The Experts below are selected from a list of 261 Experts worldwide ranked by ideXlab platform

Fumihiko Sato - One of the best experts on this subject based on the ideXlab platform.

  • a multidrug and toxic compound extrusion transporter mediates berberine accumulation into vacuoles in Coptis Japonica
    Phytochemistry, 2017
    Co-Authors: Kojiro Takanashi, Yasuyuki Yamada, Fumihiko Sato, Takayuki Sasaki, Yoko Yamamoto, Kazufumi Yazaki
    Abstract:

    Abstract Plants produce a large variety of alkaloids, which have diverse chemical structures and biological activities. Many of these alkaloids accumulate in vacuoles. Although some membrane proteins on tonoplasts have been identified as alkaloid uptake transporters, few have been characterized to date, and relatively little is known about the mechanisms underlying alkaloid transport and accumulation in plant cells. Berberine is a model alkaloid. Although all genes involved in berberine biosynthesis, as well as the master regulator, have been identified, the gene responsible for the final accumulation of berberine at tonoplasts has not been determined. This study showed that a m ultidrug a nd t oxic compound e xtrusion protein 1 (CjMATE1) may act as a berberine transporter in cultured Coptis Japonica cells. CjMATE1 was found to localize at tonoplasts in C. Japonica cells and, in intact plants, to be expressed preferentially in rhizomes, the site of abundant berberine accumulation. Cellular transport analysis using a yeast expression system showed that CjMATE1 could transport berberine. Expression analysis showed that RNAi suppression of CjbHLH1 , a master transcription factor of the berberine biosynthetic pathway, markedly reduced the expression of CjMATE1 in a manner similar to the suppression of berberine biosynthetic genes. These results strongly suggest that CjMATE1 is the transporter that mediates berberine accumulation in vacuoles.

  • characterization of the promoter region of biosynthetic enzyme genes involved in berberine biosynthesis in Coptis Japonica
    Frontiers in Plant Science, 2016
    Co-Authors: Yasuyuki Yamada, Tadashi Yoshimoto, Sayumi T Yoshida, Fumihiko Sato
    Abstract:

    The presence of alkaloids is rather specific to certain plant species. However, berberine, an isoquinoline alkaloid, is relatively broadly distributed in the plant kingdom. Thus, berberine biosynthesis has been intensively investigated, especially using Coptis Japonica cell cultures. Almost all biosynthetic enzyme genes have already been characterized at the molecular level. Particularly, two transcription factors (TFs), a plant-specific WRKY-type transcription factor, CjWRKY1, and a basic helix-loop-helix (bHLH) transcription factor, CjbHLH1, were shown to comprehensively regulate berberine biosynthesis in C. Japonica cells. In this study, we characterized the promoter region of some biosynthetic enzyme genes and associated cis-acting elements involved in the transcriptional regulation via two TFs. The promoter regions of three berberine biosynthetic enzyme genes (CYP80B2, 4’OMT and CYP719A1) were isolated, and their promoter activities were dissected by a transient assay involving the sequentially truncated promoter::luciferase (LUC) reporter constructs. Furthermore, transactivation activities of CjWRKY1 were determined using the truncated promoter::LUC reporter constructs or constructs with mutated cis-elements. These results suggest the involvement of a putative W-box in the regulation of biosynthetic enzyme genes. Direct binding of CjWRKY1 to the W-box DNA sequence was also confirmed by an electrophoresis mobility shift assay (EMSA) and by a chromatin immunoprecipitation (ChIP) assay. In addition, CjbHLH1 also activated transcription from truncated 4’OMT and CYP719A1 promoters independently of CjWRKY1, suggesting the involvement of a putative E-box. Unexpected transcriptional activation of biosynthetic enzyme genes via a non-W-box sequence and by CjWRKY1 as well as the possible involvement of a GCC-box in berberine biosynthesis in C. Japonica are discussed.

  • Asymmetric synthesis of tetrahydroisoquinolines by enzymatic Pictet-Spengler reaction.
    Bioscience biotechnology and biochemistry, 2014
    Co-Authors: Masakatsu Nishihachijo, Hiromichi Minami, Yoshinori Hirai, Shigeru Kawano, Akira Nishiyama, Takane Katayama, Yoshihiko Yasohara, Fumihiko Sato, Fumihiko Sato, Hidehiko Kumagai
    Abstract:

    Norcoclaurine synthase (NCS) catalyzes the stereoselective Pictet-Spengler reaction between dopamine and 4-hydroxyphenylacetaldehyde as the first step of benzylisoquinoline alkaloid synthesis in plants. Recent studies suggested that NCS shows relatively relaxed substrate specificity toward aldehydes, and thus, the enzyme can serve as a tool to synthesize unnatural, optically active tetrahydroisoquinolines. In this study, using an N-terminally truncated NCS from Coptis Japonica expressed in Escherichia coli, we examined the aldehyde substrate specificity of the enzyme. Herein, we demonstrate the versatility of the enzyme by synthesizing 6,7-dihydroxy-1-phenethyl-1,2,3,4-tetrahydroisoquinoline and 6,7-dihydroxy-1-propyl-1,2,3,4-tetrahydroisoquinoline in molar yields of 86.0 and 99.6% and in enantiomer excess of 95.3 and 98.0%, respectively. The results revealed the enzyme is a promising catalyst that functions to stereoselectively produce various 1-substituted-1,2,3,4-tetrahydroisoquinolines.

