Abstract
Artemisia annua is established as an efficient crop for the production of the anti-malarial compound artemisinin, a sesquiterpene lactone synthesized and stored in Glandular Secretory Trichomes (GSTs) located on the leaves and inflorescences. Amorpha-4,11-diene synthase (AMS) catalyzes the conversion of farnesyl pyrophosphate (FPP) to amorpha-4,11-diene and diphosphate, which is the first committed step in the synthesis of artemisinin. FPP is the precursor for sesquiterpene and sterol biosynthesis in the plant. This work aimed to investigate the effect of blocking the synthesis of artemisinin in the GSTs of a high artemisinin yielding line, Artemis, by down regulating AMS. We determined that there are up to 12 AMS gene copies in Artemis, all expressed in GSTs. We used sequence homology to design an RNAi construct under the control of a GST specific promoter that was predicted to be effective against all 12 of these genes. Stable transformation of Artemis with this construct resulted in over 95% reduction in the content of artemisinin and related products, and a significant increase in the FPP pool. The Artemis AMS silenced lines showed no morphological alterations, and metabolomic and gene expression analysis did not detect any changes in the levels of other major sesquiterpene compounds or sesquiterpene synthase genes in leaf material. FPP also acts as a precursor for squalene and sterol biosynthesis but levels of these compounds were also not altered in the AMS silenced lines. Four unknown oxygenated sesquiterpenes were produced in these lines, but at extremely low levels compared to Artemis non-transformed controls (NTC). This study finds that engineering A. annua GSTs in an Artemis background results in endogenous terpenes related to artemisinin being depleted with the precursor FPP actually accumulating rather than being utilized by other endogenous enzymes. The challenge now is to establish if this precursor pool can act as substrate for production of alternative sesquiterpenes in A. annua.
Highlights
Artemisia annua (A. annua) is a herbaceous plant from the Asteraceae family native to Asia, known to synthesize the leading antimalarial compound artemisinin a sesquiterpene lactone, within its glandular secretory trichomes (GSTs) (Duke et al, 1994; Olsson et al, 2009)
The Amorpha-4,11-diene synthase (AMS) enzyme responsible for catalyzing the first committed step in artemisinin production is encoded by a small gene family averaging 12 copies in the Artemis F1 hybrid variety (Figure 2)
This was mirrored by a dramatic decrease in artemisinin concentration in young, mature and dry leaves of the AMS silenced lines compared to the non-transformed controls (NTC) (Figure 3C)
Summary
Artemisia annua (A. annua) is a herbaceous plant from the Asteraceae family native to Asia, known to synthesize the leading antimalarial compound artemisinin a sesquiterpene lactone, within its glandular secretory trichomes (GSTs) (Duke et al, 1994; Olsson et al, 2009). The secretory sac is bounded by a cuticle proximal to the three apical pairs of cells. This arrangement allows phytotoxic compounds such as artemisinin to be sequestered away from the plant, preventing autotoxicity (Duke and Paul, 1993; Duke et al, 1994; Ferreira and Janick, 1995). The efficiency of the A. annua production system, which can yield artemisinin at greater than 1% lead dry weight and 41.3 Kg per Ha (Ferreira et al, 2005) has meant that it persists as the most efficient and economically feasible platform for production of artemisinin today. A desire to both stabilize and reduce costs in the supply chain has driven research into yield improvement through modern marker assisted plant breeding and genetic engineering methods and through engineering artemisinin (or precursor) synthesis in heterologous hosts (Ferreira et al, 2005; Han et al, 2006; Graham et al, 2010; Zhang et al, 2011; Paddon and Keasling, 2014; Tang et al, 2014; Pulice et al, 2016)
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