has all three catalytic residues of the short-chain dehydrogenases/reductases family and the hydrophobic wall for the enantioselective reduction of 3-quinuclidinone as well as RrQR. An additional residue on the α7 helix, Glu197, exists near the active site of AtQR. This acidic residue is considered to form a direct interaction with the amine part of 3-quinuclidinone, which contributes to substrate orientation and enhancement of substrate-binding affinity. Mutational analyses also support that Glu197 is an indispensable residue for the activity.
    Biochemical and Biophysical Research Communications 04/2014; 446(4). DOI:10.1016/j.bbrc.2014.03.030 · 2.30 Impact Factor
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    ABSTRACT: Chiral molecule (R)-3-quinuclidinol, a valuable compound for the production of various pharmaceuticals, is efficiently synthesized from 3-quinuclidinone by using NADPH-dependent 3-quinuclidinone reductase (RrQR) from Rhodotorula rubra. Here, we report the crystal structure of RrQR and the structure-based mutational analysis. The enzyme forms a tetramer, in which the core of each protomer exhibits the alpha/beta Rossmann fold and contains one molecule of NADPH, whereas the characteristic substructures of a small lobe and a variable loop are localized around the substrate-binding site. Modeling and mutation analyses of the catalytic site indicated that the hydrophobicity of two residues, I167 and F212, determines the substrate-binding orientation as well as the substrate-binding affinity. Our results revealed that the characteristic substrate-binding pocket composed of hydrophobic amino acid residues ensures substrate docking for the stereospecific reaction of RrQR in spite of its loose interaction with the substrate.
    AMB Express 02/2014; 4(1):6. DOI:10.1186/2191-0855-4-6
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    ABSTRACT: Conjugated polyketone reductase (CPR-C1) from Candida parapsilosis IFO 0708 is a member of the aldo-keto reductase (AKR) superfamily and reduces ketopantoyl lactone to D-pantoyl lactone in a NADPH-dependent and stereospecific manner. We determined the crystal structure of CPR-C1.NADPH complex at 2.20 Å resolution. CPR-C1 adopted a triose-phosphate isomerase (TIM) barrel fold at the core of the structure in which Thr25 and Lys26 of the GXGTX motif bind uniquely to the adenosine 2'-phosphate group of NADPH. This finding provides a novel structural basis for NADPH binding of the AKR superfamily. © Proteins 2013;. © 2013 Wiley Periodicals, Inc.
    Proteins Structure Function and Bioinformatics 11/2013; 81(11). DOI:10.1002/prot.24363 · 2.63 Impact Factor
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    ABSTRACT: In the representative gut bacterium Lactobacillus plantarum, we identified genes encoding the enzymes involved in a saturation metabolism of polyunsaturated fatty acids and revealed in detail the metabolic pathway that generates hydroxy fatty acids, oxo fatty acids, conjugated fatty acids, and partially saturated trans-fatty acids as intermediates. Furthermore, we observed these intermediates, especially hydroxy fatty acids, in host organs. Levels of hydroxy fatty acids were much higher in specific pathogen-free mice than in germ-free mice, indicating that these fatty acids are generated through polyunsaturated fatty acids metabolism of gastrointestinal microorganisms. These findings suggested that lipid metabolism by gastrointestinal microbes affects the health of the host by modifying fatty acid composition.
    Proceedings of the National Academy of Sciences 10/2013; 110(44). DOI:10.1073/pnas.1312937110 · 9.67 Impact Factor
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    ABSTRACT: Some Prototheca spp. were previously reported to convert n-hexadecane to 5-hexadecanol and then to 5-hexadecanone through a unique subterminal oxidation pathway. Further analysis of derivatives derived from n-hexadecane indicated that Prototheca zopfii oxidized n-alkanes with C11 to C17 chain lengths at not only the 5th but also the 4th, 3rd and 2nd positions.
    Journal of Bioscience and Bioengineering 10/2013; 117(3). DOI:10.1016/j.jbiosc.2013.09.004 · 1.88 Impact Factor
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    ABSTRACT: Asymmetric sulfoxidation of sulfur-containing L-amino acids was successfully achieved through bioconversion using IDO, which is an Fe(II)/alpha-ketoglutarate-dependent dioxygenase previously found in Bacillus thuringiensis strain 2e2. The IDO catalyzed sulfoxidation of L-methionine, L-ethionine, S-methyl-L-cysteine, S-ethyl-L-cysteine, and S-allyl-L-cysteine into the corresponding (S)-configured sulfoxides such as (+)-methiin and (+)-alliin, which are responsible for valuable physiological activities in mammals, and have high stereoselectivity. Herein we have established an effective preparative laboratory scale production method to obtain enantiomerically pure chiral sulfoxides using an IDO biocatalyst.
