Generation of the potent anti-malarial drug artemisinin in tobacco

Farhi, Moran; Marhevka, Elena; Ben-Ari, Julius; Algamas-Dimantov, Anna; Liang, Zhuobin; Zeevi, Vardit; Edelbaum, Orit; Spitzer-Rimon, Ben; Abeliovich, Hagai; Schwartz, Betty; Tzfira, Tzvi; Vainstein, Alexander

doi:10.1038/nbt.2054

Cited by 140 publications

(98 citation statements)

References 17 publications

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“…Whereas the role of metabolite repair in metabolism and its potential implementation in metabolic engineering was recently proposed (29), our results present, to our knowledge, the first successful example of using an endogenous metabolite recycling strategy for increasing terpenoid formation. Such an approach is especially attractive considering the limitations of using the cytosolic MVA pathway for high yield terpenoid production in plants (30)(31)(32). Thus, our results open a new target for metabolic engineering that, in combination with efforts to increase fluxes through upstream and downstream terpenoid metabolic pathways, will result in high production of economically important terpenoid compounds.…”

Section: Discussionmentioning

confidence: 99%

Orthologs of the archaeal isopentenyl phosphate kinase regulate terpenoid production in plants

Henry

Gutensohn

Thomas

et al. 2015

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

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Terpenoids, compounds found in all domains of life, represent the largest class of natural products with essential roles in their hosts. All terpenoids originate from the five-carbon building blocks, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), which can be derived from the mevalonic acid (MVA) and methylerythritol phosphate (MEP) pathways. The absence of two components of the MVA pathway from archaeal genomes led to the discovery of an alternative MVA pathway with isopentenyl phosphate kinase (IPK) catalyzing the final step, the formation of IPP. Despite the fact that plants contain the complete classical MVA pathway, IPK homologs were identified in every sequenced green plant genome. Here, we show that IPK is indeed a member of the plant terpenoid metabolic network. It is localized in the cytosol and is coexpressed with MVA pathway and downstream terpenoid network genes. In planta, IPK acts in parallel with the MVA pathway and plays an important role in regulating the formation of both MVA and MEP pathway-derived terpenoid compounds by controlling the ratio of IP/DMAP to IPP/DMAPP. IP and DMAP can also competitively inhibit farnesyl diphosphate synthase. Moreover, we discovered a metabolically available carbon source for terpenoid formation in plants that is accessible via IPK overexpression. This metabolite reactivation approach offers new strategies for metabolic engineering of terpenoid production.A ll living organisms produce terpenoids, directing a considerable amount of their available carbon to the biosynthesis of one of the most structurally and functionally diverse classes of primary and secondary metabolites in nature (1). These molecules play essential and specialized roles in their hosts in photosynthesis and respiration (the phytyl side-chain of chlorophyll, quinones), modulating membrane fluidity (sterols), regulating growth and development (hormones), photoprotection and energy transfer (carotenoids), and communication, environmental adaptation, and chemical defense (mono-, sesqui-, and diterpenes) (2, 3). Some compounds, like quinones, chlorophylls, and certain proteins, which require targeting to membranes for their functions, are anchored by terpenoid structures. In addition to their vital biological roles, terpenoids are also widely used by humans as nutritional supplements, flavors, fragrances, biofuels, and pharmaceuticals (4-6). Multiple metabolic pathways operate in parallel, often intersect, and routinely exchange intermediates, leading to one of the most chemically complex groups of natural products in the biosphere. Therefore, achieving a complete understanding at both genetic and biochemical levels of the underlying regulatory networks that coordinate and homeostatically govern these biosynthetic systems is of paramount importance to fully capitalize on the utility of terpenoids to natural ecosystems and humans.All terpenoids originate from C5 building blocks, isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), which are ...

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Section: Discussionmentioning

confidence: 99%

Orthologs of the archaeal isopentenyl phosphate kinase regulate terpenoid production in plants

Henry

Gutensohn

Thomas

et al. 2015

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

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“…81) Artemisinic acid and artemisinin were produced in tobacco by plant metabolic engineering. 82,83) The leaves of Nicotiana benthamiana infected by agrobacteria transformed by the cDNAs encoding ADS, hydroxymethylglutaryl (HMG)-CoA reductase and farnesyl pyrophosphate synthase accumulated amorpha-4,11-diene, and co-infiltration of the agrobacteria containing CYP71AV1 cDNA caused an accumulation of artemisinic acid-12-β-diglucoside (39.5 mg/kg fr. wt).…”

Section: Metabolic Engineering For Production Of Useful Phytochemicalsmentioning

confidence: 99%

“…wt). Bioactive artemisinin was also produced in N. tabacum 83) by transfection with a mega-vector carrying the cDNAs of CYP71AV1, P450 reductase, ADS, artemisinic aldehyde reductase and HMGCoA reductase. This transgenic tobacco accumulated artemisinin (6 µg/g dry weight), indicating that dihydroartemisinic aldehyde produced by the mega-vector is non-enzymatically converted to the final metabolite artemisinin.…”

