Expression of (s)-scoulerine 9-O-methyltransferase in Coptis japonica plants

Fujiwara, Hiroyuki; Takeshita, Norimatsu; Terano, Yoshitake; Fitchen, John H.; Tsujita, Tetsuya; Katagiri, Yasuyuki; Sato, Fumihiko; Yamada, Yasuyuki

doi:10.1016/s0031-9422(00)90692-7

Cited by 30 publications

(15 citation statements)

References 9 publications

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“…RNA gel blot and in situ hybridization analyses showed the abundant and preferential expression of Cjmdr1 in the rhizome (Yazaki et al, 2001) and suggested the localization of gene transcripts to xylem tissues (Shitan et al, 2003). In C. japonica, berberine accumulates mainly in the rhizome, whereas alkaloid biosynthetic enzymes have been associated with roots (Fujiwara et al, 1993). CjMDR1 was localized to the plasma membrane and suggested to function as an influx pump in the transport of berberine from roots to rhizomes by unloading protoberberine alkaloids from the xylem (Shitan et al, 2003).…”

Section: Discussionmentioning

confidence: 98%

“…The accumulation of protoberberine alkaloids in the endodermis and pericycle of T. flavum roots also implicates a different transport mechanism compared with C. japonica. It is not known whether berberine biosynthesis in C. japonica roots is associated with endodermis and pericycle, but the products mostly accumulate in the rhizome (Fujiwara et al, 1993). Modulations in the expression and substrate specificity of transporters, such as CjMDR1, might be a major force in the evolution of secondary metabolism in addition to the recruitment of new catalytic functions after gene duplication events (Pichersky and Gang, 2000).…”

Section: Discussionmentioning

confidence: 99%

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Cell Type–Specific Localization of Transcripts Encoding Nine Consecutive Enzymes Involved in Protoberberine Alkaloid Biosynthesis

2005

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Molecular clones encoding nine consecutive biosynthetic enzymes that catalyze the conversion of L-dopa to the protoberberine alkaloid (S)-canadine were isolated from meadow rue (Thalictrum flavum ssp glaucum). The predicted proteins showed extensive sequence identity with corresponding enzymes involved in the biosynthesis of related benzylisoquinoline alkaloids in other species, such as opium poppy (Papaver somniferum). RNA gel blot hybridization analysis showed that gene transcripts for each enzyme were most abundant in rhizomes but were also detected at lower levels in roots and other organs. In situ RNA hybridization analysis revealed the cell type-specific expression of protoberberine alkaloid biosynthetic genes in roots and rhizomes. In roots, gene transcripts for all nine enzymes were localized to immature endodermis, pericycle, and, in some cases, adjacent cortical cells. In rhizomes, gene transcripts encoding all nine enzymes were restricted to the protoderm of leaf primordia. The localization of biosynthetic gene transcripts was in contrast with the tissue-specific accumulation of protoberberine alkaloids. In roots, protoberberine alkaloids were restricted to mature endodermal cells upon the initiation of secondary growth and were distributed throughout the pith and cortex in rhizomes. Thus, the cell type-specific localization of protoberberine alkaloid biosynthesis and accumulation are temporally and spatially separated in T. flavum roots and rhizomes, respectively. Despite the close phylogeny between corresponding biosynthetic enzymes, distinct and different cell types are involved in the biosynthesis and accumulation of benzylisoquinoline alkaloids in T. flavum and P. somniferum. Our results suggest that the evolution of alkaloid metabolism involves not only the recruitment of new biosynthetic enzymes, but also the migration of established pathways between cell types.

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Section: Discussionmentioning

confidence: 98%

Section: Discussionmentioning

confidence: 99%

Cell Type–Specific Localization of Transcripts Encoding Nine Consecutive Enzymes Involved in Protoberberine Alkaloid Biosynthesis

2005

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“…4). A previous study of SMT expression indicated that cultured C. japonica cells with high berberine productivity showed a high expression of SMT, and moderate expression was seen in the root, which is the primary biosynthetic organ in C. japonica (33). When the cDNA expression for CYP719 was compared with three biosynthetic genes (CYP80B2, 6-OMT, and 4Ј-OMT), all four genes showed similar expression profiles: very high expression in cultured cells, moderate expression in root, and scarce expression in other tissues.…”

Section: Fig 5 Lc-ms Analysis Of Cyp719 Reaction Product (A) Authementioning

confidence: 99%

Molecular Cloning and Characterization of CYP719, a Methylenedioxy Bridge-forming Enzyme That Belongs to a Novel P450 Family, from cultured Coptis japonica Cells