  • characterization of Coptis Japonica cjabcb2 an atp binding cassette protein involved in alkaloid transport
    Phytochemistry, 2013
    Co-Authors: Nobuhiko Kato, Nobukazu Shitan, Fumihiko Sato, Kazumitsu Ueda, Fabien Dalmas, Cyrille Forestier, Kazufumi Yazaki
    Abstract:

    Abstract Higher plants produce a large number of secondary metabolites. Among these are the alkaloids, a group of small nitrogen-containing molecules. Alkaloids often have strong biological activity that protects alkaloid-producing plants from herbivores, and often accumulate to high concentrations in a specific organelle of a particular organ in the producing plant. However, knowledge of the membrane transport mechanism of alkaloids is still limited. Coptis Japonica , a perennial Ranunculaceous plant, produces the benzylisoquinoline alkaloid berberine. This alkaloid, though biosynthesized in root tissues, accumulates in the rhizome, suggesting translocation of the molecule via xylem. In this study, a gene encoding a ATP-binding cassette (ABC) protein of B-type, Cjabcb2 , was isolated from C. Japonica. Northern analysis showed that Cjabcb2 was preferentially expressed in the rhizome, which is the sink organ of berberine. Functional analysis of CjABCB2 using yeast suggested that CjABCB2 transports berberine in an inward direction. Membrane separation and in situ hybridization data indicated that CjABCB2 might be involved in translocation of berberine from the root to the rhizome by transporting berberine at the plasma membrane of cells around the xylem of the rhizome.

  • isoquinoline alkaloid biosynthesis is regulated by a unique bhlh type transcription factor in Coptis Japonica
    Plant and Cell Physiology, 2011
    Co-Authors: Yasuyuki Yamada, Nobuhiko Kato, Yasuhisa Kokabu, Sayumi Yoshida, Kaori Chaki, Tadashi Yoshimoto, Mai Ohgaki, Tomotsugu Koyama, Fumihiko Sato
    Abstract:

    : Specific plant species produce unique isoquinoline alkaloids (IQAs); however, the mechanism of their evolution and the regulation of their biosynthesis are largely unknown. We report here the isolation of a novel basic helix-loop-helix protein, CjbHLH1, from IQA-producing Coptis Japonica. A BLAST search indicated that CjbHLH1 homologs were only found in plant species that produce IQAs. Transient RNA interference (RNAi) and overexpression of CjbHLH1 in C. Japonica protoplasts revealed the activity of CjbHLH1 in transcription of IQA biosynthetic genes, and little activity in the transcription of genes involved in primary metabolism or the stress response. A chromatin immunoprecipitation experiment using CjbHLH1-specific antibodies revealed the direct interaction of CjbHLH1 with promoter sequences of IQA biosynthetic genes in vivo. We discuss the unique role of CjbHLH1 in IQA biosynthesis.

Kazufumi Yazaki - One of the best experts on this subject based on the ideXlab platform.

  • a multidrug and toxic compound extrusion transporter mediates berberine accumulation into vacuoles in Coptis Japonica
    Phytochemistry, 2017
    Co-Authors: Kojiro Takanashi, Yasuyuki Yamada, Fumihiko Sato, Takayuki Sasaki, Yoko Yamamoto, Kazufumi Yazaki
    Abstract:

    Abstract Plants produce a large variety of alkaloids, which have diverse chemical structures and biological activities. Many of these alkaloids accumulate in vacuoles. Although some membrane proteins on tonoplasts have been identified as alkaloid uptake transporters, few have been characterized to date, and relatively little is known about the mechanisms underlying alkaloid transport and accumulation in plant cells. Berberine is a model alkaloid. Although all genes involved in berberine biosynthesis, as well as the master regulator, have been identified, the gene responsible for the final accumulation of berberine at tonoplasts has not been determined. This study showed that a m ultidrug a nd t oxic compound e xtrusion protein 1 (CjMATE1) may act as a berberine transporter in cultured Coptis Japonica cells. CjMATE1 was found to localize at tonoplasts in C. Japonica cells and, in intact plants, to be expressed preferentially in rhizomes, the site of abundant berberine accumulation. Cellular transport analysis using a yeast expression system showed that CjMATE1 could transport berberine. Expression analysis showed that RNAi suppression of CjbHLH1 , a master transcription factor of the berberine biosynthetic pathway, markedly reduced the expression of CjMATE1 in a manner similar to the suppression of berberine biosynthetic genes. These results strongly suggest that CjMATE1 is the transporter that mediates berberine accumulation in vacuoles.

