Papers - HAYASHI Masahiro
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PUFA synthase-independent DHA synthesis pathway in Parietichytrium sp. and its modification to produce EPA and n-3DPA Reviewed
Yohei Ishibashi, Hatsumi Goda, Rie Hamaguchi, Keishi Sakaguchi, Takayoshi Sekiguchi, Yuko Ishiwata, Yuji Okita, Seiya Mochinaga , Shingo Ikeuchi , Takahiro Mizobuchi , Yoshitake Takao, Kazuki Mori, Kosuke Tashiro, Nozomu Okino, Daiske Honda, Masahiro Hayashi, Makoto Ito
Communications Biology 2021.10
Language：English Publishing type：Research paper (scientific journal)
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Lipid Dloplet Degradation Through Lipophagy in Aurantiochytrium limacinum mh0186 Reviewed
Sheng Wu, Yohei Ishibashi, Masahiro Hayashi, Nozomu Okino
Marine Biotechnology 27 121 - 136 2025.8
Language：English Publishing type：Research paper (scientific journal)
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Solitary and Synergistic Effects of Different Hydrophilic and Hydrophobic Phospholipid Moieties on Rat Behaviors Reviewed International coauthorship
Kikuchi S., Iwasaki Y., Yoshioka M., Hino K., Morita S.Y., Tada R., Uchimura Y., Kubo Y., Kobayashi T., Kinoshita Y., Hayashi M., Furusho Y., Tamiaki H., Ishiyama H., Kuroda M., Udagawa J.
Pharmaceutics 16 ( 6 ) 2024.6
Language：English Publishing type：Research paper (scientific journal) Publisher：Pharmaceutics
Glycerophospholipids have hydrophobic and hydrophilic moieties. Previous studies suggest that phospholipids with different moieties have different effects on rodent behavior; however, the relationship between chemical structures and behavioral effects remains unclear. To clarify the functions of phospholipid moieties, we injected male rats with phospholipids with different moieties and conducted behavioral tests. Exploratory activity was reduced by phosphatidylethanolamine (PE)(18:0/22:6) but not PE(18:0/18:0) or PE(18:0/20:4). Conversely, exploratory activity was increased by plasmanyl PE(16:0/22:6), which harbors an alkyl–ether linkage, but not by phosphatidylcholine (PC)(16:0/22:6) or plasmanyl PC(16:0/22:6). Docosahexaenoic acid (DHA)(22:6) and an alkyl–ether linkage in PE were thus postulated to be involved in exploratory activity. Anxiety-like behavior was reduced by plasmenyl PC(18:0/20:4), which harbors a vinyl–ether linkage, but not by PC(18:0/20:4) or plasmanyl PC(18:0/20:4), suggesting the anxiolytic effects of vinyl–ether linkage. The activation of social interaction was suppressed by PE(18:0/18:0), PE(18:0/22:6), PC(16:0/22:6), plasmanyl PE(16:0/22:6), and plasmanyl PC(16:0/22:6) but not by PE(18:0/20:4), plasmenyl PE(18:0/20:4), or plasmanyl PC(18:0/22:6). DHA may suppress social interaction, whereas arachidonic acid(20:4) or a combination of alkyl–ether linkage and stearic acid(18:0) may restore social deficits. Our findings indicate the characteristic effects of different phospholipid moieties on rat behavior, and may help to elucidate patterns between chemical structures and their effects.
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The Utilization of Heterotrophic Cultivation for Large Scale Production of Microalgae Invited Reviewed
HAYASHI Masahiro
31 ( 1 ) 4 - 7 2024.4
Authorship：Lead author, Corresponding author Language：Japanese Publishing type：Research paper (scientific journal)
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Fukuda M., Kinkawa M., Hayashi M.
Bioscience, Biotechnology and Biochemistry 88 ( 2 ) 206 - 211 2024.4
Authorship：Last author, Corresponding author Language：English Publishing type：Research paper (scientific journal) Publisher：Bioscience, Biotechnology and Biochemistry
Paramylon, a starch-like carbohydrate accumulated in Euglena gracilis cells, could be a potential source as a raw material for chemical products; its high-yield production would thus be highly desired. Although the molecular weight and polymerization degree of paramylon are important properties for its use as a raw material for chemical products, the available information about paramylon molecular weight remains insufficient. Therefore, in this study, we investigated a high-density E. gracilis culture approach and how culture conditions affect paramylon molecular weight. The nitrogen source, cultivation temperature, and nutrient feeding were optimized for maximum biomass and paramylon productivity. The maximum dry cell weight and paramylon content yields reached 108.9 g/L and 87.2%, respectively. Paramylon molecular weight was in the range of 220 000–320 000 Da. Our gel permeation chromatography analysis showed that the cells with a higher paramylon content tended to contain paramylon of a higher molecular weight.
