An integrate approach for secondary metabolites investigation and production: the case of artemisinin

The Artemisia genus, belonging to the Asteraceae family, is known for its wide distribution and extensive use in traditional medicine due to its anti-inflammatory, antimicrobial, and antiparasitic properties. Plants from Artemisia genus contain a huge and various number of secondary metabolites, which contribute to a broad spectrum of bioactivities and therapeutic applications. One of the most significant uses of Artemisia species is in the treatment of malaria, where the compound artemisinin, isolated from A. annua leaves in 1972, has emerged as the most potent antimalarial drug. Despite its effectiveness, artemisinin is not widely available in many malaria-endemic countries, and the cost of the drug can be prohibitively high for many people living in these areas. In the past decades, many efforts have been attempted to increase the global production of this molecule, ranging from traditional methods, as intensive breeding programs, to the use of more advanced biotechnological tools, such as genetic engineering in plants, use of tissue culture and semisynthesis production through the use of microorganism, e.g. yeast and bacteria. In this PhD thesis, different strategies to address this issue have been explored: • Although A. annua plants remain the major natural source of artemisinin, different studies found out that other Artemisia species can synthetize this metabolite. We selected five Artemisia species, with ascribed medicinal properties, i.e. A. absinthium L., A. alba Turra, A. annua L., A. verlotiorum Lamotte and A. vulgaris L., and characterized their phytochemical profile. The occurrence of the lead drug compound artemisinin was detected only in A. annua plants. However, the presence of its precursors was observed in A. absinthium, A. alba and A. verlotiorum. Parallelly, untargeted metabolomics was employed to explore the overall richness of their secondary metabolites. The data obtained from this analysis were correlated to those from two antioxidant assays (DPPH and FRAP), allowing the identification of metabolites putatively responsible for the antioxidant activity observed. • Artemisinin biosynthesis has been deeply characterized trough the past years and the main genes of its metabolic pathway have been identified. Despite several works focused on the transformation of A. annua plants, transient expression has been poorly explored. Using the reporter gene GFP we established a protocol for transient gene expression in in vitro A. annua plants, with the use of A. tumefaciens infiltration. This protocol was then used to study the effect of the overexpression of different combination of artemisinin biosynthetic genes, i.e Farnesyl diphosphate synthase (FPS), Amorphadiene synthase (ADS), Cytochrome P450 (CYP71AV1), Artemisinic aldehyde reductase (DBR2), Aldehyde dehydrogenase (ALDH1). Although no significative increase in artemisinin accumulation was detected, we observed a sharply reduction in the accumulation of artemisinin precursors when all the five genes were coinfiltrated simultaneously. Thus, the transient protocol we developed may represent a useful tool to investigate the complex network behind artemisinin biosynthesis in A. annua plants. • Plant cell suspension cultures provide a cost-effective substitute to conventional cultivation methods in the production of secondary metabolites with a pharmaceuticals interest. Artemisinin production trough tissue culture, e.g. hairy roots culture, and in cell suspension culture has been reported in the literature. In this work, different lines of cell culture from Artemisia spp. plants were generated to investigate their capability in producing the lead drug artemisinin and to characterize their phytochemical profile. Although different growing media and hormonal combination were applied, we were not able to detect any trace of artemisinin or its precursor. In addition, Artemisia spp. cell cultures showed a simplified metabolic when compared to those of the plants they were derived, suggesting a loss of complexity in molecular pathways as consequences of cellular dedifferentiation. • Vertical farming systems allow for the precise manipulation of growth conditions, which can be used to improve the phytochemical profile of medicinal plants. In this work, we collaborated with a company in order to test the suitability of A. annua plants to be employed in a vertical farming setup and the opportunity of increasing artemisinin yield through the application of different growing conditions. Our results demonstrated that A. annua is suitable to be grown in a VF system, with good results in artemisinin and leaf biomass yield if compared to what observed in field condition. However, these findings are very preliminary, and the actual cost of production would be extremely high for commercial purposes.