Recent work in our lab has been focused on elucidation of the biosynthetic pathways of structurally complex diterpenoids, cyanogenic glucosides and vanillin. P450s catalyze key steps in these pathways. The pathway discovery process has been guided by transcriptomics and proteomics and by functional characterization of gene candidates using transient expression in tobacco and stable expression in yeast followed by LC-MS-NMR based structural identification of products obtained. Mass spectrometry-based bio-imaging technologies have been optimized for plant tissues reaching a 5 micrometer resolution level to enable visualization of the localization of the natural products at the cellular level, the importance of endogenous recycling of auto-toxic natural products, and the storage of natural products like dhurrin and vanillin glucoside in dense bio-condensates – “black holes” – at molar concentrations based on natural deep eutectic solvents. Using the styrene-maleic acid polymer-based “cookie cutter” technology, the possibility to isolate membrane bound enzyme complexes (metabolons) catalyzing entire pathways has been demonstrated and the dynamic organization of the complexes demonstrated using fluorescence lifetime imaging microscopy. Using the approaches of synthetic biology for combinatorial biosynthesis, the functional modules identified have been assembled in new combinations to expand the natural product landscape. Successful transfer of entire pathways into chloroplasts, the power house of the photosynthetic cell, demonstrate the potential of this organelle as a direct production and storage site for different classes of phytochemicals pointing towards development of a light driven synthetic biology platform for high value natural products based on carbon dioxide from the atmosphere as the sole carbon source. 1. Laursen, T, Borch, J., Knudsen, C., Bavishi, K., Torta, F. et al. (2016). Characterization of a dynamic metabolon producing the defense compound dhurrin in sorghum. Science 354: 890-893. 2. Knudsen, C., Gallage, N.J., Hansen, C.C., Møller, B.L. & Laursen, T. (2018). Dynamic metabolic solutions to the sessile life style of plants. Natural Product Reports 35: 1140-1155. 3. Thodberg, S., Del Cueto, J., Mazzeo, R., Pavan, S., Lotti, C. et al. (2018). Elucidation of the amygdalin pathway reveals the metabolic basis of bitter and sweet almonds (Prunus dulcis). Plant Physiology 178: 1096-1111. 4. Hansen, C.C., Sørensen, M., Veiga, T.A.M., Zibrandtsen, J.F.S., Heskes, A.M. et al. (2018). Reconfigured cyanogenic glucoside biosynthesis in Eucalyptus cladocalyx involves a cytochrome P450, CYP706C55. Plant Physiology 178: 1081-1095. 5. Pateraki, I., Andersen-Ranberg, J., Jensen, N.B., Wubshet, S.G., Heskes, A.M. et al. (2017). Total biosynthesis of the cyclic AMP booster forskolin from Coleus forskohlii. eLIFE 6: e23001. 6. Bassard, J.-E., Møller, B.L., Laursen, T. (2017). Assembly of dynamic P450-mediated metabolons – order versus chaos. Current Molecular Biology Reports 3: 37-51. 7. Bjarnholt, N., Neilson, E., Crocoll, C., Jørgensen, K., Motawie, M., et al. (2018). Glutathione transferases catalyze recycling of auto-toxic cyanogenic glucosides in sorghum. The Plant Journal 94: 1109-1125.