Papers - HAYASHI Masahiro
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ミドリムシプラスチックの開発 Invited Reviewed
芝上基成、林 雅弘
機能材料 33 ( 8 ) 9 - 13 2013.8
Authorship：Corresponding author Language：Japanese Publishing type：Research paper (other academic)
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Polysaccharide nanofiber made from euglenoid alga Reviewed
Motonari Shibakami, Gen Tsubouchi, Makoto Nakamura, Masahiro Hayashi
Carbohydrate Polymers 98 ( 3 ) 499 - 505 2013.4
Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)
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毎洋性微生物資源を活用するオメガ3脂防酸素材の生産 Invited Reviewed
林 雅弘
Bioindustry 30 ( 2 ) 25 - 31 2013.2
Authorship：Lead author, Corresponding author Language：Japanese Publishing type：Research paper (scientific journal)
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Preparation of carboxylic acid-bearing polysaccharide nanofiber made from euglenoid beta-1,3-glucans Reviewed
Motonari Shibakami, Gen Tsubouchi, Makoto Nakamura, Masahiro Hayashi
Carbohydrate Polymers 98 ( 1 ) 95 - 101 2013.1
Authorship：Corresponding author Language：English Publishing type：Research paper (scientific journal)
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藻類を活用する食品素材開発(<特集>美味しい健康生活は微生物が作る～作物生産,食品素材開発,健康支援～)
林 雅弘
生物工学会誌 : seibutsu-kogaku kaishi 91 ( 11 ) 621 - 624 2013
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：日本生物工学会
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シオミズツボワムシ中の DHA 量がマダイ種苗生産飼育成績に与える効果
小谷 知也, 伏見 浩, 太田 有香, 宮嶋 暁, 須藤 健介, 林 雅弘, 佐藤 信光, 佐藤 秀一
水産増殖 61 ( 4 ) 321 - 330 2013
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：水産増殖談話会
本研究はマダイ種苗飼育成績改善を目的として，ワムシ栄養強化剤中の DHA 量について検討した。DHA 量の異なる4種類の栄養強化剤（0～60%）および市販強化剤を用いてワムシを強化した。強化剤中の総脂肪酸中 DHA 量が20%以上の時，ワムシ単独給餌期の仔魚の成長を改善できたが，アルテミアの給餌が始まった後，強化剤中 DHA 量が60%であった試験区の魚の成長が低下した。したがって，マダイ種苗生産ではワムシ強化剤中の DHA 量は総脂肪酸中20あるいは40%が適していると考えられる。
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Matsuda T., Sakaguchi K., Hamaguchi R., Kobayashi T., Abe E., Hama Y., Hayashi M., Honda D., Okita Y., Sugimoto S., Okino N., Ito M.
Journal of Lipid Research 53 ( 6 ) 1210 - 1222 2012.6
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Lipid Research
Thraustochytrids are known to synthesize PUFAs such as docosahexaenoic acid (DHA). Accumulating evidence suggests the presence of two synthetic pathways of PUFAs in thraustochytrids: the polyketide synthase-like (PUFA synthase) and desaturase/elongase (standard) pathways. It remains unclear whether the latter pathway functions in thraustochytrids. In this study, we report that the standard pathway produces PUFA in Thraustochytrium aureum ATCC 34304. We isolated a gene encoding a putative Δ12-fatty acid desaturase (TauΔ12des) from T. aureum. Yeasts transformed with the tauΔ12des converted endogenous oleic acid (OA) into linoleic acid (LA). The disruption of the tauΔ12des in T. aureum by homologous recombination resulted in the accumulation of OA and a decrease in the levels of LA and its downstream PUFAs. However, the DHA content was increased slightly in tauΔ12des -disruption mutants, suggesting that DHA is primarily produced in T. aureum via the PUFA synthase pathway. The transformation of the tauΔ12des -disruption mutants with a tauΔ12des expression cassette restored the wild-type fatty acid profiles. These data clearly indicate that TauΔ12des functions as Δ12-fatty acid desaturase in the standard pathway of T. aureum and demonstrate that this thraustochytrid produces PUFAs via both the PUFA synthase and the standard pathways. Copyright © 2012 by the American Society for Biochemistry and Molecular Biology, Inc.
DOI： 10.1194/jlr.M024935
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Sakaguchi K., Matsuda T., Kobayashi T., Ohara J., Hamaguchi R., Abe E., Nagano N., Hayashi M., Ueda M., Honda D., Okita Y., Taoka Y., Sugimoto S., Okino N., Ito M.
