| Yohei Ishibashi | Last modified date：2024.04.15 |
Assistant Professor /
Molecular Biosciences /
Department of Bioscience and Biotechnology /
Faculty of Agriculture
Papers
| 1. | Yohei Ishibashi, Shohei Sadamitsu, Yoshitomo Fukahori, Yuki Yamamoto, Rin Tanogashira, Takashi Watanabe, Masahiro Hayashi, Makoto Ito, Nozomu Okino, Characterization of thraustochytrid-specific sterol O-acyltransferase: modification of DGAT2-like enzyme to increase the sterol production in Aurantiochytrium limacinum mh0186., Applied and environmental microbiology, 10.1128/aem.01001-23, e0100123, 2023.10, 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. IMPORTANCE Since the global market for sterols and vitamin D are grown with a high compound annual growth rate, a sustainable source of these compounds is required to keep up with the increasing demand. Thraustochytrid is a marine oleaginous microorganism that can synthesize several sterols, which are stored as SE in lipid droplets. DGAT2C is an unconventional SE synthase specific to thraustochytrids. Although the primary structure of DGAT2C shows high similarities with that of DGAT, DGAT2C utilizes sterol as an acceptor substrate instead of diacylglycerol. In this study, we examined more detailed enzymatic properties, intracellular localization, and structure-activity relationship of DGAT2C. Furthermore, we successfully developed a method to increase sterol and provitamin D3 productivity of thraustochytrid by more than threefold in the process of elucidating the function of the DGAT2C-specific N-terminal region. Our findings could lead to sustainable sterol and vitamin D production using thraustochytrid.. |
| 2. | Shizuka Fukuda, Yushi Kono, Yohei Ishibashi, Mitsuaki Tabuchi, Motohiro Tani, Impaired biosynthesis of ergosterol confers resistance to complex sphingolipid biosynthesis inhibitor aureobasidin A in a PDR16-dependent manner., Scientific reports, 10.1038/s41598-023-38237-z, 13, 1, 11179-11179, 2023.07, Complex sphingolipids and sterols are coordinately involved in various cellular functions, e.g. the formation of lipid microdomains. Here we found that budding yeast exhibits resistance to an antifungal drug, aureobasidin A (AbA), an inhibitor of Aur1 catalyzing the synthesis of inositolphosphorylceramide, under impaired biosynthesis of ergosterol, which includes deletion of ERG6, ERG2, or ERG5 involved in the final stages of the ergosterol biosynthesis pathway or miconazole; however, these defects of ergosterol biosynthesis did not confer resistance against repression of expression of AUR1 by a tetracycline-regulatable promoter. The deletion of ERG6, which confers strong resistance to AbA, results in suppression of a reduction in complex sphingolipids and accumulation of ceramides on AbA treatment, indicating that the deletion reduces the effectiveness of AbA against in vivo Aur1 activity. Previously, we reported that a similar effect to AbA sensitivity was observed when PDR16 or PDR17 was overexpressed. It was found that the effect of the impaired biosynthesis of ergosterol on the AbA sensitivity is completely abolished on deletion of PDR16. In addition, an increase in the expression level of Pdr16 was observed on the deletion of ERG6. These results suggested that abnormal ergosterol biosynthesis confers resistance to AbA in a PDR16-dependent manner, implying a novel functional relationship between complex sphingolipids and ergosterol.. |