Section: Metabolic Engineering For Production Of Useful Phytochemicalsmentioning

confidence: 99%

Impacts of Diversification of Cytochrome P450 on Plant Metabolism

Mizutani

2012

Biological & Pharmaceutical Bulletin

100

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Cytochrome P450 monooxygenases (P450s) catalyze a wide variety of monooxygenation reactions in primary and secondary metabolism in plants. The share of P450 genes in each plant genome is estimated to be up to 1%. This implies that the diversification of P450 has made a significant contribution to the ability to acquire the emergence of new metabolic pathways during land plant evolution. The P450 families conserved universally in land plants contribute to their chemical defense mechanisms. Several P450s are involved in the biosynthesis and catabolism of plant hormones. Species-specific P450 families are essential for the biosynthetic pathways of phytochemicals such as terpenoids and alkaloids. Genome wide analysis of the gene clusters including P450 genes will provide a clue to defining the metabolic roles of orphan P450s. Metabolic engineering with plant P450s is an important technology for large-scale production of valuable phytochemicals such as medicines.Key words P450; metabolic engineering; terpenoid; phytohormone P450S IN THE PLANT KINGDOMMore than 5000 sequences have been stored in a plant P450 database.1) The number of P450 genes in Arabidopsis (Arabidopsis thaliana) is 245, and P450 is the third biggest family gene in Arabidopsis.1) The numbers of P450 genes in several other plants are 316 in grapevine (Vitis vinifera), 332 in soybean (Glycine max), 312 in poplar (Populus trichocarpa), 334 in rice (Oryza sativa), and 372 in sorghum (Sorghum bicolor), which are estimated to represent up to 1% of the total gene annotations of each plant species .2,3) Transcriptome analyses are also contributing to the vast increase in identified P450 sequences from various plant species (http://compbio.dfci. harvard.edu/tgi/plant.html). However, the functions of most of these are still unknown. For instance, in Arabidopsis, the number of functionally characterized P450 genes is about 60, which means that more than 70% of its 245 P450 genes still remain to be characterized. 4)Plant P450s have been shown to participate in a variety of biochemical pathways to produce primary and secondary metabolites such as phenylpropanoids, alkaloids, terpenoids, lipids, cyanogenic glycosides, and glucosinolates, as well as plant hormones.4) The diversification of P450 has had a significant biochemical impact on the emergence of new metabolic pathways during the evolutionary process of land plants. DIVERSIFICATION OF P450 GENES IN LAND PLANTSThe driving force of plant P450 diversification is closely linked to the survival strategy of land plants, conferring advantages in the continuous evolutionary competition and mutualism with other organisms. The role of plant P450 can be discussed in relation to the following categorized reactions and metabolisms: category I, essential reactions conserved in the plant kingdom; category II, core metabolisms conserved in all land plants; category III, essential metabolisms which emerged during the evolution of flowering plants (e.g., plant hormone homeostasis); category IV, secondary metabolisms unique t...

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“…It is believed that the glycosylation of artemisinic acid prohibits its further conversion to artemisinin. The most exciting news is that an entire artemisinin biosynthesis pathway has been re-established in N. tabacum by introducing a truncated HMGR from yeast, and ADS, CYP71AV1, DBR2, and cytochrome P450 reductase (CPR) from A. annua, from which artemisinin was successfully produced albeit only in a trace amount of 0.0007% in the dry weight (Farhi et al 2011).…”

Section: Introductionmentioning

confidence: 99%

An ecological implication of glandular trichome-sequestered artemisinin: as a sink of biotic/abiotic stress-triggered singlet oxygen

Jiang

Gao

Liao

et al. 2015

Preprint

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Artemisinin is accumulated in wormwood (Artemisia annua) with uncertain ecological implications. Here, we suggest that artemisinin is generated in response to biotic/abiotic stress, during which dihydroartemisinic acid, a direct artemisinin precursor, quenches singlet oxygen ( 1 O2), one kind of reactive oxygen species. Evidence supporting artemisinin as a sink of 1 O2 emerges from that volatile isoprenoids protect plants from biotic/abiotic stress; biotic/abiotic stress induces artemisinin biosynthesis; and stress signaling pathways are involved in the biosynthesis of volatile isoprenoids among plants as well as the biosynthesis of artemisinin in A. annua. In this review, we address the ecological implication of glandular trichome-sequestered artemisinin as a sink sink of biotic/abiotic stress-triggered 1 O2, and also summarize the cumulating data on the transcriptomic and metabolic profiling of stress-enhanced artemisinin production upon eliciting 1 O2 omission from chloroplasts and initiating retrograde 1 O2 signaling from chloroplasts to nuclei.

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Generation of the potent anti-malarial drug artemisinin in tobacco

Cited by 140 publications

References 17 publications

Orthologs of the archaeal isopentenyl phosphate kinase regulate terpenoid production in plants

Orthologs of the archaeal isopentenyl phosphate kinase regulate terpenoid production in plants

Impacts of Diversification of Cytochrome P450 on Plant Metabolism

An ecological implication of glandular trichome-sequestered artemisinin: as a sink of biotic/abiotic stress-triggered singlet oxygen

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