2007 Fiscal Year Final Research Report Summary
Characterizafion of gene network in isoquinoline alkaloid biosynthesis and its application
| Project/Area Number |
16208011
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied biochemistry
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| Research Institution | Kyoto University |
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Principal Investigator |
SATO Fumihiko Kyoto University, Graduate School of Biostudies, Professor (10127087)
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| Co-Investigator(Kenkyū-buntansha) |
IFUKU Kentaro Kyoto University, Graduate School of Biostudies, Assistant Professor (50359783)
YOSHIMATSU Kayo National Institute of Biomedical Innovation, Research Center for Medicinal Plant Resources, Laboratory Chief (20184418)
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| Project Period (FY) |
2004 – 2007
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| Keywords | isoquinoline alkaloids / metabolic network / transcriptional regulation / metabolic engineering / transient RNAI / reconstruction of biosynthesis in microbe |
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| Research Abstract |
1. Isolation and characterization of biosynthetic enzyme genes
(1) Identification and characterization of norcoclaurine synthase and tetrahydroberberine oxidase in berberine biosynthesis from Coptis japonica
(2) Identification and characterization of corytuberine synthase (CYP80G2) from Coptis japonica in magnoflorine biosynthesis
(3) Identification and characterization of stylopine synthase (CYP719A2/A3) from California poppy in sanguinarine biosynthesis
2. Metabolic engineering of isoquinoline alkaloid biosynthesis using biosynthetic genes isolated
(1) Increase of the isoquinoline alkaloid production by the overexpression of rate-limiting enzyme gene; e.g., overexpression of norcoclaurine 6-O-methyltransferase isolated from Coptis japonica in California poppy cells
(2) Accumulation of intermediate by the metabolic block using gene silencing with RNAi technology; e.g., reticuline accumulation in transgenic California poppy cells transformed with RNAi vector for berberine bridge enzyme
(3) Pr
… More
oduction of novel metabolites by the introduction of new branch pathway ; e.g., introduction of scoulerine 9-O- methyltransferase isolated from Coptis japonica in California poppy cells. Modification of metabolite profiles in transgenic cells has been characterized with LC-MS, LC-NMR.
(4) Increase of isoquinolie alkaloid accumulation by the overexpression of biosynthetic gene (3'-hydroxy N-methylcoclaurine 4' O-methyltransferase) was determined in transgenic Coptis japonica plants.
3. Characterization of transcriptional regulation system in isoquinoline alkaloid biosynthesis
(1) Development of novel screening system to isolate transcriptional factor genes using transient RNAi in Coptis japonica protoplasts.
(2) Identification of WRKY1 and bHLH1 as the comprehensive gene regulator specific for isoquinoline alkaloid biosynthesis in Coptis japonica cells
(3) Isolation of promoter sequences for some biosynthetic genes in isoquinoline alkaloid biosynthesis in Coptis japonica and characterization of promoter activities. Minimum promoter sequences for the expression in Coptis japonica protoplasts were identified.
4. Reconstruction of isoquinoline alkaloid biosynthesis in microbe
(1) Using isolated biosynthetic genes, isoquinoline alkaloid biosynthetic pathway from dopamine to reticuline was reconstructed in Escherichia coli.
(2) Using budding yeast with CYP80G2 gene and above transgenic E. coli, magnoflorine was produced from dopamine Less
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[Journal Article] Microbial production of plant benzylisoquinoline alkaloids2008
Author(s)
Minami, N., Kim, J-S., Ikezawa, N., Takemura, T., Katayama, T., Kumagai, H., and Sato, F.
