Transient RNA Silencing of Scoulerine 9-<i>O</i>-Methyltransferase Expression by Double Stranded RNA in<i>Coptis japonica</i>Protoplasts

Dubouzet, Joseph G.; Morishige, Takashi; Fujii, Nanae; An, Chung-Il; Fukusaki, Eiichiro; Ifuku, Kentaro; Sato, Fumihiko

doi:10.1271/bbb.69.63

Cited by 20 publications

(17 citation statements)

References 19 publications

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“…Immunoprecipitation with antiserum against CjNCS1 clearly indicated that native NCS in C. japonica cells consisted of CjNCS1 and not CjPR10A. Our preliminary characterization of the function of CjNCS1 using the transient RNA interference in C. japonica protoplasts (35) showed that accumulation of berberine clearly decreased with double-stranded RNA of CjNCS1, supporting the involvement of CjNCS1 in berberine biosynthesis. 6 Although recombinant CjNCS1 expressed in E. coli showed NCS activity, this activity was lower than that of native enzyme on a protein basis as estimated by immunoblot analysis.…”

Section: Discussionmentioning

confidence: 59%

Functional Analysis of Norcoclaurine Synthase in Coptis japonica

Minami

Dubouzet

Iwasa

et al. 2007

Journal of Biological Chemistry

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(S)-Norcoclaurine is the entry compound in benzylisoquinoline alkaloid biosynthesis and is produced by the condensation of dopamine and 4-hydroxyphenylacetaldehyde (4-HPAA) by norcoclaurine synthase (NCS) (EC 4.2.1.78). Although cDNA of the pathogenesis-related (PR) 10 family, the translation product of which catalyzes NCS reaction, has been isolated from Thalictrum flavum, its detailed enzymological properties have not yet been characterized. We report here that a distinct cDNA isolated from Coptis japonica (CjNCS1) also catalyzed NCS reaction as well as a PR10 homologue of C. japonica (CjPR10A). Both recombinant proteins stereo-specifically produced (S)-norcoclaurine by the condensation of dopamine and 4-HPAA. Because a CjNCS1 cDNA that encoded 352 amino acids showed sequence similarity to 2-oxoglutarate-dependent dioxygenases of plant origin, we characterized the properties of the native enzyme. Sequence analysis indicated that CjNCS1 only contained a Fe 2؉ -binding site and lacked the 2-oxoglutarate-binding domain. In fact, NCS reaction of native NCS isolated from cultured C. japonica cells did not depend on 2-oxoglutarate or oxygen, but did require ferrous ion. On the other hand, CjPR10A showed no specific motif. The addition of o-phenanthroline inhibited NCS reaction of both native NCS and recombinant CjNCS1, but not that of CjPR10A. In addition, native NCS and recombinant CjNCS1 accepted phenylacetaldehyde and 3,4-dihydroxyphenylacetaldehyde, as well as 4-HPAA, for condensation with dopamine, whereas recombinant CjPR10A could use 4-hydroxyphenylpyruvate and pyruvate in addition to the above aldehydes. These results suggested that CjNCS1 is the major NCS in C. japonica, whereas native NCS extracted from cultured C. japonica cells was more active and formed a larger complex compared with recombinant CjNCS1.Higher plants produce divergent chemicals such as alkaloids, terpenoids, and phenolic compounds in secondary metabolism. Among these chemicals, alkaloids are very important in medicine because of their high biological activities. Alkaloids are low molecular weight, nitrogen-containing compounds that are found in ϳ20% of plant species. Most alkaloids are derived from amines produced by the decarboxylation of amino acids such as histidine, lysine, ornithine, tryptophan, and tyrosine. Although the coupling of amines to other products is the first important step in producing diverse alkaloids, this entry reaction has been poorly characterized. (S)-Strictosidine is a central intermediate for indole alkaloids. Strictosidine synthase, which catalyzes the formation of (S)-strictosidine from tryptamine and secologanin, is a rare exception in that its cDNA has been cloned from Catharanthus roseus and Rauvolfia serpentine (1, 2).Benzylisoquinoline alkaloids are a large and diverse group of pharmaceutical alkaloids with ϳ2,500 defined structures. Norcoclaurine produced from tyrosine is a key entry compound from which various benzylisoquinoline alkaloids such as analgesic morphine, colchicines, antibacterial berberine...

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Section: Discussionmentioning

confidence: 59%

Functional Analysis of Norcoclaurine Synthase in Coptis japonica

Minami

Dubouzet

Iwasa

et al. 2007

Journal of Biological Chemistry

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126

111

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“…4B, C). [81][82][83][84] The latter approach in particular can be very useful for inducing an accumulation of key intermediates by reducing main or side reaction, and then redirecting the flow to produce the desired metabolites, with additional expression of a branch-or key-pathway enzyme. In fact, the power of RNAi in suppressing gene expression and the accumulation of key intermediates, e.g., reticuline, has been demonstrated with RNAi of the berberine-bridge enzyme gene (BBE) (Fig.…”

Section: Metabolic Engineering In Cultured Cellsmentioning

confidence: 99%

Characterization of Plant Functions Using Cultured Plant Cells, and Biotechnological Applications

Sato¹

2013

Bioscience, Biotechnology, and Biochemistry

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“…The recent identification of the transcription factors CjWRKY1 and CjbHLH1 as possible general regulators in IQA biosynthesis using a transient RNAi screening system could represent a significant discovery in the field and provide significant improvements in the yields of IQAs by enabling the regulation of the transcription processes associated with their production. 42–44) …”

Section: Clarification Of the Molecular Basis Of The Iqa Biosyntheticmentioning

confidence: 99%

Microbial production of isoquinoline alkaloids as plant secondary metabolites based on metabolic engineering research

Sato

Kumagai

2013

Proceedings of the Japan Academy. Ser. B: Physical and Biologic

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Plants produce a variety of secondary metabolites that possess strong physiological activities. Unfortunately, however, their production can suffer from a variety of serious problems, including low levels of productivity and heterogeneous quality, as well as difficulty in raw material supply. In contrast, microorganisms can be used to produce their primary and some of their secondary metabolites in a controlled environment, thus assuring high levels of efficiency and uniform quality. In an attempt to overcome the problems associated with secondary metabolite production in plants, we developed a microbial platform for the production of plant isoquinoline alkaloids involving the unification of the microbial and plant metabolic pathways into a single system. The potential applications of this system have also been discussed.

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Transient RNA Silencing of Scoulerine 9-O-Methyltransferase Expression by Double Stranded RNA inCoptis japonicaProtoplasts

Cited by 20 publications

References 19 publications

Functional Analysis of Norcoclaurine Synthase in Coptis japonica

Functional Analysis of Norcoclaurine Synthase in Coptis japonica

Characterization of Plant Functions Using Cultured Plant Cells, and Biotechnological Applications

Microbial production of isoquinoline alkaloids as plant secondary metabolites based on metabolic engineering research

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