Engineering Plant Alkaloid Biosynthetic Pathways: Progress and Prospects

Kutchan, Toni M.; Frick, Susanne; Weid, Marion

doi:10.1016/s1755-0408(07)01010-7

Cited by 13 publications

(10 citation statements)

References 107 publications

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“…However, the molecular mechanism underlying QA biosynthesis is poorly understood, unlike the mechanisms of biosynthesis of other plant alkaloids, such as tropane, isoquinoline, and indole alkaloids (De Luca and St Pierre, 2000;Facchini, 2001;Sato et al, 2007;Kutchan et al, 2008). Very few studies have aimed to characterize the enzymes and regulatory mechanisms involved in QA biosynthesis.…”

Section: Introductionmentioning

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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Section: Introductionmentioning

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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“…Whilst tropane alkaloids of W. somnifera , despite being considered its important secondary metabolites, have not been studied for the alkaloid biosynthesis. Biosynthetic studies on other tropane alkaloid producer Solanaceae plants like Hyoscyamus muticus , Atropa belledona , Hysocyamus niger have led to the concept of roots-limited synthesis of these compounds followed by their translocation (acropetal transport) through xylem to leaves for sequestration [37]. This prompted us to initiate investigation of the tropane alkaloid biosynthesis in W. somnifera through classical molecular biology approach.…”

Section: Introductionmentioning

confidence: 99%

Tropine Forming Tropinone Reductase Gene from Withania somnifera (Ashwagandha): Biochemical Characteristics of the Recombinant Enzyme and Novel Physiological Overtones of Tissue-Wide Gene Expression Patterns

et al. 2013

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Withania somnifera is one of the most reputed medicinal plants of Indian systems of medicine synthesizing diverse types of secondary metabolites such as withanolides, alkaloids, withanamides etc. Present study comprises cloning and E. coli over-expression of a tropinone reductase gene (WsTR-I) from W. somnifera, and elucidation of biochemical characteristics and physiological role of tropinone reductase enzyme in tropane alkaloid biosynthesis in aerial tissues of the plant. The recombinant enzyme was demonstrated to catalyze NADPH-dependent tropinone to tropine conversion step in tropane metabolism, through TLC, GC and GC-MS-MS analyses of the reaction product. The functionally active homodimeric ∼60 kDa enzyme catalyzed the reaction in reversible manner at optimum pH 6.7. Catalytic kinetics of the enzyme favoured its forward reaction (tropine formation). Comparative 3-D models of landscape of the enzyme active site contours and tropinone binding site were also developed. Tissue-wide and ontogenic stage-wise assessment of WsTR-I transcript levels revealed constitutive expression of the gene with relatively lower abundance in berries and young leaves. The tissue profiles of WsTR-I expression matched those of tropine levels. The data suggest that, in W. somnifera, aerial tissues as well possess tropane alkaloid biosynthetic competence. In vivo feeding of U-[14C]-sucrose to orphan shoot (twigs) and [14C]-chasing revealed substantial radiolabel incorporation in tropinone and tropine, confirming the de novo synthesizing ability of the aerial tissues. This inherent independent ability heralds a conceptual novelty in the backdrop of classical view that these tissues acquire the alkaloids through transportation from roots rather than synthesis. The TR-I gene expression was found to be up-regulated on exposure to signal molecules (methyl jasmonate and salicylic acid) and on mechanical injury. The enzyme's catalytic and structural properties as well as gene expression profiles are discussed with respect to their physiological overtones.

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“…The benzylisoquinoline alkaloids comprise a group of secondary metabolites with roughly 2500 structures. Some of them possess important pharmacological effects such as the vasodilator papaverine, the antimicrobial sanguinarine, the antitussive codeine or the analgesic morphine (Kutchan, 1998; Kutchan et al. , in press).…”

Section: Introductionmentioning

confidence: 99%

Comparative transcript and alkaloid profiling in Papaver species identifies a short chain dehydrogenase/reductase involved in morphine biosynthesis

Ziegler¹,

et al. 2006

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Plants of the order Ranunculales, especially members of the species Papaver, accumulate a large variety of benzylisoquinoline alkaloids with about 2500 structures, but only the opium poppy (Papaver somniferum) and Papaver setigerum are able to produce the analgesic and narcotic morphine and the antitussive codeine. In this study, we investigated the molecular basis for this exceptional biosynthetic capability by comparison of alkaloid profiles with gene expression profiles between 16 different Papaver species. Out of 2000 expressed sequence tags obtained from P. somniferum, 69 show increased expression in morphinan alkaloid-containing species. One of these cDNAs, exhibiting an expression pattern very similar to previously isolated cDNAs coding for enzymes in benzylisoquinoline biosynthesis, showed the highest amino acid identity to reductases in menthol biosynthesis. After overexpression, the protein encoded by this cDNA reduced the keto group of salutaridine yielding salutaridinol, an intermediate in morphine biosynthesis. The stereoisomer 7-epi-salutaridinol was not formed. Based on its similarities to a previously purified protein from P. somniferum with respect to the high substrate specificity, molecular mass and kinetic data, the recombinant protein was identified as salutaridine reductase (SalR; EC 1.1.1.248). Unlike codeinone reductase, an enzyme acting later in the pathway that catalyses the reduction of a keto group and which belongs to the family of the aldo-keto reductases, the cDNA identified in this study as SalR belongs to the family of short chain dehydrogenases/reductases and is related to reductases in monoterpene metabolism.

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Engineering Plant Alkaloid Biosynthetic Pathways: Progress and Prospects

Cited by 13 publications

References 107 publications

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

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

Tropine Forming Tropinone Reductase Gene from Withania somnifera (Ashwagandha): Biochemical Characteristics of the Recombinant Enzyme and Novel Physiological Overtones of Tissue-Wide Gene Expression Patterns

Comparative transcript and alkaloid profiling in Papaver species identifies a short chain dehydrogenase/reductase involved in morphine biosynthesis

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