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Journal Title
Proc. Natl. Acad. Sci. USA 105
Pages: 7393-7398
Description
「研究成果報告書概要(和文)」より
Peer Reviewed
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[Journal Article] Microbial production of plant benzylisoquinoline alkaloids2008
Author(s)
Minami, N., Kim, J-S., Ikezawa, N., Takemura, T., Katayama, T., Kumagai, H., Sato, F
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Journal Title
Proc. Natl. Acad. Sci., USA 105
Pages: 7393-7398
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Identification of a WRKY protein as a transcriptional regulator of benzylisoquinoline alkaloid biosynthesis in Coptis japonica2007
Author(s)
Kato, N., Dubouzet, E., Kokabu, Y., Yoshida, S., Dubouzet, J., Yazaki, K., Sato, F
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Journal Title
Plant Cell Physiol 48
Pages: 8-18
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Characterization of vacuolar transport of the endogenous alkaloid berberine in Coptis japonica.2005
Author(s)
Ohtani, M., Shitan, N., Sakai, K., M artinoia, E., Sato, F., Yazaki, K.
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Journal Title
Plant Physiology 138
Pages: 1939-1946
Description
「研究成果報告書概要(和文)」より
Peer Reviewed
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[Journal Article] Transient RNA Silencing of Scoulerine 9-O-Methyltransferase Expression by Double Stranded RNA in Coptis japonica Protoplasts.2005
Author(s)
Dubouzet, J. G., Morishige, T., Fujii, N., An, C-L., Fukusaki, E., Ifuku, K. and Sato, F.
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Journal Title
Biosci. Biotech. Biochem. 69
Pages: 63-70
Description
「研究成果報告書概要(和文)」より
Peer Reviewed
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[Journal Article] Characterization of vacuolar transport of the endogenous alkaloid berberine in Coptis japonica2005
Author(s)
Ohtani, M., Shitan, N., Sakai, K., Martinoia, E., Sato, F., Yazaki, K
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Journal Title
Plant Physiol 138
Pages: 1939-1946
Description
「研究成果報告書概要(欧文)」より
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[Journal Article] Transient RNA Silencing of Scoulerine 9-O-Methyltransferase Expression by Double Stranded RNA in Coptis japonica Protoplasts2005
Author(s)
Dubouzet, J.G., Morishige, T., Fujii, N., An, C-I., Fukusaki, E., Ifuku, K., Sato,F
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Journal Title
Biosci. Biotech. Biochem 69
Pages: 63-70
Description
「研究成果報告書概要(欧文)」より
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[Presentation] Functional analysis of WRKY transcription factor in isoquinoline alkaloid biosynthesis2006
Author(s)
Kato, N., Kokabu, Y., Yoshida, S., Taniguchi, Y., Dubouzet, E., Dubouzet, J., and Sato, F.
Organizer
20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress
Place of Presentation
Kyoto, Japan
Year and Date
20060618-23
Description
「研究成果報告書概要(和文)」より
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[Presentation] Functional analysis of WRKY transcription factor in isoquinoline alkaloid biosynthesis (poster presentation)2006
Author(s)
Kato. N., Kokabu, Y., Yoshida, S., Taniguchi, Y., Dubouzet, E., Dubouzet, J., Sato, F
Organizer
20th IUBMB International Congress of Biochemistry and Molecular Biology and 11th FAOBMB Congress
Place of Presentation
Kyoto, japan
Year and Date
20060618-23
Description
「研究成果報告書概要(欧文)」より
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[Presentation] Molecular doning of methylenedioxy bridge-forming enzymes that belong to a novel P450 family from isoqwinoline-alkaloid producing cells2004
Author(s)
Ikezawa, N., Tanaka, M., Shinkyo, R., Sakaki, T., Inouye, K., and Sato, F.
Organizer
7th International Symposium on Cytochrome P450 Biodiver slty and Blotechnology
Place of Presentation
Awaji Yumebutai International Conference Center, Hyogo
Year and Date
20040801-05
Description
「研究成果報告書概要(和文)」より
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[Presentation] オウレンの分子育種の効率化2004
Author(s)
吉松嘉代、士反伸和、木内文之、佐藤文彦、矢崎一史
Organizer
第22回日本植物細胞分子生物学会秋田大会
Place of Presentation
秋田県立大学
Year and Date
2004-08-10
Description
「研究成果報告書概要(和文)」より
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