  • characterization of Coptis Japonica cjabcb2 an atp binding cassette protein involved in alkaloid transport
    Phytochemistry, 2013
    Co-Authors: Nobuhiko Kato, Nobukazu Shitan, Fumihiko Sato, Kazumitsu Ueda, Fabien Dalmas, Cyrille Forestier, Kazufumi Yazaki
    Abstract:

    Abstract Higher plants produce a large number of secondary metabolites. Among these are the alkaloids, a group of small nitrogen-containing molecules. Alkaloids often have strong biological activity that protects alkaloid-producing plants from herbivores, and often accumulate to high concentrations in a specific organelle of a particular organ in the producing plant. However, knowledge of the membrane transport mechanism of alkaloids is still limited. Coptis Japonica , a perennial Ranunculaceous plant, produces the benzylisoquinoline alkaloid berberine. This alkaloid, though biosynthesized in root tissues, accumulates in the rhizome, suggesting translocation of the molecule via xylem. In this study, a gene encoding a ATP-binding cassette (ABC) protein of B-type, Cjabcb2 , was isolated from C. Japonica. Northern analysis showed that Cjabcb2 was preferentially expressed in the rhizome, which is the sink organ of berberine. Functional analysis of CjABCB2 using yeast suggested that CjABCB2 transports berberine in an inward direction. Membrane separation and in situ hybridization data indicated that CjABCB2 might be involved in translocation of berberine from the root to the rhizome by transporting berberine at the plasma membrane of cells around the xylem of the rhizome.

  • improvement of benzylisoquinoline alkaloid productivity by overexpression of 3 hydroxy n methylcoclaurine 4 o methyltransferase in transgenic Coptis Japonica plants
    Biological & Pharmaceutical Bulletin, 2012
    Co-Authors: Takayuki Inui, Nobukazu Shitan, Noriaki Kawano, Nobuo Kawahara, Fumiyuki Kiuchi, Kazufumi Yazaki, Kayo Yoshimatsu
    Abstract:

    : Coptis Japonica (Cj) rhizomes are used as a crude drug for gastroenteritis, since they accumulate antimicrobial berberine. Berberine also shows various useful bioactivities, including cholesterol-lowering activity. Unfortunately, Cj is a slow-growing plant and more than 5 years are required to obtain a crude drug suitable for the Japanese Pharmacopoeia. To improve alkaloid productivity, we overexpressed the 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'OMT) gene in Cj. We established the transgenic plant (named CjHE4') by introducing one copy of Cj4'OMT by Agrobacterium-mediated transformation. The successful overexpression of 4'OMT was confirmed in all tissues of CjHE4' by real-time polymerase chain reaction (PCR) analysis. HPLC analysis revealed that the berberine content of CjHE4' leaves and roots cultivated for 4 months was increased to 2.7- and 2.0-fold, respectively, compared with non-transgenic wild-type (CjWT), and these inductions of alkaloids were stable for at least 20 months. Furthermore, in CjHE4' cultivated for 20 months, the berberine content in medicinal parts, stems and rhizomes was significantly increased (1.6-fold). As a consequence, increased amounts of alkaloids in CjHE4' resulted in the improvement of berberine yields (1.5-fold), whereas CjHE4' showed slower growth than CjWT. These results indicated that 4'OMT is one of the key-step enzymes in berberine biosynthesis and is useful for metabolic engineering in Cj.

  • galactinol synthase gene of Coptis Japonica is involved in berberine tolerance
    Bioscience Biotechnology and Biochemistry, 2008
    Co-Authors: Kojiro Takanashi, Nobukazu Shitan, Akifumi Sugiyama, Yoshihisa Kamimoto, Masafumi Hamamoto, Tomoko Iwaki, Kaoru Takegawa, Kazufumi Yazaki
    Abstract:

    Many plant secondary metabolites show strong biological activities and are potentially also toxic to plants, while plants producing such active compounds are usually insensitive to their own metabolites, suggesting that they have species-specific detoxification mechanisms. In order to clarify the detoxification mechanism of alkaloids, we used cultured cells of Coptis Japonica, which are capable of producing a yellow benzylisoquinoline alkaloid, berberine, and accumulate it in the vacuole. Unlike other plant cells that do not produce berberine, C. Japonica shows strong tolerance to this alkaloid. We established a fission yeast strain that was sensitive to berberine and performed functional screening using a C. Japonica cDNA library. One cDNA clone, which conferred clear berberine tolerance, encoded galactinol synthase (CjGolS). The possible role of CjGolS in berberine tolerance is discussed.