DOI： 10.1093/bbb/zbad152
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Characterization of thraustochytrid-specific sterol O-acyltransferase: modification of DGAT2-like enzyme to increase the sterol production in Aurantiochytrium limacinum mh0186 Reviewed
石橋 洋平, 定光 翔平, 深堀 義朝, タノガシラ リン, 渡邉 昂, 林 雅弘, 伊東 信, 沖野 望
Applied and Environmental Microbiology 89 ( 11 ) 2023.11
Language：English Publishing type：Research paper (scientific journal) Publisher：American Society for Microbiology
Thraustochytrids are marine microorganisms expected to produce useful lipids. They synthesize polyunsaturated fatty acids and sterols and store them in lipid droplets as a form of triacylglycerol (TG) and sterol ester (SE), respectively. TG is synthesized by diacylglycerol O-acyltransferase (DGAT). There are several DGAT2 homologs in Aurantiochytrium limacinum. This study indicated that DGAT2C and DGAT2D are SE synthase and TG synthase, respectively, by disrupting their corresponding genes in A. limacinum mh0186. DGAT2C is revealed as thraustochytrid-specific acyl-CoA:sterol- O-acyltransferase by performing in vivo and in vitro assays after heterologous expression in Saccharomyces cerevisiae. DGAT2C and DGAT2D localized mainly to the endoplasmic reticulum (ER) and the lipid droplet, respectively, and the two of the N-terminal domains unique to DGAT2C were essential for ER localization and SE synthesis in A. limacinum mh0186. Interestingly, the study also found that deletion of the first eight transmembrane domains in the unique N-terminal region of DGAT2C increased SE and total sterol productivity when expressed in DGAT2C-deficient A. limacinum mh0186. In addition, intermediates such as Δ7-cholesterol (provitamin D3) also accumulated in SE by expression of the N-terminal-truncated DGAT2C, along with cholesterol and Δ7-stigmasterol, a major phytosterol in A. limacinum. This study proves that DGAT2C is an ER-localized SE synthase and that its suitable N-terminal deletion can increase sterol production in thraustochytrids.
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Mizuki Fukuda, Manato Kinkawa and Masahiro Hayashi
Bioscience, Biotechnology and Biochemistry 88 ( 2 ) 206 - 211 2023.11
Authorship：Last author, Corresponding author Language：English 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.
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The Utilization of Heterotrophic Cultivation for Large Scale Production of Microalgae Invited Reviewed
HAYASHI Masahiro, MATSUDA Ayako
20 ( 6 ) 37 - 42 2023.9
Authorship：Lead author, Corresponding author Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Japan Oil Chemists' Society
In various industries, practical utilizations of many microalgae are investigated recently. Many problems are recognized for a practical realization of microalgal industry, such as, a choice of cultivation method, autotroph or heterotroph, an effect of sunlight cycle on algal growth, a balance between cell density and efficacy of photosynthesis, an effects of carbon concentration on the growth, a shape and function of culture tank, practical use of extraction residue, molecular breeding and sterilization of the strains.Euglena has unique properties for the cultivation among various microalgae. Euglena can grow autotrophically, heterotrophically, or photoheterotrophically. Although each cultivation method has some merit and demerit, the authors focused on the heterotrophy cultivation, Euglena is industrially cultivated in large scale tanks in the dark.
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The Utilization of Heterotrophic Cultivation for Large Scale Production of<i> Euglena gracilis</i> Invited Reviewed
HAYASHI Masahiro, MATSUDA Ayako, TOKAI Shouta, UKEGAWA Yuino, UDAKA Takaki
Oleoscience 23 ( 2 ) 65 - 72 2023.2
Authorship：Lead author, Corresponding author Language：Japanese Publishing type：Research paper (scientific journal) Publisher：Japan Oil Chemists' Society
In various industries, practical utilizations of many microalgae are investigated recently. Many problems are recognized for a practical realization of microalgal industry, such as, a choice of cultivation method, autotroph or heterotroph, an effect of sunlight cycle on algal growth, a balance between cell density and efficacy of photosynthesis, an effects of carbon concentration on the growth, a shape and function of culture tank, practical use of extraction residue, molecular breeding and sterilization of the strains.Euglena has unique properties for the cultivation among various microalgae. Euglena can grow autotrophically, heterotrophically, or photoheterotrophically. Although each cultivation method has some merit and demerit, the authors focused on the heterotrophy cultivation, Euglena is industrially cultivated in large scale tanks in the dark.
