Sakayu Shimizu

Kyoto Gakuen University, Kioto, Kyōto, Japan

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Publications (284)591.13 Total impact

  • [Show abstract] [Hide abstract]
    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; · 2.20 Impact Factor
  • Advanced Synthesis & Catalysis 11/2014; · 5.54 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; · 1.74 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.
    Journal of lipid research. 07/2014;
  • Journal of Molecular Catalysis B Enzymatic 07/2014; · 2.82 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. · 12.67 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.
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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 01/2014; · 2.28 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; · 9.81 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; · 1.74 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; · 1.27 Impact Factor
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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 07/2013; · 3.34 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; · 3.81 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; · 1.74 Impact Factor
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    ABSTRACT: Some Prototheca sp. are known to be involved in n-hexadecane degradation. Two derivatives derived from n-hexadecane in such Prototheca sp. were identified as 5-hexadecanone and 5-hexadecanol. n-Hexadecane was assumed to be converted to 5-hexadecanol and then to 5-hexadecanone through a unique subterminal oxidation pathway in such Prototheca sp.
    Journal of Bioscience and Bioengineering 05/2013; · 1.74 Impact Factor
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    ABSTRACT: Researches related with the application of functional lipids such as polyunsaturated fatty acids (PUFAs) have been conducted in various fields with a view to health and dietary requirements. Novel rich sources other than known natural sources such as plant seeds and fish oils are required for increasing demands of PUFAs. The filamentous fungus Mortierella alpina 1S-4 produces triacylglycerols rich in arachidonic acid, i.e., ones reaching 20 g/l in concentration and containing 30-70% arachidonic acid as total fatty acids. Various mutants derived from M. alpina 1S-4 have led to the production of oils containing various PUFAs. Molecular breeding of M. alpina strains by means of manipulation of the genes involved in PUFA biosynthesis facilitates improvement of PUFA productivity and elucidation of the functions of their enzymes. This review describes practical PUFA production through mutant breeding, functional analyses of the genes of the enzymes involved in PUFA biosynthesis, and recent advances in unique PUFA production through molecular breeding.
    Journal of Bioscience and Bioengineering 05/2013; · 1.74 Impact Factor
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    ABSTRACT: A unique operon structure has been identified in the genomes of several plant- and insect-associated bacteria. The distinguishing feature of this operon is the presence of tandem hilA and hilB genes encoding dioxygenases belonging to the PF13640 and PF10014 (BsmA) Pfam families, respectively. The genes encoding HilA and HilB from Pantoea ananatis AJ13355 were cloned and expressed in Escherichia coli. The culturing of E. coli cells expressing hilA (E. coli-HilA) or both hilA and hilB (E. coli-HilAB) in the presence of l-isoleucine resulted in the conversion of l-isoleucine into two novel biogenic compounds: l-4'-isoleucine and l-4,4'-dihydroxyisoleucine, respectively. In parallel, two novel enzymatic activities were detected in the crude cell lysates of the E. coli-HilA and E. coli-HilAB strains: l-isoleucine, 2-oxoglutarate: oxygen oxidoreductase (4'-hydroxylating) (HilA) and l-4'-hydroxyisoleucine, 2-oxoglutarate: oxygen oxidoreductase (4-hydroxylating) (HilB), respectively. Two hypotheses regarding the physiological significance of C-4(4')-hydroxylation of l-isoleucine in bacteria are also discussed. According to first hypothesis, the l-isoleucine dihydroxylation cascade is involved in synthesis of dipeptide antibiotic in P. ananatis. Another unifying hypothesis is that the C-4(4')-hydroxylation of l-isoleucine in bacteria could result in the synthesis of signal molecules belonging to two classes: 2(5H)-furanones and analogs of N-acyl homoserine lactone.
    MicrobiologyOpen. 04/2013;
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    ABSTRACT: Through the screening of about 300 strains of lactic acid bacteria, Pediococcus sp. AKU 1080 was selected as a strain with the ability to hydrate linoleic acid (cis-9,cis-12-octadecadienoic acid) to three hydroxy fatty acids, i.e., 10-hydroxy-cis-12-octadecenoic acid, 13-hydroxy-cis-9-octadecenoic acid, and 10,13-dihydroxyoctadecanoic acid. The strain hydrated one of two cis double bonds at Δ9 and Δ12 positions to produce 10-hydroxy-cis-12-octadecenoic acid and 13-hydroxy-cis-9-octadecenoic acid, respectively, then further hydrated these two mono-hydroxy fatty acids to 10,13-dihydroxyoctadecanoic acid. The growing cells of this strain were applied to the production of 13-hydroxy-cis-9-octadecenoic acid, that is potential as polymer substrates and functional foods but its specific and efficient production was not established. Under the optimum conditions, 2.3 mg/mL of 13-hydroxy-cis-9-octadecenoic acid was produced from 12.3 mg/mL of linoleic acid with 0.04 mg/mL 10-hydroxy-cis-12-octadecenoic acid (HYA) and 0.05 mg/mL 10,13-dihydroxyoctadecanoic acid in the cultivation medium. Specific production of 13-hydroxy-cis-9-octadecenoic acid was attained using cell-free extracts of the strain as the catalyst. Under the optimum conditions, 0.4 mg/mL of 13-hydroxy-cis-9-octadecenoic acid was produced from 2.0 mg/mL of linoleic acid without HYA and 10,13-dihydroxyoctadecanoic acid. Practical applications: Hydroxy fatty acids are useful as starting materials for industrial chemicals, functional foods, and pharmaceuticals. Regioselective introduction of hydroxyl group to unsaturated fatty acids by microorganisms was applied to hydroxy fatty acid production. Especially, specific production of 13-hydroxy-cis-9-octadecenoic acid, which is useful for the production of 13-oxo-fatty acids with anti-obesity activity, was established in this work.
    European Journal of Lipid Science and Technology 04/2013; 115(4). · 2.27 Impact Factor
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    ABSTRACT: (R)-3-Quinuclidinol is a useful chiral building block for the synthesis of various pharmaceuticals and can be produced from 3-quinuclidinone by asymmetric reduction. A novel 3-quinuclidinone reductase from Agrobacterium tumefaciens (AtQR) catalyzes the stereospecific reduction of 3-quinuclidinone to (R)-3-quinuclidinol with NADH as a cofactor. Recombinant AtQR was overexpressed in Escherichia coli, purified and crystallized with NADH using the sitting-drop vapour-diffusion method at 293 K. Crystals were obtained using a reservoir solution containing PEG 3350 as a precipitant. X-ray diffraction data were collected to 1.72 Å resolution on beamline BL-5A at the Photon Factory. The crystal belonged to space group P2(1), with unit-cell parameters a = 62.0, b = 126.4, c = 62.0 Å, β = 110.5°, and was suggested to contain four molecules in the asymmetric unit (V(M) = 2.08 Å(3) Da(-1)).
    Acta Crystallographica Section F Structural Biology and Crystallization Communications 10/2012; 68(Pt 10):1237-9. · 0.55 Impact Factor
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    ABSTRACT: Microorganisms are promising as producers of various polyunsaturated fatty acids (PUFAs) and as catalysts transforming them into unique molecular species beyond common PUFAs. This article describes PUFA production through chemical mutant- and molecular-breeding of an oleaginous filamentous fungus Mortierella alpina 1S-4 and PUFA transformation by anaerobic bacteria. M. alpina 1S-4 and its mutants and transformants produce oils containing not only common n − 6 and n − 3 PUFAs but also rare PUFAs. Unique PUFA-transforming activities were found in anaerobic bacteria. They isomerized PUFA to conjugated fatty acids and further transformed to partially saturated fatty acids with hydroxyl fatty acids as intermediates. The functions of these unique PUFAs have been attracting much attention for improving our health and for developing new chemical materials.
    European Journal of Lipid Science and Technology 10/2012; 114(10). · 2.27 Impact Factor