  • identification of a wrky protein as a transcriptional regulator of benzylisoquinoline alkaloid biosynthesis in Coptis Japonica
    Plant and Cell Physiology, 2007
    Co-Authors: Nobuhiko Kato, Yasuhisa Kokabu, Sayumi Yoshida, Yoshimasa Taniguchi, Joseph G Dubouzet, Emilyn G Dubouzet, Kazufumi Yazaki
    Abstract:

    Selected cultured Coptis Japonica cells produce a large amount of the benzylisoquinoline alkaloid berberine. Previous studies have suggested that berberine productivity is controlled at the transcript level of biosynthetic genes. We have identified a regulator of transcription in berberine biosynthesis using functional genomics with a transient RNA interference (RNAi) and overexpression of the candidate gene. The 24 primary candidate clones were selected from 1,014 expressed sequence tags (ESTs) that were obtained from a C. Japonica cell line producing high levels of berberine. Further characterization of the expression profiles of these ESTs suggested that five ESTs would be good candidates as regulators of berberine production. A newly developed transient RNAi system with C. Japonica protoplasts indicated that double-stranded RNA of an EST clone significantly reduced the level of transcripts of 3 0 -hydroxy N-methylcoclaurine 4 0 -O-methyltransferase. Sequence analysis showed that this EST encoded a group-II WRKY, and we named it CjWRKY1. When the effects of double-stranded RNA of the CjWRKY1 gene were examined in detail, a marked reduction in the transcripts of all genes involved in berberine biosynthesis was detected, whereas little effect was found in the transcript levels of glyceraldehyde3-phosphate dehydrogenase (GAPDH) and chorismate mutase (CM) that are associated with primary metabolism. Ectopic expression of CjWRKY1 cDNA in C. Japonica protoplasts clearly increased the level of transcripts of all berberine biosynthetic genes examined compared with control treatment, whereas the levels of GAPDH and CM were not affected. The functional role of CjWRKY1 as a specific and comprehensive regulator of berberine biosynthesis is discussed.

Yasuyuki Yamada - One of the best experts on this subject based on the ideXlab platform.

  • a multidrug and toxic compound extrusion transporter mediates berberine accumulation into vacuoles in Coptis Japonica
    Phytochemistry, 2017
    Co-Authors: Kojiro Takanashi, Yasuyuki Yamada, Fumihiko Sato, Takayuki Sasaki, Yoko Yamamoto, Kazufumi Yazaki
    Abstract:

    Abstract Plants produce a large variety of alkaloids, which have diverse chemical structures and biological activities. Many of these alkaloids accumulate in vacuoles. Although some membrane proteins on tonoplasts have been identified as alkaloid uptake transporters, few have been characterized to date, and relatively little is known about the mechanisms underlying alkaloid transport and accumulation in plant cells. Berberine is a model alkaloid. Although all genes involved in berberine biosynthesis, as well as the master regulator, have been identified, the gene responsible for the final accumulation of berberine at tonoplasts has not been determined. This study showed that a m ultidrug a nd t oxic compound e xtrusion protein 1 (CjMATE1) may act as a berberine transporter in cultured Coptis Japonica cells. CjMATE1 was found to localize at tonoplasts in C. Japonica cells and, in intact plants, to be expressed preferentially in rhizomes, the site of abundant berberine accumulation. Cellular transport analysis using a yeast expression system showed that CjMATE1 could transport berberine. Expression analysis showed that RNAi suppression of CjbHLH1 , a master transcription factor of the berberine biosynthetic pathway, markedly reduced the expression of CjMATE1 in a manner similar to the suppression of berberine biosynthetic genes. These results strongly suggest that CjMATE1 is the transporter that mediates berberine accumulation in vacuoles.

  • characterization of the promoter region of biosynthetic enzyme genes involved in berberine biosynthesis in Coptis Japonica
    Frontiers in Plant Science, 2016
    Co-Authors: Yasuyuki Yamada, Tadashi Yoshimoto, Sayumi T Yoshida, Fumihiko Sato
    Abstract:

    The presence of alkaloids is rather specific to certain plant species. However, berberine, an isoquinoline alkaloid, is relatively broadly distributed in the plant kingdom. Thus, berberine biosynthesis has been intensively investigated, especially using Coptis Japonica cell cultures. Almost all biosynthetic enzyme genes have already been characterized at the molecular level. Particularly, two transcription factors (TFs), a plant-specific WRKY-type transcription factor, CjWRKY1, and a basic helix-loop-helix (bHLH) transcription factor, CjbHLH1, were shown to comprehensively regulate berberine biosynthesis in C. Japonica cells. In this study, we characterized the promoter region of some biosynthetic enzyme genes and associated cis-acting elements involved in the transcriptional regulation via two TFs. The promoter regions of three berberine biosynthetic enzyme genes (CYP80B2, 4’OMT and CYP719A1) were isolated, and their promoter activities were dissected by a transient assay involving the sequentially truncated promoter::luciferase (LUC) reporter constructs. Furthermore, transactivation activities of CjWRKY1 were determined using the truncated promoter::LUC reporter constructs or constructs with mutated cis-elements. These results suggest the involvement of a putative W-box in the regulation of biosynthetic enzyme genes. Direct binding of CjWRKY1 to the W-box DNA sequence was also confirmed by an electrophoresis mobility shift assay (EMSA) and by a chromatin immunoprecipitation (ChIP) assay. In addition, CjbHLH1 also activated transcription from truncated 4’OMT and CYP719A1 promoters independently of CjWRKY1, suggesting the involvement of a putative E-box. Unexpected transcriptional activation of biosynthetic enzyme genes via a non-W-box sequence and by CjWRKY1 as well as the possible involvement of a GCC-box in berberine biosynthesis in C. Japonica are discussed.