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Ishibashi Y., Sadamitsu S., Fukahori Y., Yamamoto Y., Tanogashira R., Watanabe T., Hayashi M., Ito M., Okino N.
Applied and Environmental Microbiology 89 ( 11 ) 2023
Publishing type：Research paper (scientific journal) Publisher：Applied and Environmental Microbiology
Thraustochytrids are marine microorganisms expected to produce useful lipids. They synthesize polyunsaturated fatty acids and sterols and store them in lipid droplets as a form of triacylglycerol (TG) and sterol ester (SE), respectively. TG is synthesized by diacylglycerol O-acyltransferase (DGAT). There are several DGAT2 homologs in Aurantiochytrium limacinum. This study indicated that DGAT2C and DGAT2D are SE synthase and TG synthase, respectively, by disrupting their corresponding genes in A. limacinum mh0186. DGAT2C is revealed as thraustochytrid-specific acyl-CoA:sterolO-acyltransferase by performing in vivo and in vitro assays after heterologous expression in Saccharomyces cerevisiae. DGAT2C and DGAT2D localized mainly to the endoplasmic reticulum (ER) and the lipid droplet, respectively, and the two of the N-terminal domains unique to DGAT2C were essential for ER localization and SE synthesis in A. limacinum mh0186. Interestingly, the study also found that deletion of the first eight transmembrane domains in the unique N-terminal region of DGAT2C increased SE and total sterol productivity when expressed in DGAT2C-deficient A. limacinum mh0186. In addition, intermediates such as Δ7-cholesterol (provitamin D3) also accumulated in SE by expression of the N-terminal-truncated DGAT2C, along with cholesterol and Δ7-stigmasterol, a major phytosterol in A. limacinum. This study proves that DGAT2C is an ER-localized SE synthase and that its suitable N-terminal deletion can increase sterol production in thraustochytrids.
DOI： 10.1128/aem.01001-23
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有用新奇海洋微生物の探索：ラビリンチュラ Invited Reviewed
林 雅弘, 松田 綾子
遺伝 76 ( 6 ) 428 - 433 2022.11
Authorship：Lead author, Corresponding author Language：Japanese Publishing type：Research paper (scientific journal) Publisher：公益社団法人 日本油化学会
様々な微細藻類の社会実装が検討される中で，独立栄養か従属栄養かの選択，太陽光による光合成への日周サイクルの影響，培養密度と光合成効率のバランス，培養液中の炭素源濃度の影響，培養槽の形状や機能の問題，目的物の抽出残渣処理，株の育種や無菌化など，実用化に向けてクリアすべき問題は多い。微細藻類の中でもユーグレナは特徴的な培養特性を持ち，光合成による独立栄養培養，光合成に加えて有機炭素源を利用した光従属栄養培養，光を利用せず培地中の有機炭素源を利用する従属栄養培養のいずれの培養による増殖も可能である。いずれの培養様式もそれぞれ特徴を持ち，目的や規模に応じて使い分ける必要があるが筆者らのグループは従属栄養培養に焦点を絞り，ユーグレナの工業レベルでの大量生産を行っている。本稿では筆者らが行っているユーグレナの工業レベルの大量培養を1つの題材に，微細藻類の社会実装に向けた大量培養技術について考える。
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海洋微生物ラビリンチュラの食品素材利用 Invited Reviewed
林 雅弘, 松田 綾子、親泊政二三
アグリバイオ 6 ( 10 ) 897 - 901 2022.9
Authorship：Lead author, Corresponding author Language：Japanese Publishing type：Research paper (scientific journal) Publisher：公益社団法人 日本油化学会
様々な微細藻類の社会実装が検討される中で，独立栄養か従属栄養かの選択，太陽光による光合成への日周サイクルの影響，培養密度と光合成効率のバランス，培養液中の炭素源濃度の影響，培養槽の形状や機能の問題，目的物の抽出残渣処理，株の育種や無菌化など，実用化に向けてクリアすべき問題は多い。微細藻類の中でもユーグレナは特徴的な培養特性を持ち，光合成による独立栄養培養，光合成に加えて有機炭素源を利用した光従属栄養培養，光を利用せず培地中の有機炭素源を利用する従属栄養培養のいずれの培養による増殖も可能である。いずれの培養様式もそれぞれ特徴を持ち，目的や規模に応じて使い分ける必要があるが筆者らのグループは従属栄養培養に焦点を絞り，ユーグレナの工業レベルでの大量生産を行っている。本稿では筆者らが行っているユーグレナの工業レベルの大量培養を1つの題材に，微細藻類の社会実装に向けた大量培養技術について考える。
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微細藻類の産業利用ー従属栄養培養の活用ー Invited Reviewed
林 雅弘, 松田 綾子
アクアネット 25 ( 8 ) 32 - 37 2022.8
Authorship：Lead author, Corresponding author Language：Japanese Publishing type：Research paper (scientific journal) Publisher：公益社団法人 日本油化学会
