Improvement of Benzylisoquinoline Alkaloid Productivity by Overexpression of 3′-Hydroxy-N-methylcoclaurine 4′-O-Methyltransferase in Transgenic Coptis japonica Plants
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概要
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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.
著者
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Yazaki Kazufumi
Research Institute For Sustainable Humanosphere (rish) Kyoto University
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SHITAN Nobukazu
Department of Natural Medicinal Chemistry, Kobe Pharmaceutical University
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Sato Fumihiko
Division Of Applied Life Sciences Graduate School Of Agriculture Kyoto University
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Shitan Nobukazu
Department Of Natural Medicinal Chemistry Kobe Pharmaceutical University
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Kawahara Nobuo
Tsukuba Div. Res. Center For Medicinal Plant Resources National Inst. Of Biomedical Innovation
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Kiuchi Fumiyuki
Tsukuba Div. Res. Center For Medicinal Plant Resources National Inst. Of Biomedical Innovation
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Yoshimatsu Kayo
Tsukuba Div. Res. Center For Medicinal Plant Resources National Inst. Of Biomedical Innovation
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Inui Takayuki
Tsukuba Division, Research Center for Medicinal Plant Resources, National Institute of Biomedical Innovation
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Kawano Noriaki
Tsukuba Division, Research Center for Medicinal Plant Resources, National Institute of Biomedical Innovation
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Kiuchi Fumiyuki
Tsukuba Division, Research Center for Medicinal Plant Resources, National Institute of Biomedical Innovation
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Yoshimatsu Kayo
Tsukuba Division, Research Center for Medicinal Plant Resources, National Institute of Biomedical Innovation
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Kawahara Nobuo
Tsukuba Division, Research Center for Medicinal Plant Resources, National Institute of Biomedical Innovation
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