Abstract
Artemisia annua is the only medicinal crop that produces artemisinin for malarial treatment. Herein, we describe the cloning of a cinnamyl alcohol dehydrogenase (AaCAD) from an inbred self-pollinating (SP) A. annua cultivar and its effects on lignin and artemisinin production. A recombinant AaCAD was purified via heterogeneous expression. Enzyme assays showed that the recombinant AaCAD converted p-coumaryl, coniferyl, and sinapyl aldehydes to their corresponding alcohols, which are key intermediates involved in the biosynthesis of lignin. Km, Vmax, and Vmax/Km values were calculated for all three substrates. To characterize its function in planta, AaCAD was overexpressed in SP plants. Quantification using acetyl bromide (AcBr) showed significantly higher lignin contents in transgenics compared with wild-type (WT) plants. Moreover, GC-MS-based profiling revealed a significant increase in coumarin contents in transgenic plants. By contrast, HPLC-MS analysis showed significantly reduced artemisinin contents in transgenics compared with WT plants. Furthermore, GC-MS analysis revealed a decrease in the contents of arteannuin B and six other sesquiterpenes in transgenic plants. Confocal microscopy analysis showed the cytosolic localization of AaCAD. These data demonstrate that AaCAD plays a dual pathway function in the cytosol, in which it positively enhances lignin formation but negatively controls artemisinin formation. Based on these data, crosstalk between these two pathways mediated by AaCAD catalysis is discussed to understand the metabolic control of artemisinin biosynthesis in plants for high production.
Highlights
Artemisinin-based combination therapy (ACT) is the first-line treatment for malaria (WHO, 2006, 2013; Maude et al, 2009)
The present study shows that understanding the function of AaCAD, a short chain oxidoreductase in A. annua, is fundamental for the metabolic engineering of this medicinal crop to achieve high production levels of artemisinin
Numerous studies have reported that Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in the biosynthesis of lignin and plays an essential role in plant development associated with plant biomass (Mitchell et al, 1994; Feuillet et al, 1995; Halpin et al, 1998; BagniewskaZadworna et al, 2014; Pan et al, 2014; Choi et al, 2016)
Summary
Artemisinin-based combination therapy (ACT) is the first-line treatment for malaria (WHO, 2006, 2013; Maude et al, 2009). In addition to research conducted in plants, fundamental successes in synthetic biology have further demonstrated the steps catalyzed by ADS, CYP71AV1, CPR1, and AaADH1 (Ro et al, 2006; Paddon et al, 2013; Turconi et al, 2014). These previous achievements have demonstrated fundamental promising methods to improve the artemisinin supply, global production from current sweet wormwood crops still lacks stability and is unable to meet the increase in medicinal demands (Paddon et al, 2013; Ma et al, 2017a). Continuous research efforts are urgently necessary to elucidate the regulatory mechanisms of artemisinin biosynthesis to improve the global yield
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