Silencing amorpha-4,11-diene synthase Genes in Artemisia annua Leads to FPP Accumulation

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.

Flavonoid Versus Artemisinin Anti-malarial Activity in Artemisia annua Whole-Leaf Extracts

“…To test for the potential antiplasmodial activity of artemisinin-unrelated compounds in A. annua , we used the artemisinin-reduced AMS silenced plant line (Catania et al, 2018). Samples from this line were prepared alongside the other genetic variants and re-suspended to match the wild-type casticin levels, which resulted in a 100-fold reduction in artemisinin levels compared to the wild type (Table 1).…”

“…We extended the bioassays to include whole-leaf extracts from A. annua silenced in amorpha-4,11-diene synthase ( AMS ), which encodes the enzyme that catalyzes the first committed step of artemisinin biosynthesis (Catania et al, 2018), and the cyp71av1-1 mutant, impaired in the second committed step of artemisinin biosynthesis (Czechowski et al, 2016). The AMS silenced line has dramatically reduced artemisinin production (5% of the wild-type levels) and accumulates the sesquiterpene precursor farnesyl pyrophosphate in trichomes (Catania et al, 2018). cyp71av1-1 completely abolishes artemisinin production and redirects the artemisinin pathway to the synthesis of arteannuin X, a novel sesquiterpene epoxide (Czechowski et al, 2016).…”

Silencing of germacrene A synthase genes reduces guaianolide oxalate content in Cichorium intybus L.

“…62 Recently, silencing of amorpha-4,11-diene synthase, which encodes the first step in artemisinin biosynthesis, was achieved in Artemisia annua. 63 The authors report a stable Agrobacterium transformation of A. annua using hpRNA constructs and selecting several lines with substantial gene silencing -25 times reduction of gene expression compared to untransformed control plants. When assessing the content of artemisinin, a cadinanolide STL (a compound synthetized several steps downstream from the silenced gene, which is comparable to our system), the authors also found a substantial reduction of 20-fold in selected clone lines.…”

“…However, when measuring amorpha-4,11-diene, a direct product of the silenced gene, no reduction could be observed, but there was a significant increase of this compound in young leaves of silenced lines compared to controls and a comparable level in mature or dry leaves of clones to controls. 63 These results suggest that the effect of silencing genes from the diverse group of terpenoid synthases can be difficult to predict and interpret. In our system, some lines displayed a reduction of gene expression and STL oxalate content of several orders of magnitude ( Table 2), suggesting that gene silencing with amiRNAs could be a good method for the production of low-bitterness chicory clones.…”

Transgenesis as a Tool for the Efficient Production of Selected Secondary Metabolites from Plant in Vitro Cultures

“…One of the key strategies enabling the overproduction of valuable plant secondary metabolites in in vitro cultures is the manipulation of existing metabolic pathways by overexpressing or silencing selected elements involved in their biosynthesis [24][25][26][27]. A wide range of plant vectors have been designed that allow the simple and quick introduction of synthetic expression cassettes, allowing easier and more effective creation of in vitro transgenic plant cultures [28][29][30].…”