Obtaining transgenic multi-biofortified plant crops could help alleviate micronutrient malnutrition, particularly in rural areas with little access to other alternatives. The development of multi-biofortified potato lines with enhanced iron, provitamin A and folate concentrations is therefore a necessary endeavour within the plant biofortification field. However, most biofortification studies usually only focus on the enhancement of the target micronutrients and neglect (possible) physiological adaptations. This comprises interactions with other micronutrient biosynthesis pathways, whole-plant metabolism feedback mechanisms and important agronomic parameters. Therefore, the main goal of this PhD project is to produce potato multi-biofortified lines that will be studied for metabolism adaptations and micronutrient interplay. This will comprehend quantitative variations of target and non-target micronutrient concentrations, together with an analysis on gene expression patterns. Therefore, we will gain information regarding any differences on the activation/repression of gene circuits operating within the multi-biofortified tubers. Furthermore, important physiological and agronomic parameters will be evaluated, as this will attest the fitness of the obtained transgenic plants. Finally, a tailored metabolic engineering strategy will be designed. This will be built on the new knowledge regarding micronutrient interplay and the plant’s physiological adaptations.