| 3. | ＃丹生谷颯人、石橋洋平、伊東信、沖野望, Significance of mitochondrial fatty acid beta-oxidation for the survivability of Aurantiochytrium limacinum ATCC MYA-1381 during sugar starvation, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 10.1093/bbb/zbac141, 86, 11, 1524-1535, 2022.10. |
| 4. | Ayano Koga, Chihiro Takayama, Yohei Ishibashi, Yushi Kono, Momoko Matsuzaki, Motohiro Tani, Loss of tolerance to multiple environmental stresses due to limitation of structural diversity of complex sphingolipids, MOLECULAR BIOLOGY OF THE CELL, 10.1091/mbc.E22-04-0117, 33, 12, 2022.10. |
| 5. | Yoneda K, Ishibashi Y, Yoshida M, Watanabe M. Ito M, Iwane S., Proteomic and lipidomic analyses of lipid droplets in Aurantiochytrium limacinum ATCC MYA-1381, Algal Research, https://doi.org/10.1016/j.algal.2022.102844, 67, 102844, 2022.09. |
| 6. | Yohei Ishibashi, Functions and applications of glycolipid-hydrolyzing microbial glycosidases, BIOSCIENCE BIOTECHNOLOGY AND BIOCHEMISTRY, 10.1093/bbb/zbac089, 86, 8, 974-984, 2022.07. |
| 7. | Urita, Atsuya; Ishibashi, Yohei; Kawaguchi, Ryotaro; Yanase, Yukimi; Tani, Motohiro, Crosstalk between protein kinase A and the HOG pathway under impaired biosynthesis of complex sphingolipids in budding yeast, FEBS JOURNAL, 10.1111/febs.16188, 289, 3, 766-786, 2022.02. |
| 8. | 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, PUFA synthase-independent DHA synthesis pathway in Parietichytrium sp. and its modification to produce EPA and n-3DPA, COMMUNICATIONS BIOLOGY, 10.1038/s42003-021-02857-w, 4, 1, 2021.12.  |
| 9. | Maruyama-Nakashita A, Ishibashi Y, Yamamoto K, Liu Z, Morikawa-Ichinose T, Kim SJ, Hayashi N, Oxygen plasma modulates glucosinolate levels without affecting lipid contents and composition in Brassica napus seeds, Biosci. Biotechnol. Biochem., 2021.12, [URL]. |
| 10. | Ikumi Endo, Takashi Watanabe, Tomofumi Miyamoto, Hatsumi Monjusho-Goda, Junichiro Ohara, Masahiro Hayashi, Yoichiro Hama, Yohei Ishibashi, Nozomu Okino, Makoto Ito, C4-monomethylsterol β-glucoside and its synthase in Aurantiochytrium limacinum mh0186, Glycobiology, 2021.11. |
| 11. | Nyunoya, Hayato; Noda, Tatsuki; Kawamoto, You; Hayashi, Yasuhiro; Ishibashi, Yohei; Ito, Makoto; Okino, Nozomu, Lack of delta 5 Desaturase Activity Impairs EPA and DHA Synthesis in Fish Cells from Red Sea Bream and Japanese Flounder, MARINE BIOTECHNOLOGY, 10.1007/s10126-021-10040-9, 23, 3, 472-481, 2021.06. |
| 12. | 石橋 洋平、伊東 信、平林 義雄, The sirtuin inhibitor cambinol reduces intracellular glucosylceramide with ceramide accumulation by inhibiting glucosylceramide synthase, Bioscience, Biotechnology, and Biochemistry, https://doi.org/10.1080/09168451.2020.1794785, 2020.07. |
| 13. | Okino N, Li M, Qu Q, Nakagawa T, Hayashi Y, Matsumoto M, Ishibashi Y, Ito M , Two bacterial glycosphingolipid synthases responsible for the synthesis of glucuronosylceramide and alpha-galactosylceramide, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.RA120.013796, 295, 31, 10709-10725, 2020.07. |
| 14. | Ishibashi Y, Aoki K, Okino N, Hayashi M, Ito M, A thraustochytrid-specific lipase/phospholipase with unique positional specificity contributes to microbial competition and fatty acid acquisition from the environment, Scientific reports, 10.1038/s41598-019-52854-7, 9, 1, 2019.12. |
| 15. | Nutahara E, Abe E, Uno S, Ishibashi Y, Watanabe T, Hayashi M, Okino N, Ito M, The glycerol-3-phosphate acyltransferase PLAT2 functions in the generation of DHA-rich glycerolipids in Aurantiochytrium limacinum F26-b., PLoS One, 14(1):e0211164., 2019.01. |
