Polyketides are one of the largest classes of secondary compounds in nature. Their complex and diverse chemical structures arise from a common biosynthetic strategy. In plants, aromatic polyketides range from compounds that act as phytoalexins, plant defense compounds, and colorants to compounds such as the bioactive ingredients in phytomedicinal crops - for example, the hypericins, a family of red anthraquinones produced by the herb St. John's wort, Hypericum perforatum. Recent work points out the roles these secondary metabolites play in defense strategies for herbivory, pathogen attack, intra-plant competition, and attraction for symbionts and pollinators. There are significant economic considerations for the phytomedicinal industry since concentrations of particular plant secondary metabolites within dried materials determine crop market value. Polyketide synthases (PKS) are enzymes that make polyketides and they share striking structural similarities and chemical strategies in synthesis of the backbone structures of polyketides, regardless of organism carrying out this synthesis. The bulk of the research on plant polyketide synthases to date has concentrated on chalcone synthase (CHS), a plant-specific PKS that synthesizes chalcone, a key intermediate for flavonoids. Because of the structural diversity of plant polyketides, it is clear that there are many more PKS enzymes yet to be discovered. The goal of this research is to use H. perforatum (St. John's Wort) as a model system to study polyketide formation because polycyclic aromatic compounds similar to the intermediates and end products of H. perforatum are produced across multiple fungal and plant species. The focus will be to determine the biosynthetic intermediates and key PKS(s) involved in the formation of hypericins by: 1) identification of putative intermediates in the polyketide pathway leading to the hypericin family of compounds, and 2) identification and characterize the PKSs responsible for hypericin biosynthesis in H. perforatum. To determine the pathway and detect intermediates leading to synthesis of hypericins, a 13C biolabeling technique will be employed followed by 1H and 13C-NMR spectroscopy analysis for structure elucidation. Genetic analyses of H. perforatum will be conducted initially to identify and characterize PKSs by using degenerate PCR primers designed to conserved regions of known PKS genes to amplify and clone unique gene fragments present in H. perforatum. These fragments will be used to probe a cDNA library derived from a clonal line that is a high producer of hypericins. Alternatively, a second approach will be to use T-DNA insertional mutagenesis. T-DNA tagged genes from these mutants will be isolated and their role in secondary metabolism will be determined.
Broader impacts: Key findings obtained during the initial stages of this project would shed light on the dynamics of plant secondary metabolism; this work will contribute toward an understanding of chemical diversity and its role in plant physiology, and contribute information related to phytomedicinal herb quality and food (dietary supplement) safety. In addition, this project will generate information concerning how organisms utilize chemical diversity to live and prosper in harsh and challenging environments. This work will have a strong educational impact since undergraduate and graduate students (MS) will be recruited to work on this research project and equipment will be used for training of students. In addition, the research will be integrated into undergraduate chemistry education at the host institution.
