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.
Key message 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.
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Acknowledgments
We thank George P. Lomonossoff for providing the pEAQ-HT vector, Mike Boehm for providing the pJL3:p19 vector, P.J.J. Hooykaas for providing the LBA4404 strain and Ingela Fridborg for providing N. benthamiana seeds. We thank Linda Olofsson for excellent suggestions on qPCR and providing ADS and ECS qPCR primers. We also thank Conny Tolf for his assistance in qPCR experimental work. This work was supported by grants from the Faculty of Natural Science and Engineering awarded to PB. SK was supported by the fund from Sparbanksstiftelsen Kronan to PB.
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Kanagarajan, S., Muthusamy, S., Gliszczyńska, A. et al. Functional expression and characterization of sesquiterpene synthases from Artemisia annua L. using transient expression system in Nicotiana benthamiana . Plant Cell Rep 31, 1309–1319 (2012). https://doi.org/10.1007/s00299-012-1250-z
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DOI: https://doi.org/10.1007/s00299-012-1250-z