Molecular Cloning and Characterization of a cDNA for Glycyrrhiza glabra Cycloartenol Synthase.

Hayashi, Hiroaki; Hiraoka, Noboru; Ikeshiro, Yasumasa; Kushiro, Toshio; Morita, Masayo; Shibuya, Masabumi; Ebizuka, Yutaka

doi:10.1248/bpb.23.231

Cited by 55 publications

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“…18) A cDNA library prepared from cultured licorice cells was screened by the DIG-labeled probe under conditions of low stringency as reported. 18) Nine positive clones, isolated from 2ϫ10 5 plaques, were subcloned into pBluescript SK (Ϫ) by in vivo excision.…”

Section: Plant Materials and Culture Conditionsmentioning

confidence: 99%

“…15) cDNAs of cycloartenol synthase, an OSC involved in sterol biosynthesis, and b-amyrin synthase, an OSC involved in both glycyrrhizin and soyasaponin biosynthesis, have been cloned from G. glabra. 18,19) In addition to these two OSCs, the third OSC, lupeol synthase, which is responsible for the biosynthesis of betulinic acid, most likely exists in cultured licorice cells. In the present study, an OSC cDNA for lupeol synthase was isolated by heterologous hybridization with that of olive lupeol synthase, 20) and the patterns of mRNA expression of the three OSCs in the cultured cells and the intact plants of G. glabra were examined.…”

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confidence: 99%

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Differential Expression of Three Oxidosqualene Cyclase mRNAs in Glycyrrhiza glabra

Hayashi

Huang

Takada

et al. 2004

Biological & Pharmaceutical Bulletin

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Higher plants produce not only sterols but also various triterpenes, which are derived from six isoprene units, 1) and some triterpenes are further glycosylated to produce various triterpene saponins.2) These triterpenes and triterpene saponins are believed to participate in the plant defence systems against microbial pathogens or insects. [3][4][5] An interesting effect that has also been reported is that an exogenous triterpene saponin stimulates the growth of roots in several plants. 6,7) In addition to the benefit for plants themselves, these triterpenes and saponins are beneficial for human beings as medicines, sweeteners, detergents, and cosmetics. Although structural elucidation of triterpenes and saponins has been extensively studied, 1,2) our understanding about the regulation of their biosyntheses is quite limited at the molecular level. 8)Oxidosqualene cyclases (OSCs) catalyze the cyclization of 2,3-oxidosqualene, a common intermediate of both sterols and triterpenes. Lanosterol synthase, playing a crucial role in cholesterol biosynthesis, is a unique OSC in mammals. In higher plants, on the other hand, some OSCs responsible for triterpene biosynthesis co-exist with cycloartenol synthase corresponding to lanosterol synthase in mammals. 8,9) Up to now, cDNAs of OSCs including cycloartenol synthase, bamyrin synthase, lupeol synthase and multifunctional triterpene synthase have been functionally characterized from various plant species. 8) After cloning of these OSC genes, our interest has shifted the regulatory roles of these OSCs in triterpene and saponin biosyntheses in higher plants. 8,[10][11][12][13] In our study, licorice (Glycyrrhiza glabra L., Fabaceae) was used as a model plant to elucidate the regulation of triterpene biosyntheses in higher plants (Fig. 1). Cultured licorice cells lacked the ability to produce glycyrrhizin, a sweet oleanane-type triterpene saponin produced in the roots of the mother plant, but produced phytosterols and two structurally different triterpenoid constituents, soyasaponins and betulinic acid. 15,16) Glycyrrhizin is localized exclusively in the woody parts of the thickened roots and stolons, whereas soyasaponins, oleanane-type triterpene saponins, are localized mainly in the seeds and rootlets of licorice.17) On the other hand, the content of betulinic acid, a lupane-type triterpene, is high in the cork layer of thickened licorice roots. 15) cDNAs of cycloartenol synthase, an OSC involved in sterol biosynthesis, and b-amyrin synthase, an OSC involved in both glycyrrhizin and soyasaponin biosynthesis, have been cloned from G. glabra. 18,19) In addition to these two OSCs, the third OSC, lupeol synthase, which is responsible for the biosynthesis of betulinic acid, most likely exists in cultured licorice cells. In the present study, an OSC cDNA for lupeol synthase was isolated by heterologous hybridization with that of olive lupeol synthase, 20) and the patterns of mRNA expression of the three OSCs in the cultured cells and the intact plants of G. glabra were examined. ...

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Section: Plant Materials and Culture Conditionsmentioning

confidence: 99%

mentioning

confidence: 99%

Differential Expression of Three Oxidosqualene Cyclase mRNAs in Glycyrrhiza glabra

Hayashi

Huang

Takada

et al. 2004

Biological & Pharmaceutical Bulletin

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“…Phytosterols, such as campesterol and sitosterol, are biosynthesized via cycloartenol and catalyzed by cycloartenol synthase (CAS) in higher plants ( Fig. 1) (4,5). Recently, however, three different laboratories have identified LAS genes from dicotyledonous plant species including Arabidopsis thaliana (6,7), Panax ginseng (6), and Lotus japonica (8) using a yeast expression system.…”

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Dual biosynthetic pathways to phytosterol via cycloartenol and lanosterol in Arabidopsis

Ohyama

Suzuki²,

Kikuchi³

et al. 2009

Proc. Natl. Acad. Sci. U.S.A.

