Genetic engineering of artemisinin production in Nicotiana tabacum trichomes

Each year, over half a million of people die from malaria. The World Health Organization has recommended artemisinin-based combination therapies as the treatment of choice. However, artemisinin, a sesquiterpene synthesized in Artemisia annua glandular trichomes, is produced in low amount. Due to a worldwide shortage of the drug, intense research has been undertaken in order to increase the yield of artemisinin in the plant or to develop alternative methods for its production. The terpenoid metabolism is highly active in the trichomes of Nicotiana tabacum, a high biomass species. We therefore investigated the possibility to target N. tabacum trichomes for metabolic engineering of artemisinin. The reconstitution of this pathway in trichomes requires trichome-specific transcription promoters. We therefore undertook a proteome comparison of trichomes and different tissues of N. tabacum and identified several trichome-specific proteins. Their specificity was validated at the RNA level by RT-PCR and their corresponding transcription promoters were isolated, fused to a reporter gene and introduced in the genome of N. tabacum. One promoter was shown to be trichome-specific and when experimentally compared to other trichome-specific promoters reported in the literature, turned out to be the most appropriate one. We fused this promoter to the genes of the artemisinin pathway and obtained transgenic plants expressing those genes. Some of these plants showed retarded development, indicating that the metabolism was disturbed. Artemisinin was not detected but other metabolic intermediates have to be analyzed. We discovered that the identified trichome-specific gene encodes a small subunit of Rubisco (RbcS), the enzyme responsible for carbon dioxide fixation during photosynthesis. However, this gene is phylogenetically distant from those expressed in the mesophyll cells and belongs to a so far uncharacterized cluster of RbcS genes which arose before the emergence of monocot and dicot species. Expression in the microalgae Chlamydomonas showed that the trichome RbcS conferred a more acidic activity vs pH profile than the mesophyll RbcS. We propose that RbcS from this particular cluster might be more adapted to a more acidic chloroplast stroma and function in secretory cells to recycle CO2 released by the active secondary metabolism.(AGRO - Sciences agronomiques et ingénierie biologique) -- UCL, 2015Accès interdit14/10/2015(AGRO - Sciences agronomiques et ingénierie biologique) -- UCL, 2015Anglai