title: Nicotiana benthamiana as a production platform for artemisinin precursors.
abstract: BACKGROUND
  Production 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.
  
  
  RESULTS
  Biosynthetic 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 x 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.
  
  
  CONCLUSIONS
  This 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.
authors.affiliation: Laboratory of Plant Physiology, Wageningen University, Wageningen, The Netherlands.
authors.forename: Teun W J M
authors.initials: TW
authors.lastname: van Herpen
authors.forename: Katarina
authors.initials: K
authors.lastname: Cankar
authors.forename: Marilise
authors.initials: M
authors.lastname: Nogueira
authors.forename: Dirk
authors.initials: D
authors.lastname: Bosch
authors.forename: Harro J
authors.initials: HJ
authors.lastname: Bouwmeester
authors.forename: Jules
authors.initials: J
authors.lastname: Beekwilder
mesh_terms: D016960:Agrobacterium tumefaciens / Q000378:metabolism; D000962:Antimalarials / Q000378:metabolism* / Q000494:pharmacology; D037621:Artemisinins / Q000378:metabolism* / Q000494:pharmacology; D002626:Chemistry, Pharmaceutical / Q000379:methods*; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D015195:Drug Design; D008401:Gas Chromatography-Mass Spectrometry; D008928:Mitochondria / Q000378:metabolism; D008956:Models, Chemical; D009841:Oligonucleotides / Q000737:chemistry; D016366:Open Reading Frames; D010100:Oxygen / Q000378:metabolism; D010936:Plant Extracts / Q000378:metabolism* / Q000494:pharmacology; D018515:Plant Leaves / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*
chemical_list: D000962:Antimalarials; D037621:Artemisinins; D009841:Oligonucleotides; D010936:Plant Extracts; D010940:Plant Proteins; D003577:Cytochrome P-450 Enzyme System; C031327:artemisinin; D010100:Oxygen