様々な微細藻類の社会実装が検討される中で，独立栄養か従属栄養かの選択，太陽光による光合成への日周サイクルの影響，培養密度と光合成効率のバランス，培養液中の炭素源濃度の影響，培養槽の形状や機能の問題，目的物の抽出残渣処理，株の育種や無菌化など，実用化に向けてクリアすべき問題は多い。微細藻類の中でもユーグレナは特徴的な培養特性を持ち，光合成による独立栄養培養，光合成に加えて有機炭素源を利用した光従属栄養培養，光を利用せず培地中の有機炭素源を利用する従属栄養培養のいずれの培養による増殖も可能である。いずれの培養様式もそれぞれ特徴を持ち，目的や規模に応じて使い分ける必要があるが筆者らのグループは従属栄養培養に焦点を絞り，ユーグレナの工業レベルでの大量生産を行っている。本稿では筆者らが行っているユーグレナの工業レベルの大量培養を1つの題材に，微細藻類の社会実装に向けた大量培養技術について考える。
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Ishibashi Yohei, Goda Hatsumi, Hamaguchi Rie, Sakaguchi Keishi, Sekiguchi Takayoshi, Ishiwata Yuko, Okita Yuji, Mochinaga Seiya, Ikeuchi Shingo, Mizobuchi Takahiro, Takao Yoshitake, Mori Kazuki, Tashiro Kosuke, Okino Nozomu, Honda Daiske, Hayashi Masahiro, Ito Makoto
Communications Biology 4 ( 1 ) 1378 2021.12
Language：English Publishing type：Research paper (scientific journal)
The demand for n-3 long-chain polyunsaturated fatty acids (n-3LC-PUFAs), such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), will exceed their supply in the near future, and a sustainable source of n-3LC-PUFAs is needed. Thraustochytrids are marine protists characterized by anaerobic biosynthesis of DHA via polyunsaturated fatty acid synthase (PUFA-S). Analysis of a homemade draft genome database suggested that Parietichytrium sp. lacks PUFA-S but possesses all fatty acid elongase (ELO) and desaturase (DES) genes required for DHA synthesis. The reverse genetic approach and a tracing experiment using stable isotope-labeled fatty acids revealed that the ELO/DES pathway is the only DHA synthesis pathway in Parietichytrium sp. Disruption of the C20 fatty acid ELO (C20ELO) and ∆4 fatty acid DES (∆4DES) genes with expression of ω3 fatty acid DES in this thraustochytrid allowed the production of EPA and n-3docosapentaenoic acid (n-3DPA), respectively, at the highest level among known microbial sources using fed-batch culture.
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C4-monomethylsterol β-glucoside and its synthase in Aurantiochytrium limacinum mh0186 Reviewed
Endo I., Watanabe T., Miyamoto T., Monjusho-Goda H., Ohara J., Hayashi M., Hama Y., Ishibashi Y., Okino N., Ito M.
Glycobiology 31 ( 10 ) 1350 - 1363 2021.10
Language：English Publishing type：Research paper (scientific journal) Publisher：Glycobiology
Thraustochytrids, unicellular marine protists, synthesize polyunsaturated fatty acids (PUFAs) and PUFA-containing phospholipids; however, little is known about their glycolipids and their associated metabolism. Here, we report two glycolipids (GL-A, B) and their synthases in Aurantiochytrium limacinum mh0186. Two glycolipids were purified from A. limacinum mh0186, and they were determined by gas chromatography, mass spectrometry and 2D nuclear magnetic resonance to be 3-O-β-D-glucopyranosyl-stigmasta-5,7,22-triene (GL-A) and 3-O-β-D-glucopyranosyl-4α-methyl-stigmasta-7,22-diene (GL-B), both of which are sterol β-glucosides (β-SGs); the structure of GL-B has not been reported thus far. Seven candidate genes responsible for the synthesis of these β-SGs were extracted from the draft genome database of A. limacinum using the yeast sterol β-glucosyltransferase (SGT; EC 2.4.1.173) sequence as a query. Expression analysis using Saccharomyces cerevisiae revealed that two gene products (AlSGT-1 and 2) catalyze the transfer of glucose from uridine diphosphate (UDP)-glucose to sterols, generating sterylglucosides (SGs). Compared to AlSGT-1, AlSGT-2 exhibited wide specificity for sterols and used C4-monomethylsterol to synthesize GL-B. The disruption of alsgt-2 but not alsgt-1 in strain mh0186 resulted in a decrease in the total SG and an almost complete loss of GL-B, indicating that AlSGT-2 is responsible for the synthesis of β-SGs in A. limacinum mh0186, especially GL-B, which possesses a unique sterol structure.
