Carotenoids and ketocarotenoids are isoprenoid molecules, which represent one of the most widespread classes of natural pigments, found in animals, plants and microorganisms. Moreover, they have valuable antioxidant properties. Their health benelfits and colorant aspects have led to attempts to elevate their level in foodstuffs. In the present study, several metabolic engineering strategies were tested in order to enhance the levels of high-value carotenoid and kctocarotenoid compounds, such as iycopcne, carotene, canthaxanthin and astaxanthin, in tomato and tobacco plants. Biosynthetic bacterial pathway genes have been ovcrexpressed independently (GGPP synthase (Cr/E), phytoene synthase (Cr/B) and phytoene desaturase (Crll)) and in combination (CrtE+B, CrtE+I, Cr/B+I and p-carotene hydroxylase and kctolase (C1'fZ+W», with different promoters (for CrIB. CrlI and CrtB+1) or In association with transcription factors (Phytochrome-interacting factor 5 (CrIZ+W+PIFS) and Arabidopsis Response Regulator 14 (Cr/Z+W+ARRI4)). The effects of these different strategies on the plant metabolism and especially on carotenoid formation, sequestration and the activation of regulation mechanisms were studied. The combination of the two genes CrIB and CrII, in their hemizygous state, had a synergistic effect on the production of carotenoids and the expression of CrtZ+W+ARR 14 increased the levels ofketocarotenoids in the plants. The important features for the design of metabolic engineering strategies were highlighted. Moreover, regulatory mechanisms that operate across multiple levels of cellular regulation, including transcription, protein localisation, metabolite levels, cell or tissue type, and organelle/sub-organelle structure and organisation were revealed. It was demonstrated how changes to chromoplast and sub-chromoplast structures, such as crystal formation, plastoglobulc and membrane composition/structures can arise in response to changes in metabolites. A new carotenoid regulat ion mechanism at the sub-organellar level was discovered and a schematic model was proposed. 3

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