Artemisinin, a sesquiterpene lactone produced by Artemisia annua glandular secretory trichomes, is the active ingredient in the most effective treatment for malaria currently available. We identified a mutation that disrupts the amorpha-4,11-diene C-12 oxidase (CYP71AV1) enzyme, responsible for a series of oxidation reactions in the artemisinin biosynthetic pathway. Detailed metabolic studies of cyp71av1-1 revealed that the consequence of blocking the artemisinin biosynthetic pathway is the redirection of sesquiterpene metabolism to a sesquiterpene epoxide, which we designate arteannuin X. This sesquiterpene approaches half the concentration observed for artemisinin in wild-type plants, demonstrating high-flux plasticity in A. annua glandular trichomes and their potential as factories for the production of novel alternate sesquiterpenes at commercially viable levels. Detailed metabolite profiling of leaf maturation time-series and precursor-feeding experiments revealed that nonenzymatic conversion steps are central to both artemisinin and arteannuin X biosynthesis. In particular, feeding studies using 13 C-labeled dihydroartemisinic acid (DHAA) provided strong evidence that the final steps in the synthesis of artemisinin are nonenzymatic in vivo. Our findings also suggest that the specialized subapical cavity of glandular secretory trichomes functions as a location for both the chemical conversion and the storage of phytotoxic compounds, including artemisinin. We conclude that metabolic engineering to produce high yields of novel secondary compounds such as sesquiterpenes is feasible in complex glandular trichomes. Such systems offer advantages over single-cell microbial hosts for production of toxic natural products.artemisinin | p450 oxidase | terpenoid | sesquiterpene | Artemisia annua

Chemical derivatives of artemisinin, a sesquiterpene lactone produced by Artemisia annua, are the active ingredient in the most effective treatment for malaria. Comprehensive phytochemical analysis of two contrasting chemotypes of A. annua resulted in the characterization of over 80 natural products by NMR, more than 20 of which are novel and described here for the first time. Analysis of high- and low-artemisinin producing (HAP and LAP) chemotypes of A. annua confirmed the latter to have a low level of DBR2 (artemisinic aldehyde Δ11(13) reductase) gene expression. Here we show that the LAP chemotype accumulates high levels of artemisinic acid, arteannuin B, epi-deoxyarteannuin B and other amorpha-4,11-diene derived sesquiterpenes which are unsaturated at the 11,13-position. By contrast, the HAP chemotype is rich in sesquiterpenes saturated at the 11,13-position (dihydroartemisinic acid, artemisinin and dihydro-epi-deoxyarteannunin B), which is consistent with higher expression levels of DBR2, and also with the presence of a HAP-chemotype version of CYP71AV1 (amorpha-4,11-diene C-12 oxidase). Our results indicate that the conversion steps from artemisinic acid to arteannuin B, epi-deoxyarteannuin B and artemisitene in the LAP chemotype are non-enzymatic and parallel the non-enzymatic conversion of DHAA to artemisinin and dihyro-epi-deoxyarteannuin B in the HAP chemotype. Interestingly, artemisinic acid in the LAP chemotype preferentially converts to arteannuin B rather than the endoperoxide bridge containing artemisitene. In contrast, in the HAP chemotype, DHAA preferentially converts to artemisinin. Broader metabolomic and transcriptomic profiling revealed significantly different terpenoid profiles and related terpenoid gene expression in these two morphologically distinct chemotypes.

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.

The STORR gene fusion event is considered essential for the evolution of the promorphinan/morphinan subclass of benzylisoquinoline alkaloids (BIAs) in opium poppy as the resulting bi-modular protein performs the isomerization of (S)- to (R)-reticuline essential for their biosynthesis. Here, we show that of the 12 Papaver species analysed those containing the STORR gene fusion also contain promorphinans/morphinans with one important exception. P. californicum encodes a functionally conserved STORR but does not produce promorphinans/morphinans. We also show that the gene fusion event occurred only once, between 16.8-24.1 million years ago before the separation of P. californicum from other Clade 2 Papaver species. The most abundant BIA in P. californicum is (R)-glaucine, a member of the aporphine subclass of BIAs, raising the possibility that STORR, once evolved, contributes to the biosynthesis of more than just the promorphinan/morphinan subclass of BIAs in the Papaveraceae.

The STORR gene fusion event is considered a key step in the evolution of benzylisoquinoline alkaloid (BIA) metabolism in opium poppy as the resulting bi-modular protein performs the isomerization of (S)- to (R)- reticuline which is required for morphinan biosynthesis. Our previous analysis of the opium poppy genome suggested the STORR gene fusion event occurred before a whole genome duplication event 7.2 million years ago. Here we use a combination of phylogenetic, transcriptomic, metabolomic, biochemical and genomic analysis to investigate the origin of the STORR gene fusion across the Papaveraceae family. The pro-morphinan/morphinan subclass of BIAs was present in a subset of 10 Papaver species including P. somniferum (opium poppy) and this correlated with the presence of the STORR gene fusion with one important exception. P. californicum does not produce morphinans but it does contain a STORR gene fusion that epimerizes (S)- to (R)- reticuline when heterologously expressed in yeast. The high similarity of the amino acid sequence linking the two modules of STORR along with phylogenetic gene tree analysis strongly suggests the gene fusion occurred only once and between 17-25 million years ago before the separation of P. californicum from the other Papaver species. We discovered that the most abundant BIA in P. californicum is (R)- glaucine, a member of the aporphine subclass of BIAs. Only the (S) isomer of this compound has previously been reported from nature. These results lead us to conclude that the function of the STORR gene fusion is not exclusive to morphinan production in the Papaveraceae.