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Ishibashi Y., Aoki K., Okino N., Hayashi M., Ito M.
Scientific Reports 9 ( 1 ) 2019.12
Language：English Publishing type：Research paper (scientific journal) Publisher：Scientific Reports
© 2019, The Author(s). Thraustochytrids are heterotrophic marine protists that are considered as important decomposers in the marine ecosystem; however, how they digest and uptake lipid nutrients from the environment is largely unknown. Genomic clustering analysis using thraustochytrid draft genome databases revealed that novel proteins with a Lipase_3 domain are commonly present in thraustochytrids, including Aurantiochytrium limacinum. After heterologous expression and His tag-based purification, protein ID: 145138 was identified as lipase/phospholipase capable of hydrolyzing triacylglycerol (TG) and phosphatidylcholine (PC). 145138 was secreted into the medium, and deletion of the 145138 gene in A. limacinum reduced the degradation of extracellular lipids. Fatty acids generated by 145138 were reused for the biosynthesis of PC and TG, and 145138 allowed A. limacinum to survive in the medium containing TG as a sole carbon source. 145138 hydrolyzed all the acyl-ester linkages of TG; however, the enzyme showed strict positional specificity toward phospholipids, generating 2-acyl lysophospholipids. The 2-acyl lysophospholipids showed stronger antimicrobial activity compared with 1-acyl lysophospholipids. These results suggested that 145138 is a bifunctional enzyme that contributes to the acquisition of lipid nutrients from the environment, as well as to generate antimicrobial lysophospholipids that are beneficial for competition with bacteria over lipid nutrients in the marine environment.
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バイオ燃料の社会実装に向けた技術的課題とおの実現可能性 Invited Reviewed
平川拓弥、林 雅弘
応用細胞生物学研究 32 ( 1 ) 11 - 34 2019.5
Authorship：Corresponding author Language：Japanese Publishing type：Research paper (scientific journal)
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物質生産ツールとしてのユーグレナの可能性および生産性向上を目指した高密度培養技術 Invited Reviewed
山嵜真彩、林 雅弘
応用細胞生物学研究 32 ( 1 ) 35 - 54 2019.5
Authorship：Corresponding author Language：Japanese Publishing type：Research paper (scientific journal)
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Eri Nutahara, Eriko Abe, Shinya Uno, Yohei Ishibashi, Takashi Watanabe, Masahiro Hayashi, Nozomu Okino, Makoto Ito
PLOS ONE ( 1 ) 2019.4
Language：English Publishing type：Research paper (scientific journal)
© 2019, The Author(s). Thraustochytrids are heterotrophic marine protists that are considered as important decomposers in the marine ecosystem; however, how they digest and uptake lipid nutrients from the environment is largely unknown. Genomic clustering analysis using thraustochytrid draft genome databases revealed that novel proteins with a Lipase_3 domain are commonly present in thraustochytrids, including Aurantiochytrium limacinum. After heterologous expression and His tag-based purification, protein ID: 145138 was identified as lipase/phospholipase capable of hydrolyzing triacylglycerol (TG) and phosphatidylcholine (PC). 145138 was secreted into the medium, and deletion of the 145138 gene in A. limacinum reduced the degradation of extracellular lipids. Fatty acids generated by 145138 were reused for the biosynthesis of PC and TG, and 145138 allowed A. limacinum to survive in the medium containing TG as a sole carbon source. 145138 hydrolyzed all the acyl-ester linkages of TG; however, the enzyme showed strict positional specificity toward phospholipids, generating 2-acyl lysophospholipids. The 2-acyl lysophospholipids showed stronger antimicrobial activity compared with 1-acyl lysophospholipids. These results suggested that 145138 is a bifunctional enzyme that contributes to the acquisition of lipid nutrients from the environment, as well as to generate antimicrobial lysophospholipids that are beneficial for competition with bacteria over lipid nutrients in the marine environment.
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食糧資源としてのユーグレナの可能性を広げるために Invited Reviewed
長岡 綾、林 雅弘
応用細胞生物学研究 30 ( 1 ) 7 - 12 2018.4
Authorship：Corresponding author Language：Japanese Publishing type：Research paper (scientific journal)