Functional expression and characterization of sesquiterpene synthases from Artemisia annua L. using transient expression system in Nicotiana benthamiana.

摘要

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 micro mol min-1 mg-1 protein for ADS and ECS, respectively. The apparent k_cat values were 2.1x10-3 s-1 and 11x10-3 s-1 for ADS and ECS, respectively.