  • isoquinoline alkaloid biosynthesis is regulated by a unique bhlh type transcription factor in Coptis Japonica
    Plant and Cell Physiology, 2011
    Co-Authors: Yasuyuki Yamada, Nobuhiko Kato, Yasuhisa Kokabu, Sayumi Yoshida, Kaori Chaki, Tadashi Yoshimoto, Mai Ohgaki, Tomotsugu Koyama, Fumihiko Sato
    Abstract:

    : Specific plant species produce unique isoquinoline alkaloids (IQAs); however, the mechanism of their evolution and the regulation of their biosynthesis are largely unknown. We report here the isolation of a novel basic helix-loop-helix protein, CjbHLH1, from IQA-producing Coptis Japonica. A BLAST search indicated that CjbHLH1 homologs were only found in plant species that produce IQAs. Transient RNA interference (RNAi) and overexpression of CjbHLH1 in C. Japonica protoplasts revealed the activity of CjbHLH1 in transcription of IQA biosynthetic genes, and little activity in the transcription of genes involved in primary metabolism or the stress response. A chromatin immunoprecipitation experiment using CjbHLH1-specific antibodies revealed the direct interaction of CjbHLH1 with promoter sequences of IQA biosynthetic genes in vivo. We discuss the unique role of CjbHLH1 in IQA biosynthesis.

  • high density culture of Coptis Japonica cells increases berberine production
    Journal of Chemical Technology & Biotechnology, 2007
    Co-Authors: Koichi Matsubara, Teijiro Morimoto, Shigekazu Kitani, Toshihiro Yoshioka, Yasuhiro Fujita, Yasuyuki Yamada
    Abstract:

    A high density culture method was devised to improve the yield of berberine from highly productive cells of Coptis Japonica. By adjusting aeration and stirring, Coptis cells were cultured at densities of up to 75 g dm−3 (dry weight) in a culture tank fitted with a hollow-paddle type stirrer. Whereas a maximum density of 30 g dm−3 of C. Japonica cells could be used in ordinary batch culture, 48 g dm−3 could be used in a fed-batch culture in which the amounts of the nutrients in the medium were made proportional to the density of the inoculum. Moreover, in fed-batch culture done with modified medium, the composition of which had been determined from the amounts of components incorporated in cells grown at the usual density for ordinary batch culture, the cell yield was improved to 55 g dm−3 and the berberine yield to 3.5 g dm−3.

  • metabolic engineering of plant alkaloid biosynthesis
    Proceedings of the National Academy of Sciences of the United States of America, 2001
    Co-Authors: Fumihiko Sato, Kum Boo Choi, Takashi Morishige, Kenichi Tamura, Takashi Hashimoto, Akira Hachiya, Hideki Fujimoto, Yasuyuki Yamada
    Abstract:

    Plant alkaloids, one of the largest groups of natural products, provide many pharmacologically active compounds. Several genes in the biosynthetic pathways for scopolamine, nicotine, and berberine have been cloned, making the metabolic engineering of these alkaloids possible. Expression of two branching-point enzymes was engineered: putrescine N-methyltransferase (PMT) in transgenic plants of Atropa belladonna and Nicotiana sylvestris and (S)-scoulerine 9-O-methyltransferase (SMT) in cultured cells of Coptis Japonica and Eschscholzia californica. Overexpression of PMT increased the nicotine content in N. sylvestris, whereas suppression of endogenous PMT activity severely decreased the nicotine content and induced abnormal morphologies. Ectopic expression of SMT caused the accumulation of benzylisoquinoline alkaloids in E. californica. The prospects and limitations of engineering plant alkaloid metabolism are discussed.

Nobukazu Shitan - One of the best experts on this subject based on the ideXlab platform.