| 16. | Yohei Ishibashi, Makoto Ito, Yoshio Hirabayashi, Regulation of glucosylceramide synthesis by Golgi-localized phosphoinositide, Biochemical and Biophysical Research Communications, 10.1016/j.bbrc.2018.04.039, 499, 4, 1011-1018, 2018.05, Phosphoinositides mediate a large number of signaling processes in mammalian cells. Here, we report that phophatidylinositol-4-phosphate (PtdIns(4)P) downregulates the cellular glucosylceramide (GlcCer) level by inhibiting the interaction between GlcCer synthase (UGCG) and UDP-glucose in the Golgi apparatus. In this study, we used two PH domain probes to bind phosphoinositides; one derived from FAPP1 for targeting to the Golgi PtdIns(4)P and the other from PLC δ for targeting to the plasma membrane PtdIns(4,5)P2. The levels of GlcCer and lactosylceramide, but not of sphingomyelin (SM), were increased following expression of the FAPP1 PH domain in cells, accompanied by an increase in UGCG activity. However, no elevated GlcCer level was observed after expression of the PLC δ PH domain. PtdIns(4)P inhibited UGCG activity, but not SMS activity, in a concentration-dependent manner, and UGCG activity was restored by the addition of UDP-glucose in the reaction mixture. These results indicate that PtdIns(4)P inhibits UGCG activity by competing with UDP-glucose. We conclude that the increase in UGCG activity due to the expression of the FAPP1 PH domain was caused by an attenuation of the inhibitory effect of PtdIns(4)P on UGCG. This study provides new insights into the regulation of GlcCer synthesis by PtdIns(4)P in the Golgi apparatus.. |
| 17. | Nozomu Okino, Hiroyoshi Wakisaka, Yohei Ishibashi, Makoto Ito, Visualization of Endoplasmic Reticulum and Mitochondria in Aurantiochytrium limacinum by the Expression of EGFP with Cell Organelle-Specific Targeting/Retaining Signals, Marine Biotechnology, 10.1007/s10126-018-9795-7, 20, 2, 182-192, 2018.04, Thraustochytrids are single cell marine eukaryotes that produce large amounts of polyunsaturated fatty acids such as docosahexaenoic acid. In the present study, we report the visualization of endoplasmic reticulum (ER) and mitochondria in a type strain of the thraustochytrid, Aurantiochytrium limacinum ATCC MYA-1381, using the enhanced green fluorescent protein (EGFP) with specific targeting/retaining signals. We expressed the egfp gene with ER targeting/retaining signals from A. limacinum calreticulin or BiP/GRP78 in the thraustochytrid, resulting in the distribution of EGFP signals at the perinuclear region and near lipid droplets. ER-Tracker™ Red, an authentic fluorescent probe for the visualization of ER in mammalian cells, also stained the same region. We observed small lipid droplets generated from the visualized ER in the early growth phase of cell culture. Expression of the egfp gene with the mitochondria targeting signal from A. limacinum cytochrome c oxidase resulted in the localization of EGFP near the plasma membrane. The distribution of EGFP signals coincided with that of MitoTracker® Red CMXRos, which is used to visualize mitochondria in eukaryotes. The ER and mitochondria of A. limacinum were visualized for the first time by EGFP with thraustochytrid cell organelle-specific targeting/retaining signals. These results will contribute to classification of the intracellular localization of proteins expressed in ER and mitochondria as well as analyses of these cell organelles in thraustochytrids.. |
