Plant Biochemistry Laboratory, Center for Synthetic Biology, University of Copenhagen, Thorvaldsensvej 40, DK-1871 Frederiksberg C, Copenhagen, Denmark; blm@plen.ku.dk
Recent work in our lab has resulted in the elucidation of the biosynthetic pathways of structurally complex diterpenoids from Coleus forskohlii, Vitex agnus-castus, Eremophila lucida and Ginkgo biloba, elucidation of the synthesis of vanillin in Vanilla planifolia and of the pathways for synthesis of cyanogenic glucosides in Hordeum vulgare, Eucalyptus cladocalyx, Prunus amygdalus and the ferns Phlebodium aureum and Pteridium aquilinum. The pathway discovery process has been guided by transcriptomics and proteomics and 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. MS bio-imaging technology has been optimized for plant cells to visualize the localization of the natural products at the cellular levels and has facilitated interpretation of the role of endogenous recycling of auto-toxic natural products. 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 based on carbon dioxide from the atmosphere as the sole carbon source.