Publication Stats

3k Citations
591.13 Total Impact Points

Institutions

  • 2010–2014
    • Kyoto Gakuen University
      Kioto, Kyōto, Japan
  • 1979–2014
    • Kyoto University
      • • Division of Applied Life Sciences
      • • Graduate School of Agriculture / Faculty of Agriculture
      • • Graduate School of Biostudies
      Kioto, Kyōto, Japan
  • 2009–2013
    • The University of Tokyo
      • Department of Applied Biological Chemistry
      Tokyo, Tokyo-to, Japan
    • NAGASE & CO., LTD.
      Edo, Tōkyō, Japan
    • Hohenheim University
      Stuttgart, Baden-Württemberg, Germany
    • AJINOMOTO CO., INC.
      Edo, Tōkyō, Japan
    • Toyota Central R & D Labs., Inc.
      Nagoya, Aichi, Japan
  • 2007–2013
    • Ajinomoto-Genetika Research Institute
      Moskva, Moscow, Russia
  • 2007–2012
    • Iwate University
      • • United Graduate School of Agricultural Sciences
      • • Department of Biological Chemistry and Food Sciences
      • • Department of Agro-bioscience
      Morioka, Iwate, Japan
  • 2008
    • Spanish National Research Council
      • Instituto de Química Orgánica General
      Madrid, Madrid, Spain
  • 2004
    • Osaka Municipal Technical Research Institute
      Ōsaka, Ōsaka, Japan
    • Toyama Prefectural University
      • Biotechnology Research Center
      Toyama-shi, Toyama-ken, Japan
  • 2003
    • Gifu University
      • Department of Chemistry and Biomolecular Science
      Gifu-shi, Gifu-ken, Japan
  • 1995–2003
    • Kinki University
      • • Department of Food and Nutrition
      • • Faculty of Agriculture
      Ōsaka, Ōsaka, Japan
  • 2002
    • University of Tsukuba
      • Institute of Applied Biochemistry
      Tsukuba, Ibaraki, Japan
  • 1999
    • Fukui Prefectural University
      Kioto, Kyōto, Japan
  • 1998–1999
    • Tottori University
      • Department of Biotechnology
      Tottori, Tottori-ken, Japan