Applied and Environmental Microbiology 78 ( 9 ) 3193 - 3202 2012.5
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Applied and Environmental Microbiology
A versatile transformation system for thraustochytrids, a promising producer for polyunsaturated fatty acids and fatty acid-derived fuels, was established. G418, hygromycin B, blasticidin, and zeocin inhibited the growth of thraustochytrids, indicating that multiple selectable marker genes could be used in the transformation system. A neomycin resistance gene (neo r ), driven with an ubiquitin or an EF-1α promoter-terminator from Thraustochytrium aureum ATCC 34304, was introduced into representatives of two thraustochytrid genera, Aurantiochytrium and Thraustochytrium. The neo r marker was integrated into the chromosomal DNA by random recombination and then functionally translated into neo r mRNA. Additionally, we confirmed that another two genera, Parietichytrium and Schizochytrium, could be transformed by the same method. By this method, the enhanced green fluorescent protein was functionally expressed in thraustochytrids. Meanwhile, T. aureum ATCC 34304 could be transformed by two 18S ribosomal DNA-targeting vectors, designed to cause single- or double-crossover homologous recombination. Finally, the fatty acid Δ5 desaturase gene was disrupted by double-crossover homologous recombination in T. aureum ATCC 34304, resulting in an increase of dihomo-γ-linolenic acid (C 20:3n-6 ) and eicosatetraenoic acid (C 20:4n-3 ), substrates for Δ5 desaturase, and a decrease of arachidonic acid ( C20:4n-6 ) and eicosapentaenoic acid (C 20:5n-3 ), products for the enzyme. These results clearly indicate that a versatile transformation system which could be applicable to both multiple transgene expression and gene targeting was established for thraustochytrids. © 2012, American Society for Microbiology.
DOI： 10.1128/AEM.07129-11
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Fabrication of Doughnut-Shaped Particles from Spheroidal Paramylon Granules of Euglena gracilis using Acetylation Reaction Reviewed
Motonari Shibakami, Mitsugu Sohma, Masahiro Hayashi
Carbohydrate Polymers 87 452 - 456 2012.4
Language：English Publishing type：Research paper (scientific journal)
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Analysis of delta-12-fatty acid desaturase function revealed that two distinct pathways are active for the synthesis of PUFAs in T. aureum ATCC 34304 Reviewed
Takanori Matsuda , Keishi Sakaguchi , Rie Hamaguchi , Takumi Kobayashi , Eriko Abe , Yoichiro Hama , Masahiro Hayashi , Daiske Honda ,Yuji Okita , Shinichi Sugimoto, Nozomu Okino,Makoto Ito
Journal of Lipid Research 53 1210 - 1222 2012.4
Language：English Publishing type：Research paper (scientific journal)
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Matsuda F., Hayashi M., Kondo A.
Bioscience, Biotechnology and Biochemistry 75 ( 11 ) 2253 - 2256 2011.11
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Bioscience, Biotechnology and Biochemistry
Comparative metabolic profiling analysis was performed to investigate light- and aeration-dependent regulation of central metabolism in Euglena gracilis. The metabolic profiles of E. gracilis were significantly altered in response to changes in aeration conditions. While many glycolytic intermediates and amino acids accumulated in aerobically grown E. gracilis, a significant reduction in these metabolites was observed for cells under anaerobic conditions, which resulted in elevated production of wax ester.
DOI： 10.1271/bbb.110482
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Matsuda Takanori, Sakaguchi Keishi, Kobayashi Takumi, ABE Eriko, KURANO Norihide, SATO Akira, OKITA Yuji, SUGIMOTO Shinichi, HAMA Yoichiro, HAYASHI Masahiro, OKINO Nozomu, ITO Makoto
The journal of biochemistry 150 ( 4 ) 375 - 383 2011.10
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Japanese Biochemical Society
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Nagano N., Sakaguchi K., Taoka Y., Okita Y., Honda D., Ito M., Hayashi M.
Journal of Oleo Science 60 ( 9 ) 475 - 481 2011.8
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Journal of Oleo Science
Heterotrophic marine protists known as thraustochytrids can synthesize polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA). The biosynthetic pathways of PUFAs in thraustochytrids are poorly understood, however. In this study, we attempted to reveal the enzymes involved in DHA synthesis in thraustochytrids. Nine thraustochytrid strains representing 3 genera (Aurantiochytrium, Schizochytrium, and Thraustochytrium) were used for PCR-based detection of the genes encoding Δ5-elongase and Δ4-desaturase and for fatty acid analysis. The degenerate primers were designed to amplify the Δ5-elongase and Δ4-desaturase genes, and the partial sequences of the enzymes were obtained from the genera Thraustochytrium and Schizochytrium. These fragments were identical to those of known Δ5-elongase and Δ4-desaturase. Neither Δ5-elongase nor Δ4-desaturase was detected in the strains belonging to the genus Aurantiochytrium, however, suggesting that this group likely synthesizes DHA not via the elongation/desaturation pathway but via an alternate pathway such as the polyketide synthase pathway. The fatty acid profiles of thraustochytrids were consistent with the presence of genes involved in PUFA biosynthesis in thraustochytrid genera. Thus, our fi ndings suggest that two biosynthetic pathways for PUFAs exist in these organisms. © 2011 by Japan Oil Chemists' Society.