    Tetrahedron Asymmetry 09/2013; 24(17):990-994. DOI:10.1016/j.tetasy.2013.07.017 · 2.16 Impact Factor
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    ABSTRACT: A succinimide-assimilating bacterium, Pseudomonas putida s52, was found to be a potent producer of pyruvate from fumarate. Using washed cells from P. putida s52 as catalyst, 400 mM pyruvate was produced from 500 mM fumarate in a 36-h reaction. Bromopyruvate, a malic enzyme inhibitor, was used for the selection of mutants with higher pyruvate productivity. A bromopyruvate-resistant mutant, P. putida 15160, was found to be an effective catalyst for pyruvate production. Moreover, under batch bioreactor conditions, 767 mM of pyruvate was successfully produced from 1,000 mM fumarate in a 72-h reaction with washed cells from P. putida 15160 as catalyst.
    Bioscience Biotechnology and Biochemistry 08/2013; 77(8). DOI:10.1271/bbb.130182 · 1.06 Impact Factor
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    ABSTRACT: Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, identified as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ketopantoyl lactone reductase, belongs to the aldo-keto reductase superfamily. This enzyme reduces ketopantoyl lactone to D-pantoyl lactone in a strictly stereospecific manner. To elucidate the structural basis of the substrate specificity, we determined the crystal structures of the apo CPR-C2 and CPR-C2/NADPH complex at 1.70 and 1.80 Å resolutions, respectively. CPR-C2 adopted a triose-phosphate isomerase barrel fold at the core of the structure. Binding with the cofactor NADPH induced conformational changes in which Thr27 and Lys28 moved 15 and 5.0 Å, respectively, in the close vicinity of the adenosine 2'-phosphate group of NADPH to form hydrogen bonds. Based on the comparison of the CPR-C2/NADPH structure with 3-α-hydroxysteroid dehydrogenase and mutation analyses, we constructed substrate binding models with ketopantoyl lactone, which provided insight into the substrate specificity by the cofactor-induced structure. The results will be useful for the rational design of CPR-C2 mutants targeted for use in the industrial manufacture of ketopantoyl lactone.
    Applied Microbiology and Biotechnology 07/2013; 98(1). DOI:10.1007/s00253-013-5073-9 · 3.34 Impact Factor
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    ABSTRACT: A ω3-fatty acid desaturase gene (maw3) which is involved in biosynthesis of n-3 polyunsaturated fatty acids (PUFAs) was previously isolated from Mortierella alpina 1S-4. In this report, we investigated the products of MAW3 catalyzing reaction with endogenous and exogenous fatty acids in the yeast transformant. Two unusual fatty acids de novo synthesized in the yeast transformant expressing maw3 gene were identified as n-4 hexadecadienoic acid (16:2(9cis,12cis)) and n-1 hexadecatrienoic acid (16:3(9cis,12cis,15)) by GC-MS and (1)H NMR analyses. In addition to the desaturation activity at the ω3-position for 18- and 20-carbon PUFAs, MAW3 in the yeast transformant inserted a double bond at Δ12-position of endogenous palmitoleic acid (16:1(9cis)) and further at Δ15-position of the resulting 16:2(9cis,12cis) to result in the formation of 16:3(9cis,12cis,15) leading to a bifunctional Δ12/Δ15-desaturase for 16-carbon fatty acids. Moreover, we evaluated the activity of MAW3 in the yeast transformant under different temperatures. The MAW3 did not have desaturation activities in M. alpina 1S-4 at 28°C but it had in the yeast transformant for various fatty acids. The MAW3 was demonstrated to be a trifunctional Δ12/Δ15/ω3-desaturase, exhibiting Δ12-desaturation for 16:1(9cis), Δ15-desaturation for 16- and 18-carbon fatty acids that had a preexisting cis-double bond at Δ12 position, and ω3-desaturation for 20-carbon fatty acids having that at Δ14-position. It is the first report that the fatty acid desaturase (MAW3) is shown to have Δ12- and Δ15-desaturation activities for a 16-carbon fatty acid, in addition to its major function, ω3-desaturation activity.
    Journal of Bioscience and Bioengineering 06/2013; 116(6). DOI:10.1016/j.jbiosc.2013.05.023 · 1.88 Impact Factor

Publication Stats

9k Citations
892.84 Total Impact Points


  • 2010-2015
    • Kyoto Gakuen University
      Kioto, Kyoto, Japan
  • 1979-2015
    • Kyoto University
      • Division of Applied Life Sciences
      Kioto, Kyōto, Japan
  • 2009
    • Hohenheim University
      Stuttgart, Baden-Württemberg, Germany
    • Georg-August-Universität Göttingen
      • Institute of Microbiology and Genetics
      Göttingen, Lower Saxony, Germany
  • 2003
    • Kinki University
      • Department of Food and Nutrition
      Ōsaka, Ōsaka, Japan
    • Gifu University
      • Department of Chemistry and Biomolecular Science
      Gifu-shi, Gifu-ken, Japan
  • 2001
    • The University of Tokushima
      • Department of Biological Science and Technology
      Tokusima, Tokushima, Japan
  • 1999-2001
    • Okayama University
      • Faculty of Agriculture
      Okayama, Okayama, Japan