Artemisinin is highly effective against drug-resistant malarial parasites, which affects nearly half of the global population and kills >500 000 people each year. The primary cost of artemisinin is the very expensive process used to extract and purify the drug from Artemisia annua. Elimination of this apparently unnecessary step will make this potent antimalarial drug affordable to the global population living in endemic regions. Here we reported the oral delivery of a non-protein drug artemisinin biosynthesized (~0.8 mg/g dry weight) at clinically meaningful levels in tobacco by engineering two metabolic pathways targeted to three different cellular compartments (chloroplast, nucleus, and mitochondria). The doubly transgenic lines showed a three-fold enhancement of isopentenyl pyrophosphate, and targeting AACPR, DBR2, and CYP71AV1 to chloroplasts resulted in higher expression and an efficient photo-oxidation of dihydroartemisinic acid to artemisinin. Partially purified extracts from the leaves of transgenic tobacco plants inhibited in vitro growth progression of Plasmodium falciparum-infected red blood cells. Oral feeding of whole intact plant cells bioencapsulating the artemisinin reduced the parasitemia levels in challenged mice in comparison with commercial drug. Such novel synergistic approaches should facilitate low-cost production and delivery of artemisinin and other drugs through metabolic engineering of edible plants.

Chloroplasts offer high-level transgene expression and transgene containment due to maternal inheritance, and are ideal hosts for biopharmaceutical biosynthesis via multigene engineering. To exploit these advantages, we have expressed 12 enzymes in chloroplasts for the biosynthesis of artemisinic acid (precursor of artemisinin, antimalarial drug) in an alternative plant system. Integration of transgenes into the tobacco chloroplast genome via homologous recombination was confirmed by molecular analysis, and biosynthesis of artemisinic acid in plant leaf tissues was detected with the help of 13C NMR and ESI-mass spectrometry. The excess metabolic flux of isopentenyl pyrophosphate generated by an engineered mevalonate pathway was diverted for the biosynthesis of artemisinic acid. However, expression of megatransgenes impacted the growth of the transplastomic plantlets. By combining two exogenous pathways, artemisinic acid was produced in transplastomic plants, which can be improved further using better metabolic engineering strategies for commercially viable yield of desirable isoprenoid products.