180

138

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The differences between the biosynthesis of sterols in higher plants and yeast/mammals are believed to originate at the cyclization step of oxidosqualene, which is cyclized to cycloartenol in higher plants and lanosterol in yeast/mammals. Recently, lanosterol synthase genes were identified from dicotyledonous plant species including Arabidopsis, suggesting that higher plants possess dual biosynthetic pathways to phytosterols via lanosterol, and through cycloartenol. To identify the biosynthetic pathway to phytosterol via lanosterol, and to reveal the contributions to phytosterol biosynthesis via each cycloartenol and lanosterol, we performed feeding experiments by using [6-13 C 2 H3]mevalonate with Arabidopsis seedlings. Applying 13 C-{ 1 H}{ 2 H} nuclear magnetic resonance (NMR) techniques, the elucidation of deuterium on C-19 behavior of phytosterol provided evidence that small amounts of phytosterol were biosynthesized via lanosterol. The levels of phytosterol increased on overexpression of LAS1, and phytosterols derived from lanosterol were not observed in a LAS1-knockout plant. This is direct evidence to indicate that the biosynthetic pathway for phytosterol via lanosterol exists in plant cells. We designate the biosynthetic pathway to phytosterols via lanosterol ''the lanosterol pathway.'' LAS1 expression is reported to be induced by the application of jasmonate and is thought to have evolved from an ancestral cycloartenol synthase to a triterpenoid synthase, such as ␤-amyrin synthase and lupeol synthase. Considering this background, the lanosterol pathway may contribute to the biosynthesis of not only phytosterols, but also steroids as secondary metabolites. metabolic diversity ͉ mevalonate ͉ sterol ͉ tracer experiment ͉ triterpene

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“…In G. glabra, three OSCs: β-amyrin synthase (bAS), lupeol synthase (LUS) and cycloartenol synthase (CAS) were cloned and characterized. These three enzymes are responsible for the branching biosynthesis step of oleanane-type triterpene saponins (glycyrrhizin and soyasaponins), lupane-type triterpene and phytosterols, respectively (Hayashi et al, 2000;Hayashi et al, 2001). Cytochrome P450s play critical roles in oxidative reactions during the biosynthesis of diverse natural plant products, including terpenoids.…”

Section: Introductionmentioning

confidence: 99%

“…Cytochrome P450s play critical roles in oxidative reactions during the biosynthesis of diverse natural plant products, including terpenoids. β-amyrin oxidation in C-11 and C-30 positions produced glycyrrhizin, C-22 and C-24 positions led to the soyasaponins production (Hayashi et al, 2000;Hayashi et al, 2001;Hayashi and Sudo, 2009;Seki et al, 2008). β-amyrin 11-oxidase catalyze two sequential oxidation steps of β -amyrin at the C-11 position to yield 11-oxo-β-amyrin (Seki et al, 2008).…”

Section: Introductionmentioning

confidence: 99%

Isolation, cloning and bioinformatics analysis of β-amyrin 11-oxidase coding sequence from licorice

Shirazi¹,

Aalami

Tohidfar³

et al. 2016

Plant Omics

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Licorice is the roots and stolons of Glycyrrhiza uralensis which have several chemical compounds. Triterpene saponins such as glycyrrhizin and glycyrrhetinic acid and flavonoids like liquiritin, isoliquiritigenin and glabron are main compounds detected in liquorice root. The plant's major constituent is a glycyrrhizin. The β-amyrin 11-oxidase catalyzes the sequential two-step oxidation of β-amyrin in C-11 to produce 11-oxo-β-amyrin, a possible biosynthetic intermediate between β-amyrin and glycyrrhizin. In this study, the total RNA was extracted from licorice roots and cDNA synthesis, then PCR products were cloned into pTZ57R/T vector. Sequencing confirmed piece length of 1482 bp that encodes a protein of 493 amino acid residues. The results of alignment showe d 99% similarity to β-amyrin sequence of Glycyrrhiza uralensis. Subcellular studies using Softberry and Psort software showed that the activity of this protein is in endoplasmic reticulum. Moreover the protein has a signal peptide and is targeted to the secretory pathway. The results of phylogenetic tree determined most similar amino acid sequence to the CYP88D subfamily of cytochrome P450. These findings can be used for nucleotide or protein manipulation and transformation.

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Molecular Cloning and Characterization of a cDNA for Glycyrrhiza glabra Cycloartenol Synthase.

Cited by 55 publications

References 0 publications

Differential Expression of Three Oxidosqualene Cyclase mRNAs in Glycyrrhiza glabra

Differential Expression of Three Oxidosqualene Cyclase mRNAs in Glycyrrhiza glabra

Dual biosynthetic pathways to phytosterol via cycloartenol and lanosterol in Arabidopsis

Isolation, cloning and bioinformatics analysis of β-amyrin 11-oxidase coding sequence from licorice

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