Improved metabolic action of a bacterial lysine decarboxylase gene in tobacco hairy root cultures by its fusion to arbcS transit peptide coding sequence

Herminghaus, S.; Tholl, Dorothea; Rügenhagen, C.; Fecker, Lothar F.; Leuschner, Carola; Berlin, Jochen

doi:10.1007/bf01969709

Cited by 36 publications

(21 citation statements)

References 25 publications

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“…Our results are in agreement with the enhanced cadaverine and anabasine levels observed after feeding lysine to hairy roots of N. tabacum expressing a bacterial LDC gene (Fecker et al 1993). It has been shown that the accumulation of cadaverine and anabasine could be further enhanced by expressing bacterial LDC fused to a chloroplast signal peptides in plant cells (Herminghaus 1996). Although the La-L/ ODC used in this study was targeted to chloroplasts, the La-L/ODC-expressing line produced only 2 times higher anabasine compared to the control line (Figure 2) (Bunsupa, et al 2012a).…”

Section: Discussionsupporting

confidence: 90%

Revisiting anabasine biosynthesis in tobacco hairy roots expressing plant lysine decarboxylase gene by using <sup>15</sup>N-labeled lysine

Bunsupa

Komastsu

Nakabayashi

et al. 2014

Plant Biotechnology

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Anabasine is an alkaloid found in a small number of Nicotiana species. The components of the anabasine biosynthetic pathway have yet to be identified. Here, we report the reinvestigation of biosynthetic pathways of anabasine and related tobacco alkaloids in genetically engineered cells. Hairy roots of N. tabacum harboring a lysine/ornithine decarboxylase gene from Lupinus angustifolius (La-L/ODC) were fed with labeled [ε-Relative to the unfed control, feeding of labeled 15 N-L-lysine greatly enhanced anabasine levels 13.5-fold in La-L/ODC-expressing line compared to 5.3-fold in the control line, suggesting that both LDC activity and substrate supplied are important factors for the efficient production of anabasine. GUS-expressing line showed preferential incorporation of [ε-15 N]-L-lysine into anabasine, indicating the main biosynthetic pathway of Δ 1 -piperideine intermediate in tobacco is asymmetrically processes. In contrast, the expression of La-L/ODC showed the symmetric labeling of 15 N atom into anabasine, implying the occurrence of free cadaverine, which is produced by La-L/ODC enzyme, during the biosynthesis of Δ 1 -piperideine intermediate. No considerable incorporation of 15 N into other tobacco alkaloids such as, nicotine, anatabine, and anatalline, was detected. Detailed analysis using ultra-high resolution mass spectrometry indicated that two 15 N atoms were incorporated into anabasine in La-L/ODC-expressing lines after feeding [ε-15 N]-L-lysine. Our results not only provide information insight into the biosynthesis of anabasine but also suggest an alternative route for the production of anabasine by genetic engineering.

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Section: Discussionsupporting

confidence: 90%

Revisiting anabasine biosynthesis in tobacco hairy roots expressing plant lysine decarboxylase gene by using <sup>15</sup>N-labeled lysine

Bunsupa

Komastsu

Nakabayashi

et al. 2014

Plant Biotechnology

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“…This localization of La-L/ODC is in agreement with the localization of ODC in chloroplasts (at least partial) (Andreadakis and Kotzabasis, 1996;Tassoni et al, 2003), supporting a hypothesis that L/ODC evolved from an ancestor ODC (as discussed below). Additionally, it was reported that the metabolic action of bacterial LDC in transgenic plants was improved by its fusion to a chloroplast-targeting signal sequence (Herminghaus et al, 1996).…”