| 18. | Takashi Watanabe, Ryo Sakiyama, Yuya Iimi, Satomi Sekine, Eriko Abe, Kazuko H. Nomura, Kazuya Nomura, Yohei Ishibashi, Nozomu Okino, Masahiro Hayashi, Makoto Ito, Regulation of TG accumulation and lipid droplet morphology by the novel TLDP1 in Aurantiochytrium limacinum F26-b, Journal of Lipid Research, 10.1194/jlr.M079897, 58, 12, 2334-2347, 2017.01, Thraustochytrids are marine single-cell protists that produce large amounts of PUFAs, such as DHA. They accumulate PUFAs in lipid droplets (LDs), mainly as constituent(s) of triacylglycerol (TG). We identified a novel protein in the LD fraction of Aurantiochytrium limacinum F26-b using 2D-difference gel electrophoresis. The protein clustered with orthologs of thraustochytrids; however, the cluster was evolutionally different from known PAT family proteins or plant LD protein; thus, we named it thraustochytrid-specific LD protein 1 (TLDP1). TLDP1 surrounded LDs when expressed as a GFP-tagged form. Disruption of the tldp1 gene decreased the content of TG and number of LDs per cell; however, irregular and unusually large LDs were generated in tldp1-deficient mutants. Although the level of TG synthesis was unchanged by the disruption of tldp1, the level of TG degradation was higher in tldp1-deficient mutants than in the WT. These phenotypic abnormalities in tldp1-deficient mutants were restored by the expression of tldp1. These results indicate that TLDP1 is a thraustochytrid-specific LD protein and regulates the TG accumulation and LD morphology in A. limacinum F26-b.—Watanabe, T., R. Sakiyama, Y. Iimi, S. Sekine, E. Abe, K. H. Nomura, K. Nomura, Y. Ishibashi, N. Okino, M. Hayashi, and M. Ito. Regulation of TG accumulation and lipid droplet morphology by the novel TLDP1 in Aurantiochytrium limacinum F26-b.. |
| 19. | Takehiro Shinoda, Naoko Shinya, Kaori Ito, Yoshiko Ishizuka-Katsura, Noboru Osawa, Takaho Terada, Kunio Hirata, Yoshiaki Kawano, Masaki Yamamoto, Taisuke Tomita, Yohei Ishibashi, Yoshio Hirabayashi, Tomomi Kimura-Someya, Mikako Shirouzu, Shigeyuki Yokoyama, Cell-free methods to produce structurally intact mammalian membrane proteins, Scientific Reports, 10.1038/srep30442, 6, 2016.07. |
| 20. | Yohei Ishibashi, Yoshio Hirabayashi, AMP-activated Protein Kinase Suppresses Biosynthesis of Glucosylceramide by Reducing Intracellular Sugar Nucleotides, JOURNAL OF BIOLOGICAL CHEMISTRY, 10.1074/jbc.M115.658948, 290, 29, 18245-18260, 2015.07. |
| 21. | Yohei Ishibashi, Yusuke Nagamatsu, Tomofumi Miyamoto, Naoyuki Matsunaga, NOZOMU OKINO, Kuniko Yamaguchi, Makoto Ito, A novel ether-linked phytol-containing digalactosylglycerolipid in the marine green alga, Ulva pertusa., Biochem Biophys Res Commun, 10.1016/j.bbrc.2014.08.056., 452, 873-80, 2014.10, Galactosylglycerolipids (GGLs) and chlorophyll are characteristic components of chloroplast in photosynthetic organisms. Although chlorophyll is anchored to the thylakoid membrane by phytol (tetramethylhexadecenol), this isoprenoid alcohol has never been found as a constituent of GGLs. We here described a novel GGL, in which phytol was linked to the glycerol backbone via an ether linkage. This unique GGL was identified as an Alkaline-resistant and Endogalactosylceramidase (EGALC)-sensitive GlycoLipid (AEGL) in the marine green alga, Ulva pertusa. EGALC is an enzyme that is specific to the R-Galα/β1-6Galβ1-structure of galactolipids. The structure of U. pertusa AEGL was determined following its purification to 1-O-phytyl-3-O-Galα1-6Galβ1-sn-glycerol by mass spectrometric and nuclear magnetic resonance analyses. AEGLs were ubiquitously distributed in not only green, but also red and brown marine algae; however, they were rarely detected in terrestrial plants, eukaryotic phytoplankton, or cyanobacteria. . |
