Plant Biochemistry Laboratory, Department of Plant and Environmental Science and
Center for Synthetic Biology, University of Copenhagen Denmark
With 12,000+ known structures, diterpenoids constitute a prime example of the vast repository of bio-active natural products produced by plants. Many of these diterpenoids are used by humans as highly valuable pharmaceuticals (e.g. taxol, artemisinin and triptolide for treatment of cancer, malaria and multiple sclerosis), as fragrances (e.g. ambergris), as natural plant growth promoters (e.g. gibberellins), or as food ingredients such as flavors (e.g. specific steviosides as natural sweeteners) or as colorants and spices (e.g. saffron obtained from the stigma of crocus flowers). The key to this diversification resides in the modular nature of the diterpenoid biosynthetic routes, where initially coupled pairs of diterpene synthases (diTPS) catalyze the cyclisation of geranylgeranyl-diphosphate into (multi)cyclic backbone structures which are then decorated by hydroxylation reactions catalyzed by a cascade of cytochrome P450s (P450s). Unfortunately such compounds are typically produced in minute amounts in plants and their structural complexity render them difficult to prepare from fossil resources using organic chemical synthesis. To harness these biosynthetic pathways for bio-sustainable production, we have developed an integrated biochemical and functional genomics approach including targeted metabolomics and single cell-type transcriptome studies to identify the genes encoding a desired pathway. Using this pipeline, the entire pathway for forskolin formation has been elucidated. Forskolin is a cyclic AMP booster used for treatment of glaucoma but also as a weight loss aid. Another target diterpenoid is ingenol-3-angelate used for treatment of squamous cell cancers where we elucidated the entire route to the intermediate jolkinol C. The plasticity of the functional parts identified in the course of these studies enabled combinatorial assembly of new-to-nature diterpenoid pathways.