DOI： 10.5650/jos.60.475
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ラビリンチュラ類の脂質蓄積関連タンパク質の単離と機能解析 Reviewed
山元悠樹、小林 巧、安部英理子、坂口圭史、沖野 望、林 雅弘、沖田裕司 、杉本愼一、伊東 信
脂質生化学論文集 2011.7
Language：Japanese Publishing type：Research paper (scientific journal)
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The Distribution of Extracellular Cellulase Activity in Marine Eukaryotes, Thraustochytrids Reviewed
Naoki Nagano, Yousuke Taoka, Yuji Okita, Hitoshi Izumida, Shinichi Sugimoto, Masahiro Hayashi
Marine Biotechnology 13 133 - 136 2011.6
Language：English Publishing type：Research paper (scientific journal)
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Molecular cloning of a Pinguiochrysis pyriformis oleate-specific microsomal Δ12-fatty acid desaturase and functional analysis in yeasts and thraustochytrids Reviewed
Matsuda T, Sakaguchi K, Kobayashi T, Abe E, Kurano N, Sato A, Okita Y, Sugimoto S, Hama Y, Hayashi M, Okino N, Ito M.
Journal of Biochemistry 150 2011.6
Language：English Publishing type：Research paper (scientific journal)
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Kobayashi T., Sakaguchi K., Matsuda T., Abe E., Hama Y., Hayashi M., Honda D., Okita Y., Sugimoto S., Okino N., Ito M.
Applied and Environmental Microbiology 77 ( 11 ) 3870 - 3876 2011.6
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Applied and Environmental Microbiology
Thraustochytrids, marine protists known to accumulate polyunsaturated fatty acids (PUFAs) in lipid droplets, are considered an alternative to fish oils as a source of PUFAs. The major fatty acids produced in thraustochytrids are palmitic acid (C 16:0 ), n - 6 docosapentaenoic acid (DPA) (C 22:5n - 6 ), and docosahexaenoic acid (DHA) (C 22:6n - 3), with eicosapentaenoic acid (EPA) (C 20:5n - 3) and arachidonic acid (AA) (C 20:4n - 6 ) as minor constituents. We attempted here to alter the fatty acid composition of thraustochytrids through the expression of a fatty acid Δ5 desaturase gene driven by the thraustochytrid ubiquitin promoter. The gene was functionally expressed in Aurantiochytrium limacinum mh0186, increasing the amount of EPA converted from eicosatetraenoic acid (ETA) (C 20:4n - 3 ) by the Δ5 desaturase. The levels of EPA and AA were also increased by 4.6and 13.2-fold in the transgenic thraustochytrids compared to levels in the mock transfectants when ETA and dihomo-γ-linolenic acid (DGLA) (C 20:3n - 6) were added to the culture at 0.1 mM. Interestingly, the amount of EPA in the transgenic thraustochytrids increased in proportion to the amount of ETA added to the culture up to 0.4 mM. The rates of conversion and accumulation of EPA were much higher in the thraustochytrids than in baker's yeasts when the desaturase gene was expressed with the respective promoters. This report describes for the first time the finding that an increase of EPA could be accomplished by introducing the Δ5 desaturase gene into thraustochytrids and indicates that molecular breeding of thraustochytrids is a promising strategy for generating beneficial PUFAs. © 2011, American Society for Microbiology.
DOI： 10.1128/AEM.02664-10
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The Distribution of Extracellular Cellulase Activity in Marine Eukaryotes, Thraustochytrids
Naoki Nagano, Yousuke Taoka, Yuji Okita, Hitoshi Izumida, Shinichi Sugimoto, Masahiro Hayashi
Marine Biotechnology 13 133 - 136 2011.6
Language：Japanese Publishing type：Research paper (scientific journal)
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Effects of cold shock teratment on total lipid content and fatty acid composition of Aurantiochytrium limacinum strain mh0186 Reviewed
Taoka, Y., Nagano, N., Okita, Y., Izumida, H., Sugimoto, S., Hayashi, M.
Journal of Oleo Science 60 ( 5 ) 217 - 220 2011.5
Language：English Publishing type：Research paper (scientific journal)
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Taoka Yousuke, Nagano Naoki, Okita Yuji, IZUMIDA Hitoshi, SUGIMOTO Shinichi, HAYASHI Masahiro
Journal of bioscience and bioengineering 111 ( 4 ) 420 - 424 2011.4
Language：Japanese Publishing type：Research paper (scientific journal) Publisher：公益社団法人日本生物工学会
Thraustochytrium aureum ATCC 34304 was grown in the presence and absence of polyoxyethylene sorbitan monooleate (Tween 80). The aim of this work was to obtain basic knowledge about the effect of Tween 80 on growth, lipid accumulation and fatty acid composition in T. aureum. The addition of Tween 80 to a culture medium significantly enhanced the growth of T. aureum, and the biomass increased with an increase of Tween 80 content. Total lipid content and total fatty acid content were significantly higher in 1.0% Tween 80 in comparison with the control (absence of Tween 80). The fatty acid profile showed that the content of C18:1n-9 (oleicacid) significantly increased as a result of the addition of Tween 80. These results indicated that part of the Tween 80 added to the medium was utilized as a carbon source or that the oleate included in Tween 80 was directly incorporated into T. aureum cells as a fatty acid. Neither the DHA content nor the percentage of DHA did not change in spite of the addition of Tween 80. However, the DHA yield significantly increased because the biomass increased due to the addition of Tween 80.