School of Natural Sciences, Linnaeus University, 39182 Kalmar, Sweden.
Plant Cell Rep. 2012 Jul;31(7):1309-19. doi: 10.1007/s00299-012-1250-z. Epub 2012 May 8.
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.
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.
青蒿属植物产生了许多倍半萜合酶，这些酶可以催化法呢基二磷酸转化为各种倍半萜。青蒿素生物合成中的关键酶 4,11-二烯合酶（ADS）和复杂倍半萜环化合酶表愈创木醇合酶（ECS）的 cDNA 被克隆到带有 Kozak 共有序列和 C 端组氨酸标签的豇豆花叶病毒载体（pEAQ-HT）中。质粒被转化到根癌农杆菌 LBA4404 中，并与含有番茄丛矮病毒转录后基因沉默抑制子的载体（pJL3：p19）一起农杆菌浸润到菸草的叶片中。定量 PCR 用于测量侵染后 0、3、6、9、12 和 15 天的转录水平。这两个基因的相对表达量在 9 天达到最高。通过 SDS-PAGE 和 Western blot 验证了 ADS 和 ECS 的重组蛋白的瞬时表达。从 9 天的叶片中提取重组蛋白，并通过固定化金属离子亲和层析用组氨酸标签纯化，分别得到 ADS 和 ECS 的产量为 90 和 96 mg kg⁻¹新鲜叶片重量。使用气相色谱-质谱联用仪对产物进行鉴定，并使用柠檬烯作为内标进行定量，测定纯化酶的活性。重组 ADS 和 ECS 将法呢基二磷酸转化为 4,11-二烯（97%）和表愈创木醇（96%）作为主要产物。纯化的酶对 ADS 和 ECS 的比活性分别为 0.002 和 0.01 μmol min⁻¹ mg⁻¹蛋白。它们的表观 k(cat)值分别为 2.1×10⁻³ s⁻¹和 11×10⁻³ s⁻¹。
