Abstract
Plant specialized metabolites provide a rich source of essential medicines. However, the availability of plant material is a major discouraging factor for the medical industry in exploring this resource further in the search for and development of new drugs. Recent advances in biotechnology have paved the way for alternative and environmentally friendly production platforms that are opening up exciting possibilities for the development of new plant derived medicines. At its core, this new technology uses genetically modified microorganism grown at large scale to produce plant metabolites. To achieve this feat requires knowledge of the underlying biosynthetic pathways that give rise to the metabolites of interest in the native plants.
This thesis presents an investigation of the biosynthetic pathways of medicinal compounds in Tripterygium wilfordii (Celastraceae), a widely used plant in Chinese traditional medicine. Specifically, the research focuses on the discovery of the molecular underpinnings of two bioactive terpenoids found in T. wilfordii with potent anti-cancer properties: the diterpenoid, triptolide, and the triterpenoid, celastrol. The research methods employed included metabolite guided transcriptomics and high-throughput transient gene (co)expression in Nicotiana benthaniana.
Multiple candidate diterpene synthases were identified in a root specific transcriptome and functional characterization showed that they could account for four of the six known diterpene backbones of T. wilfordii as well as a diterpene backbone not yet isolated from the plant. The major result of this work was the discovery of a terpene synthase from the TPS-b subfamily (generally associated with monoterpene biosynthesis) that is involved in the formation of the diterpene miltiradiene, a possible precursor of triptolide (Chapter 2, doi: 10.1111/tpj.13410). Attempts to identify downstream oxidative steps in triptolide biosynthesis resulted in the testing of 49 cytochrome P450 (CYP) candidates of T. wilfordii. One CYP (CYP82D60) was shown to facilitate incomplete aromatization of miltiradiene to dehydroabitadiene (Chapter 3; unpublished results).
Expected first steps in celastrol biosynthesis were identified in another study of the 49 candidate CYPs when they were tested for conversion of triterpenoid backbones. Specifically three CYPs (CYP712K1 to 3) were shown to form 3-oxofriedelan-29-oic acid and its precursor 29-hydroxyfriedelin from friedelin, which was formed by the identified oxidosqualene cyclase TwOSC4 (Chapter 5; manuscript in preparation).
A different study probed residues important for product identity in two functionally distinct but closely related class II diterpene synthases of T. wilfordii. Two residues located in the predicted active site of the enzymes were shown to control product outcome (Chapter 4; doi: 10.1016/j.phytochem.2017.02.022).
Finally, an unrelated study of the sole diterpene synthase of Physcomitrella patens; copalyl diphosphate/kaurene synthase, demonstrated additional diterpenes formed by this enzyme (Chapter 6; doi: 10.1016/j.plaphy.2015.07.011).
The results of this PhD project define steps in T. wilfordii terpenoid biosynthesis of importance for future biotechnological production of triptolide and celastrol. Furthermore, the results shed new light on the evolutionary substrate flexibility within the plant TPS family and contribute to our understanding of molecular factors underlying diterpenoid diversity.
This thesis presents an investigation of the biosynthetic pathways of medicinal compounds in Tripterygium wilfordii (Celastraceae), a widely used plant in Chinese traditional medicine. Specifically, the research focuses on the discovery of the molecular underpinnings of two bioactive terpenoids found in T. wilfordii with potent anti-cancer properties: the diterpenoid, triptolide, and the triterpenoid, celastrol. The research methods employed included metabolite guided transcriptomics and high-throughput transient gene (co)expression in Nicotiana benthaniana.
Multiple candidate diterpene synthases were identified in a root specific transcriptome and functional characterization showed that they could account for four of the six known diterpene backbones of T. wilfordii as well as a diterpene backbone not yet isolated from the plant. The major result of this work was the discovery of a terpene synthase from the TPS-b subfamily (generally associated with monoterpene biosynthesis) that is involved in the formation of the diterpene miltiradiene, a possible precursor of triptolide (Chapter 2, doi: 10.1111/tpj.13410). Attempts to identify downstream oxidative steps in triptolide biosynthesis resulted in the testing of 49 cytochrome P450 (CYP) candidates of T. wilfordii. One CYP (CYP82D60) was shown to facilitate incomplete aromatization of miltiradiene to dehydroabitadiene (Chapter 3; unpublished results).
Expected first steps in celastrol biosynthesis were identified in another study of the 49 candidate CYPs when they were tested for conversion of triterpenoid backbones. Specifically three CYPs (CYP712K1 to 3) were shown to form 3-oxofriedelan-29-oic acid and its precursor 29-hydroxyfriedelin from friedelin, which was formed by the identified oxidosqualene cyclase TwOSC4 (Chapter 5; manuscript in preparation).
A different study probed residues important for product identity in two functionally distinct but closely related class II diterpene synthases of T. wilfordii. Two residues located in the predicted active site of the enzymes were shown to control product outcome (Chapter 4; doi: 10.1016/j.phytochem.2017.02.022).
Finally, an unrelated study of the sole diterpene synthase of Physcomitrella patens; copalyl diphosphate/kaurene synthase, demonstrated additional diterpenes formed by this enzyme (Chapter 6; doi: 10.1016/j.plaphy.2015.07.011).
The results of this PhD project define steps in T. wilfordii terpenoid biosynthesis of importance for future biotechnological production of triptolide and celastrol. Furthermore, the results shed new light on the evolutionary substrate flexibility within the plant TPS family and contribute to our understanding of molecular factors underlying diterpenoid diversity.
| Original language | Danish |
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| Publisher | Department of Plant and Environmental Sciences, Faculty of Science, University of Copenhagen |
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| Publication status | Published - 2017 |