title: Functional expression and characterization of sesquiterpene synthases from Artemisia annua L. using transient expression system in Nicotiana benthamiana.
abstract: Artemisia annua L. produces a number of sesquiterpene synthases, which catalyze the conversion of farnesyl diphosphate to various sesquiterpenes. The cDNAs encoding amorpha-4,11-diene synthase (ADS), a key enzyme in the artemisinin biosynthesis, and epi-cedrol synthase (ECS), a complex sesquiterpene cyclization synthase, were cloned into Cowpea mosaic virus-based viral vector (pEAQ-HT) with Kozak consensus motif and C-terminal histidine tag. The plasmids were transformed into Agrobacterium LBA4404 and, agroinfiltrated into Nicotiana benthamiana leaves along with vector (pJL3:p19) containing Tomato bushy stunt virus post-transcriptional gene silencing suppressor. Quantitative PCR was carried out to measure the transcript levels at 0, 3, 6, 9, 12 and 15 days post-infiltration (dpi). The highest relative expression was observed at 9 dpi for both genes. Transiently expressed recombinant proteins of ADS and ECS were confirmed by SDS-PAGE and western blot. Recombinant proteins were extracted from 9 dpi leaves and purified by immobilized metal ion affinity chromatography using histidine tag, which produced yields of 90 and 96 mg kg⁻¹ fresh weight of leaves for ADS and ECS, respectively. Activities of the purified enzymes were assayed using gas chromatography-mass spectrometry for product identification and quantification using valencene as internal standard. The recombinant ADS and ECS converted farnesyl diphosphate into amorpha-4,11-diene (97 %) and epi-cedrol (96 %) as the major products, respectively. The purified enzymes exhibited the specific activity of 0.002 and 0.01 μmol min⁻¹ mg⁻¹ protein for ADS and ECS, respectively. The apparent k(cat) values were 2.1 × 10⁻³ s⁻¹ and 11 × 10⁻³ s⁻¹ for ADS and ECS, respectively.
  
  
  CONCLUSIONS
  Agroinfiltration of leaves of Nicotiana bentamiana can be used to produce recombinant biosynthetic enzymes as exemplified by two sesquiterpene synthases from Artemisia annua in relatively high yields.
authors.affiliation: School of Natural Sciences, Linnaeus University, 39182 Kalmar, Sweden.
authors.forename: Selvaraju
authors.initials: S
authors.lastname: Kanagarajan
authors.forename: Saraladevi
authors.initials: S
authors.lastname: Muthusamy
authors.forename: Anna
authors.initials: A
authors.lastname: Gliszczyńska
authors.forename: Anneli
authors.initials: A
authors.lastname: Lundgren
authors.forename: Peter E
authors.initials: PE
authors.lastname: Brodelius
mesh_terms: D060054:Agrobacterium; D019883:Alkyl and Aryl Transferases / Q000096:biosynthesis* / Q000235:genetics; D031172:Artemisia annua / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D005822:Genetic Vectors; D010940:Plant Proteins / Q000096:biosynthesis* / Q000235:genetics; D030821:Plants, Genetically Modified / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D012717:Sesquiterpenes / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D012717:Sesquiterpenes; D019883:Alkyl and Aryl Transferases; C403714:amorpha-4,11-diene synthase