  • characterization of Coptis Japonica cjabcb2 an atp binding cassette protein involved in alkaloid transport
    Phytochemistry, 2013
    Co-Authors: Nobuhiko Kato, Nobukazu Shitan, Fumihiko Sato, Kazumitsu Ueda, Fabien Dalmas, Cyrille Forestier, Kazufumi Yazaki
    Abstract:

    Abstract Higher plants produce a large number of secondary metabolites. Among these are the alkaloids, a group of small nitrogen-containing molecules. Alkaloids often have strong biological activity that protects alkaloid-producing plants from herbivores, and often accumulate to high concentrations in a specific organelle of a particular organ in the producing plant. However, knowledge of the membrane transport mechanism of alkaloids is still limited. Coptis Japonica , a perennial Ranunculaceous plant, produces the benzylisoquinoline alkaloid berberine. This alkaloid, though biosynthesized in root tissues, accumulates in the rhizome, suggesting translocation of the molecule via xylem. In this study, a gene encoding a ATP-binding cassette (ABC) protein of B-type, Cjabcb2 , was isolated from C. Japonica. Northern analysis showed that Cjabcb2 was preferentially expressed in the rhizome, which is the sink organ of berberine. Functional analysis of CjABCB2 using yeast suggested that CjABCB2 transports berberine in an inward direction. Membrane separation and in situ hybridization data indicated that CjABCB2 might be involved in translocation of berberine from the root to the rhizome by transporting berberine at the plasma membrane of cells around the xylem of the rhizome.

  • improvement of benzylisoquinoline alkaloid productivity by overexpression of 3 hydroxy n methylcoclaurine 4 o methyltransferase in transgenic Coptis Japonica plants
    Biological & Pharmaceutical Bulletin, 2012
    Co-Authors: Takayuki Inui, Nobukazu Shitan, Noriaki Kawano, Nobuo Kawahara, Fumiyuki Kiuchi, Kazufumi Yazaki, Kayo Yoshimatsu
    Abstract:

    : Coptis Japonica (Cj) rhizomes are used as a crude drug for gastroenteritis, since they accumulate antimicrobial berberine. Berberine also shows various useful bioactivities, including cholesterol-lowering activity. Unfortunately, Cj is a slow-growing plant and more than 5 years are required to obtain a crude drug suitable for the Japanese Pharmacopoeia. To improve alkaloid productivity, we overexpressed the 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase (4'OMT) gene in Cj. We established the transgenic plant (named CjHE4') by introducing one copy of Cj4'OMT by Agrobacterium-mediated transformation. The successful overexpression of 4'OMT was confirmed in all tissues of CjHE4' by real-time polymerase chain reaction (PCR) analysis. HPLC analysis revealed that the berberine content of CjHE4' leaves and roots cultivated for 4 months was increased to 2.7- and 2.0-fold, respectively, compared with non-transgenic wild-type (CjWT), and these inductions of alkaloids were stable for at least 20 months. Furthermore, in CjHE4' cultivated for 20 months, the berberine content in medicinal parts, stems and rhizomes was significantly increased (1.6-fold). As a consequence, increased amounts of alkaloids in CjHE4' resulted in the improvement of berberine yields (1.5-fold), whereas CjHE4' showed slower growth than CjWT. These results indicated that 4'OMT is one of the key-step enzymes in berberine biosynthesis and is useful for metabolic engineering in Cj.

  • galactinol synthase gene of Coptis Japonica is involved in berberine tolerance
    Bioscience Biotechnology and Biochemistry, 2008
    Co-Authors: Kojiro Takanashi, Nobukazu Shitan, Akifumi Sugiyama, Yoshihisa Kamimoto, Masafumi Hamamoto, Tomoko Iwaki, Kaoru Takegawa, Kazufumi Yazaki
    Abstract:

    Many plant secondary metabolites show strong biological activities and are potentially also toxic to plants, while plants producing such active compounds are usually insensitive to their own metabolites, suggesting that they have species-specific detoxification mechanisms. In order to clarify the detoxification mechanism of alkaloids, we used cultured cells of Coptis Japonica, which are capable of producing a yellow benzylisoquinoline alkaloid, berberine, and accumulate it in the vacuole. Unlike other plant cells that do not produce berberine, C. Japonica shows strong tolerance to this alkaloid. We established a fission yeast strain that was sensitive to berberine and performed functional screening using a C. Japonica cDNA library. One cDNA clone, which conferred clear berberine tolerance, encoded galactinol synthase (CjGolS). The possible role of CjGolS in berberine tolerance is discussed.

  • bowman birk proteinase inhibitor confers heavy metal and multiple drug tolerance in yeast
    Plant and Cell Physiology, 2007
    Co-Authors: Nobukazu Shitan, Fumihiko Sato, Kenichi Horiuchi, Kazufumi Yazaki
    Abstract:

    Cultured Coptis Japonica cells show tolerance to various toxic compounds. By yeast functional screening of cadmium (Cd) plates with its cDNA library, we isolated a gene encoding Bowman-Birk proteinase inhibitor (CjBBI). The yeast transformant of CjBBI showed multiple tolerance to various drugs adding to Cd, and revealed reduced Cd accumulation in cells. Preferential organs for Cjbbi expression were aerial parts of intact plants, and the subcellular localization of CjBBI was shown, using its green fluorescent protein fusion, to be the apoplast. Induction of Cjbbi expression by Cd treatment suggested that CjBBI was responsible for the tolerance to Cd observed in C. Japonica cells.