Ikezawa

Tanaka

Nagayoshi

et al. 2003

Journal of Biological Chemistry

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180

152

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Two cytochrome P450 (P450) cDNAs involved in the biosynthesis of berberine, an antimicrobial benzylisoquinoline alkaloid, were isolated from cultured Coptis japonica cells and characterized. A sequence analysis showed that one C. japonica P450 (designated CYP719) belonged to a novel P450 family. Further, heterologous expression in yeast confirmed that it had the same activity as a methylenedioxy bridge-forming enzyme (canadine synthase), which catalyzes the conversion of ( S)-tetrahydrocolumbamine ((S)-THC) to (S)-tetrahydroberberine ((S)-THB, (S)-canadine). The other P450 (designated CYP80B2) showed high homology to California poppy (S)-N-methylcoclaurine-3-hydroxylase (CYP80B1), which converts (S)-N-methylcoclaurine to (S)-3-hydroxy-N-methylcoclaurine. Recombinant CYP719 showed typical P450 properties as well as high substrate affinity and specificity for (S)-THC. (S)-Scoulerine was not a substrate of CYP719, indicating that some other P450, e.g. (S)-cheilanthifoline synthase, is needed in (S)-stylopine biosynthesis. All of the berberine biosynthetic genes, including CYP719 and CYP80B2, were highly expressed in selected cultured C. japonica cells and moderately expressed in root, which suggests coordinated regulation of the expression of biosynthetic genes.

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“…Previous findings suggest that SMT activity in Coptis cells was not correlated with the berberine͞coptisine ratio, suggesting that SMT at the branch point (Fig. 2) cannot effectively compete for a rate-limiting intermediate in the biosynthesis of berberine and coptisine (25). To directly evaluate the contribution of SMT at this branch point for the regulation of metabolic flow in berberine and coptisine biosynthesis, we overexpressed Coptis SMT in cultured Coptis 156-1 cells that had been selected as high berberine producers (19).…”

Section: Resultsmentioning

confidence: 88%

Inaugural Article: Metabolic engineering of plant alkaloid biosynthesis

Sato

Hashimoto²,

Hachiya³

et al. 2000

Proceedings of the National Academy of Sciences

102

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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-Omethyltransferase (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.berberine ͉ nicotine ͉ polyamine ͉ sanguinarine ͉ scopolamine H igher plants constitute one of our most important natural resources. They provide not only foodstuffs, fibers, and woods, but many chemicals, such as oils, flavorings, dyes, and pharmaceuticals. Although plants are renewable resources, some species are becoming more difficult to obtain in sufficient amounts to meet increasing demands. Destruction of natural habitats and technical difficulties in cultivation also are driving the drastic reductions in plant availability. For example, it is claimed that the demand for paclitaxel, a potent anticancer compound, could endanger forests of Taxus brevifolia (Pacific yew) because of the low paclitaxel content (40-100 mg͞kg of bark) in and slow growth of the trees (1).For many natural chemicals it is possible to synthesize alternatives from petroleum, coal, or both. The economic limitations of chemical synthesis and the pollution that accompanies this type of chemical synthesis, however, have led to the development of cell culture and molecular engineering of plants for the production of important and commodity chemicals. Plant cell and organ culture offer promising alternatives for the production of chemicals because totipotency enables plant cells and organs to produce useful secondary metabolites in vitro (2). Cell culture is also advantageous in that useful metabolites are obtained under a controlled environment, independent of climatic changes and soil conditions. In addition, the products are free of microbe and insect contamination. Fermentation technology also can be used to produce desired metabolites and can be optimized to maintain high and stable yields of known quality by cellular and molecular breeding techniques to further improve productivity and quality. After extensive empirical trials, some metabolites are now being produced by large-scale cell culture (e.g., shikonin and berberine; ref. 2), but the numbers of compounds that are producible commercially by cell culture technology are still very few. The main limitations are low productivity and the necessity of the down-stream pr...

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Expression of (s)-scoulerine 9-O-methyltransferase in Coptis japonica plants

Cited by 30 publications

References 9 publications

Cell Type–Specific Localization of Transcripts Encoding Nine Consecutive Enzymes Involved in Protoberberine Alkaloid Biosynthesis

Cell Type–Specific Localization of Transcripts Encoding Nine Consecutive Enzymes Involved in Protoberberine Alkaloid Biosynthesis

Molecular Cloning and Characterization of CYP719, a Methylenedioxy Bridge-forming Enzyme That Belongs to a Novel P450 Family, from cultured Coptis japonica Cells

Inaugural Article: Metabolic engineering of plant alkaloid biosynthesis

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