Improvement of benzylisoquinoline alkaloid productivity by overexpression of 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase in transgenic Coptis japonica plants

Takayuki Inui, Noriaki Kawano, Nobukazu Shitan, Kazufumi Yazaki, Fumiyuki Kiuchi, Nobuo Kawahara, Fumihiko Sato, Kayo Yoshimatsu

研究成果: Article

10 引用 (Scopus)

抄録

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.

元の言語English
ページ(範囲)650-659
ページ数10
ジャーナルBiological and Pharmaceutical Bulletin
35
発行部数5
DOI
出版物ステータスPublished - 2012 5

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Coptis
Benzylisoquinolines
Berberine
Methyltransferases
Alkaloids
Rhizome
Metabolic Engineering
Agrobacterium
Pharmacopoeias
Genetically Modified Plants
Gastroenteritis
Pharmaceutical Preparations
Real-Time Polymerase Chain Reaction
Cholesterol
High Pressure Liquid Chromatography
Enzymes
Growth

ASJC Scopus subject areas

  • Pharmaceutical Science
  • Pharmacology

これを引用

Improvement of benzylisoquinoline alkaloid productivity by overexpression of 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase in transgenic Coptis japonica plants. / Inui, Takayuki; Kawano, Noriaki; Shitan, Nobukazu; Yazaki, Kazufumi; Kiuchi, Fumiyuki; Kawahara, Nobuo; Sato, Fumihiko; Yoshimatsu, Kayo.

:: Biological and Pharmaceutical Bulletin, 巻 35, 番号 5, 05.2012, p. 650-659.

研究成果: Article

Inui, Takayuki ; Kawano, Noriaki ; Shitan, Nobukazu ; Yazaki, Kazufumi ; Kiuchi, Fumiyuki ; Kawahara, Nobuo ; Sato, Fumihiko ; Yoshimatsu, Kayo. / Improvement of benzylisoquinoline alkaloid productivity by overexpression of 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase in transgenic Coptis japonica plants. :: Biological and Pharmaceutical Bulletin. 2012 ; 巻 35, 番号 5. pp. 650-659.
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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.",
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T1 - Improvement of benzylisoquinoline alkaloid productivity by overexpression of 3′-hydroxy-N-methylcoclaurine 4′-O-methyltransferase in transgenic Coptis japonica plants

AU - Inui, Takayuki

AU - Kawano, Noriaki

AU - Shitan, Nobukazu

AU - Yazaki, Kazufumi

AU - Kiuchi, Fumiyuki

AU - Kawahara, Nobuo

AU - Sato, Fumihiko

AU - Yoshimatsu, Kayo

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N2 - 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.

AB - 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.

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KW - Coptis japonica

KW - Metabolic engineering

KW - Transgenic plant

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