Section: Discussion Physiological Roles Of Ldc In Qa-producing Plantsmentioning

confidence: 99%

Lysine Decarboxylase Catalyzes the First Step of Quinolizidine Alkaloid Biosynthesis and Coevolved with Alkaloid Production in Leguminosae

et al. 2012

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Lysine decarboxylase (LDC) catalyzes the first-step in the biosynthetic pathway of quinolizidine alkaloids (QAs), which form a distinct, large family of plant alkaloids. A cDNA of lysine/ornithine decarboxylase (L/ODC) was isolated by differential transcript screening in QA-producing and nonproducing cultivars of Lupinus angustifolius. We also obtained L/ODC cDNAs from four other QA-producing plants, Sophora flavescens, Echinosophora koreensis, Thermopsis chinensis, and Baptisia australis. These L/ODCs form a phylogenetically distinct subclade in the family of plant ornithine decarboxylases. Recombinant L/ODCs from QA-producing plants preferentially or equally catalyzed the decarboxylation of L-lysine and L-ornithine. L. angustifolius L/ODC (La-L/ODC) was found to be localized in chloroplasts, as suggested by the transient expression of a fusion protein of La-L/ODC fused to the N terminus of green fluorescent protein in Arabidopsis thaliana. Transgenic tobacco (Nicotiana tabacum) suspension cells and hairy roots produced enhanced levels of cadaverine-derived alkaloids, and transgenic Arabidopsis plants expressing (La-L/ODC) produced enhanced levels of cadaverine, indicating the involvement of this enzyme in lysine decarboxylation to form cadaverine. Site-directed mutagenesis and protein modeling studies revealed a structural basis for preferential LDC activity, suggesting an evolutionary implication of L/ODC in the QAproducing plants. INTRODUCTIONAlkaloids are one of the most diverse groups of natural products and are found in ;20% of plant species. Many of the ;12,000 known alkaloids produced by plants display potent pharmacological activities and several are widely used as pharmaceuticals (Croteau et al., 2000;De Luca and St Pierre, 2000). Alkaloids are derived from the products of primary metabolism with amino acids such as Phe, Tyr, Trp, Orn, and Lys serving as their main precursors (Facchini, 2001). Quinolizidine alkaloids (QAs) are derived from cadaverine, and several hundred structurally related compounds have been identified that are distributed mostly within the Leguminosae (Ohmiya et al., 1995;Michael, 2008). Some QAs exhibit beneficial pharmacological properties, such as cytotoxic, antiarrhythmic, oxytocic, hypoglycemic, and antipyretic activities, and thus can be used as drugs (Ohmiya et al., 1995). They can therefore serve as potential starting compounds in the development of new drugs and in pest control for plants .QAs are synthesized through the cyclization of the cadaverine unit (Golebiewski and Spenser, 1988), which is produced through the action of Lys decarboxylase (LDC; EC 4.1.1.18) (Figure 1), via a postulated enzyme-bound intermediate . The various skeletons of QAs [e.g., bicyclic alkaloids such as (2)-lupinine and (+)-epilupinine or tetracyclic alkaloids such as (+)-multiflorine, (+)-lupanine, and (+)-matrine] are then further modified by tailoring reactions (e.g., dehydrogenation, oxygenation, esterification, and glycosylation) to yield hundreds of structurally related alkaloids (Ohmiya ...

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“…Tryptophan and the terpenoid precursor geraniol are synthesized in the plastids, tryptophan is then decarboxylated in the cytosol, and the two moieties are condensed in the vacuole (De Luca and St-Pierre, 2000). This compartmentation is particularly important to consider as incorrect targeting of transgenes will leave them without the necessary substrates (Herminghaus et al, 1996).…”

Section: Considerations For Plant Metabolic Engineeringmentioning

confidence: 99%

Metabolic Engineering of Plants for Alkaloid Production

Hughes

Shanks

2002

Metabolic Engineering

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Improved metabolic action of a bacterial lysine decarboxylase gene in tobacco hairy root cultures by its fusion to arbcS transit peptide coding sequence

Cited by 36 publications

References 25 publications

Revisiting anabasine biosynthesis in tobacco hairy roots expressing plant lysine decarboxylase gene by using <sup>15</sup>N-labeled lysine

Revisiting anabasine biosynthesis in tobacco hairy roots expressing plant lysine decarboxylase gene by using <sup>15</sup>N-labeled lysine

Lysine Decarboxylase Catalyzes the First Step of Quinolizidine Alkaloid Biosynthesis and Coevolved with Alkaloid Production in Leguminosae

Metabolic Engineering of Plants for Alkaloid Production

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