  • characterization of vacuolar transport of the endogenous alkaloid berberine in Coptis Japonica
    Plant Physiology, 2005
    Co-Authors: Mihoko Otani, Nobukazu Shitan, Fumihiko Sato, Kyoko Sakai, Enrico Martinoia, Kazufumi Yazaki
    Abstract:

    Alkaloids comprise one of the largest groups of plant secondary metabolites. Many of them exhibit strong biological activities, and, in most cases, they are accumulated in the central vacuole of alkaloid-producing plants after synthesis. However, the mechanisms involved in alkaloid transport across the tonoplast are only poorly understood. In this study, we analyzed the vacuolar transport mechanism of an isoquinoline alkaloid, berberine, which is produced and accumulated in the vacuole of cultured cells of Coptis Japonica. The characterization of berberine transport using intact vacuoles and a tonoplast vesicle system showed that berberine uptake was stimulated by Mg/ATP, as well as GTP, CTP, UTP, and Mg/pyrophosphate. Berberine uptake was strongly inhibited by NH4+ and bafilomycin A1, while vanadate, which is commonly used to inhibit ATP-binding cassette transporters, had only a slight effect, which suggests the presence of a typical secondary transport mechanism. This is contrary to the situation in the plasma membrane of this plant cell, where the ATP-binding cassette transporter is involved in berberine transport. Model experiments with liposomes demonstrated that an ion-trap mechanism was hardly implicated in berberine transport. Further studies suggested that berberine was transported across the tonoplast via an H+/berberine antiporter, which has a Km value of 43.7 μm for berberine. Competition experiments using various berberine analogs, as well as other classes of alkaloids, revealed that this transporter is fairly specific, but not exclusive, for berberine.

Takashi Morishige - One of the best experts on this subject based on the ideXlab platform.

  • molecular characterization of o methyltransferases involved in isoquinoline alkaloid biosynthesis in Coptis Japonica
    Proceedings of the Japan Academy. Series B Physical and biological sciences, 2010
    Co-Authors: Takashi Morishige, Masanori Tamakoshi, Tomoya Takemura, Fumihiko Sato
    Abstract:

    O-Methyltransferases, which catalyze the production of small molecules in plants, play a crucial role in determining biosynthetic pathways in secondary metabolism because of their strict substrate specificity. Using three O-methyltransferase (OMT) cDNAs that are involved in berberine biosynthesis, we investigated the structure that was essential for this substrate specificity and the possibility of creating a chimeric enzyme with novel substrate specificity. Since each OMT has a relatively well-conserved C-terminal putative S-adenosyl-L-methionine-binding domain, we first exchanged the N-terminal halves of different OMTs. Among the 6 combinations that we tested for creating chimeric OMTs, 5 constructs produced detectable amounts of recombinant proteins, and only one of these with an N-terminal half of 6-OMT and a C-terminal half of 4'-OMT (64'-OMT) showed methylation activity with isoquinoline alkaloids as a substrate. Further enzymological analysis of 64'-OMT reaction product indicated that 64'-OMT retained the regio-specificity of 6-OMT. Further examination of the N-terminal region of 64'-OMT showed that about 90 amino acid residues in the N-terminal half were critical for reaction specificity. The creation of OMTs with novel reactivity is discussed.

  • overexpression of Coptis Japonica norcoclaurine 6 o methyltransferase overcomes the rate limiting step in benzylisoquinoline alkaloid biosynthesis in cultured eschscholzia californica
    Plant and Cell Physiology, 2007
    Co-Authors: Takayuki Inui, Nanae Fujii, Kenichi Tamura, Takashi Morishige
    Abstract:

    Benzylisoquinoline alkaloids are one of the most important secondary metabolite groups, and include the economically important analgesic morphine and the antimicrobial agent berberine. To improve the production of these alkaloids, we investigated the effect of the overexpression of putative rate-limiting step enzymes in benzylisoquinoline alkaloid biosynthesis. We introduced two O-methyltransferase [Coptis Japonica norcoclaurine 6-O-methyltransferase (6OMT) and 3 0 -hydroxy-N-methylcoclaurine 4 0 -O-methyltransferase (4 0 OMT)] expression vectors into cultured California poppy cells to avoid the gene silencing effect of endogenous genes. We established 20 independent lines for 6OMT transformants and 15 independent lines for 4 0 OMT transformants. HPLC/ liquid chromatography-mass spectrometry (LC-MS) analysis revealed that the overexpression of C. Japonica 6OMT was associated with an average alkaloid content 7.5 times greater than that in the wild type, whereas the overexpression of C. Japonica 4 0 OMT had only a marginal effect. Further characterization of 6OMT in California poppy cells indicated that a 6OMT-specific gene is missing and 4OMT catalyzes the 6OMT reaction with low activity in California poppy, which supports the notion that the 6OMT reaction is important for alkaloid biosynthesis in this plant species. We discuss the importance of 6OMT in benzylisoquinoline alkaloid biosynthesis and the potential for using a rate-limiting step gene to improve alkaloid production.