| 22. | Yohei Ishibashi, Ayako Kohyama-Koganeya, Yoshio Hirabayashi, New insights on glucosylated lipids: metabolism and functions., Biochim Biophys Acta, 10.1016/j.bbalip.2013.06.001, 1831, 1475-1485, 2013.09, Ceramide, cholesterol, and phosphatidic acid are major basic structures for cell membrane lipids. These lipids are modified with glucose to generate glucosylceramide (GlcCer), cholesterylglucoside (ChlGlc), and phosphatidylglucoside (PtdGlc), respectively. Glucosylation dramatically changes the functional properties of lipids. For instance, ceramide acts as a strong tumor suppressor that causes apoptosis and cell cycle arrest, while GlcCer has an opposite effect, downregulating ceramide activities. All glucosylated lipids are enriched in lipid rafts or microdomains and play fundamental roles in a variety of cellular processes. In this review, we discuss the biological functions and metabolism of these three glucosylated lipids. . |
| 23. | Yohei Ishibashi, Utaro Kobayashi, Atsushi Hijikata, Keishi Sakaguchi, Hstsumi M Goda, Tomohiro Tamura, NOZOMU OKINO, Makoto Ito, Preparation and characterization of EGCase I, applicable to the comprehensive analysis of GSLs, using a rhodococcal expression system, J. Lipid Res., 10.1194/jlr.D028951, 53, 10, 2242-2251, 2012.10. |
| 24. | Yohei Ishibashi, Kazutaka Ikeda, Keishi Sakaguchi, NOZOMU OKINO, Ryo Taguchi, Makoto Ito, Quality control of fungus-specific glucosylceramide in Cryptococcus neoformans by endoglycoceramidase-related protein 1 (EGCrP1)., J. Biol. Chem., 287, 1, 368-381, 2012.01. |
| 25. | Naoki Fujitani, Yasuhiro Takegawa, Yohei Ishibashi, Kayo Araki, Jun-ichi Furukawa, Susumu Mitsutake, Yasuyuki Igarashi, Makoto Ito, Yasuro Shinohara, Qualitative and Quantitative Cellular Glycomics of Glycosphingolipids Based on Rhodococcal Endoglycosylceramidase-assisted Glycan Cleavage, Glycoblotting-assisted Sample Preparation, and Matrix-assisted Laser Desorption Ionization Tandem Time-of-flight Mass Spectrometry Analysis, J. Biol. Chem., 10.1074/jbc.M111.301796, 286, 48, 41669-41679, 2011.12. |
| 26. | Yu-Teh Li, Chau-Wen Chou, Su-Chen Li, Utaro Kobayashi, Yohei Ishibashi, Makoto Ito, Preparation of homogenous oligosaccharide chains from glycosphingolipids, Glycoconjugate J., 10.1007/s10719-008-9125-9, 26, 8, 929-933, 2009.11. |
| 27. | Yohei Ishibashi, Yusuke Nagamatsu, Sandra Meyer, Akihiro Imamura, Hideharu Ishida, Makoto Kiso, NOZOMU OKINO, Rudolf Geyer, Makoto Ito, Transglycosylation-based fluorescent labeling of 6-gala series glycolipids by endogalactosylceramidase, Glycobiology, 10.1093/glycob/cwp051, 19, 7, 797-807, 2009.07. |
| 28. | Yohei Ishibashi, Masahi Kiyohara, NOZOMU OKINO, Makoto Ito, Synthesis of fluorescent glycosphingolipids and neoglycoconjugates which contain 6-gala oligosaccharides using the transglycosylation reaction of a novel endoglycoceramidase (EGALC)., J. Biochem., 2007.08. |
| 29. | Yohei Ishibashi, Toru Nakasone, Masashi Kiyohara, Yasuhiro Horibata, Keishi Sakaguchi, Atsushi Hijikata, Sachiyo Ichinose, Akira Omori, Yasuyuki Yasui, Akihiro Imamura, Hideharu Ishida, Makoto Kiso, NOZOMU OKINO, Makoto Ito, A novel endoglycoceramidase hydrolyzes oligogalactosylceramides to produce galactooligosaccharides and ceramides., J. Biol. Chem., 2007.04. |
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