Nicotiana benthamiana as a Production Platform for Artemisinin Precursors

Abstract

BackgroundProduction of pharmaceuticals in plants provides an alternative for chemical synthesis, fermentation or natural sources. Nicotiana benthamiana is deployed at commercial scale for production of therapeutic proteins. Here the potential of this plant is explored for rapid production of precursors of artemisinin, a sesquiterpenoid compound that is used for malaria treatment.Methodology/Principal FindingsBiosynthetic genes leading to artemisinic acid, a precursor of artemisinin, were combined and expressed in N. benthamiana by agro-infiltration. The first committed precursor of artemisinin, amorpha-4,11-diene, was produced upon infiltration of a construct containing amorpha-4,11-diene synthase, accompanied by 3-hydroxy-3-methylglutaryl-CoA reductase and farnesyl diphosphate synthase. Amorpha-4,11-diene was detected both in extracts and in the headspace of the N. benthamiana leaves. When the amorphadiene oxidase CYP71AV1 was co-infiltrated with the amorphadiene-synthesizing construct, the amorpha-4,11-diene levels strongly decreased, suggesting it was oxidized. Surprisingly, no anticipated oxidation products, such as artemisinic acid, were detected upon GC-MS analysis. However, analysis of leaf extracts with a non-targeted metabolomics approach, using LC-QTOF-MS, revealed the presence of another compound, which was identified as artemisinic acid-12-β-diglucoside. This compound accumulated to 39.5 mg.kg−1 fwt. Apparently the product of the heterologous pathway that was introduced, artemisinic acid, is further metabolized efficiently by glycosyl transferases that are endogenous to N. benthamiana.Conclusion/SignificanceThis work shows that agroinfiltration of N. bentamiana can be used as a model to study the production of sesquiterpenoid pharmaceutical compounds. The interaction between the ectopically introduced pathway and the endogenous metabolism of the plant is discussed.

The re-emergence of natural products for drug discovery in the genomics era

“…The tobacco plant Nicotiana benthamiana has also been explored as a production platform for precursors of artemisinin, which is usually extracted from Artemesia annua, for the treatment of malaria 177 . Genes for three biosynthetic enzymes necessary to produce artemisinic acid ( The Keasling group in University of California, Berkeley, USA, used the combined expression of A.…”

PPR2263, a DYW-Subgroup Pentatricopeptide Repeat Protein, Is Required for Mitochondrial nad5 and cob Transcript Editing, Mitochondrion Biogenesis, and Maize Growth

“…The binary construct, in which the PPR2263:GFP chimera was under the control of the cauliflower mosaic virus 35S promoter, was transferred into Agrobacterium tumefaciens strain C58pMP90. Using established protocols (van Herpen et al, 2010), the resulting strain was coinfiltrated into Nicotiana benthamiana leaves with a strain harboring the 35S:P19 plasmid expressing the silencing suppressor p19 of tomato bushy stunt virus (Voinnet et al, 2003) and a third strain containing the mitochondrial marker construct FDH:RFP2 (courtesy of Catherine Colas des Francs-Small). In the latter construct, formate dehydrogenase from potato (Solanum tuberosum; Ambard-Bretteville et al, 2003) had been amplified with primers ATG1 and FHD-R (see Supplemental Table 1 online) and cloned into pGreen (Vain et al, 2003).…”

Impacts of Diversification of Cytochrome P450 on Plant Metabolism

“…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).…”