  • transient rna silencing of scoulerine 9 o methyltransferase expression by double stranded rna in Coptis Japonica protoplasts
    Bioscience Biotechnology and Biochemistry, 2005
    Co-Authors: Joseph G Dubouzet, Nanae Fujii, Takashi Morishige, A N Chungil, Eiichiro Fukusaki, Kentaro Ifuku, Fumihiko Sato
    Abstract:

    RNAi (RNA interference, RNA silencing) is a powerful tool for functional genomics, but the construction of an RNAi vector(s) and the establishment of stable transformants are time-consuming and laborious. Here we report the transient RNAi of endogenous biosynthetic genes involved in isoquinoline alkaloid biosynthesis in Coptis Japonica protoplasts. Double stranded (ds) RNA fragments of various lengths prepared from several different positions of the coding sequence of scoulerine 9-O-methyltransferase (SMT) were introduced into C. Japonica protoplasts by polyethylene glycol-mediated transformation, and their effects were monitored by reverse transcription-polymerase chain reaction. Substantial silencing of SMT gene expression was obtained by the introduction of these SMT dsRNAs. A significant reduction in SMT protein levels was also observed. The potentials of this transient RNAi system to evaluate the functions of biosynthetic genes in Coptis alkaloid research are discussed.

  • Molecular cloning of columbamine O-methyltransferase from cultured Coptis Japonica cells.
    FEBS Journal, 2002
    Co-Authors: Takashi Morishige, Kum Boo Choi, Emilyn G Dubouzet, Kazufumi Yazaki
    Abstract:

    To identify all of the O-methyltransferase genes involved in isoquinoline alkaloid biosynthesis in Coptis Japonica cells, we sequenced 1014 cDNA clones isolated from high-alkaloid-producing cultured cells of C. Japonica. Among them, we found all three reported O-methyltransferases and an O-methyltransferase-like cDNA clone (CJEST64). This cDNA was quite similar to S-adenosyl-l-methionine:coclaurine 6-O-methyltransferase and S-adenosyl-l-methionine:isoflavone 7-O-methyltransferase. As S-adenosyl-l-methionine:columbamine O-methyltransferase, which catalyzes the conversion of columbamine to palmatine, is one of the remaining unelucidated components in isoquinoline alkaloid biosynthesis in C. Japonica, we heterologously expressed the protein in Escherichia coli and examined the activity of columbamine O-methyltransferase. The recombinant protein clearly showed O-methylation activity using columbamine, as well as (S)-tetrahydrocolumbamine, (S)-, (R,S)-scoulerine and (R,S)-2,3,9,10-tetrahydroxyprotoberberine as substrates. This result clearly indicated that EST analysis was useful for isolating the candidate gene in a relatively well-characterized biosynthetic pathway. The relationship between the structure and substrate recognition of the O-methyltransferases involved in isoquinoline alkaloid biosynthesis, and a reconsideration of the biosynthetic pathway to palmatine are discussed.

  • molecular cloning and characterization of coclaurine n methyltransferase from cultured cells of Coptis Japonica
    Journal of Biological Chemistry, 2002
    Co-Authors: Kum Boo Choi, Takashi Morishige, Nobukazu Shitan, Kazufumi Yazaki, Fumihiko Sato
    Abstract:

    S-adenosyl-L-methionine:coclaurine N-methyltransferase (CNMT) converts coclaurine to N-methylcoclaurine in isoquinoline alkaloid biosynthesis. The N-terminal amino acid sequence of Coptis CNMT was used to amplify the corresponding cDNA fragment and later to isolate full-length cDNA using 5'- and 3'-rapid amplification of cDNA ends (RACE). The nucleotide sequence and predicted amino acid sequence showed that the cDNA encoded 358 amino acids, which contained a putative S-adenosyl-L-methionine binding domain and showed relatively high homology to tomato phosphoethanolamine-N-methyltransferase. A recombinant protein was expressed in Escherichia coli, and its CNMT activity was confirmed. Recombinant CNMT was purified to homogeneity, and enzymological characterization confirmed that Coptis CNMT has quite broad substrate specificity, i.e. not only for 6-O-methylnorlaudanosoline and norreticuline but also for 6,7-dimethoxy-1,2,3,4-tetrahydroisoquinoline. The evolution of N-methyltransferases in secondary metabolism is discussed based on sequence similarity.