UBC Theses and Dissertations
Elucidation and characterization of genes associated with montbretin a biosynthesis within crocosmia x crocosmiiflora and white pine weevil defense in picea sitchensis Roach, Christopher Robert
Plant-specialized metabolites have long been utilized as medicines, cosmetics, flavours, and industrial raw materials. To explore the biosynthesis of a specialized metabolite in a non-model system and utilize the biosynthetic genes for future application, genomics-informed research typically flows through three phases: i) development of genomic or transcriptomic resources, ii) discovery and characterization of biosynthetic genes, and iii) application of the genes and enzymes for improved production of the specialized metabolite. This thesis describes hypothesis-driven research along these three phases in two different plant species and two different metabolic systems. My research with Crocosmia x crocosmiiflora focused on resource development and discovery of biosynthetic genes of a specialized metabolite of interest, montbretin A (MbA). I developed new resources for this system including metabolite-profiles and transcriptome sequences and annotations. This work resulted in insight into the spatial and temporal patterns of MbA accumulation in C. x crocosmiiflora and a first reference transcriptome with annotation for this species. Using these resources, I functionally characterized four UDP-xylose synthases and five UDP-rhamnose synthases. I discuss the application of these genes for possible use in an improved MbA production system and provide a proof of concept for using these genes to enable characterization of downstream MbA biosynthetic genes. I also identified 14 UDP-glycosyltransferases as candidate MbA biosynthetic genes through a guilt-by-association analysis; however, their functional characterization did not support a role in MbA biosynthesis. In the second biological system, Sitka spruce (Picea sitchensis), I performed a detailed characterization of a set of monoterpene synthases involved in the biosynthesis of the (+)-3-carene. Using domain swapping and site-directed mutagenesis, I demonstrated the catalytic plasticity of monoterpene synthases across a family of (+)-3-carene synthase-like genes associated with P. sitchensis resistance against the white pine weevil (Pissodes strobi). This work identified a single amino acid as most critical in determining both product profile and enzyme kinetics. Furthermore, I described mechanisms by which this amino acid directs product profiles through differential stabilization of the reaction intermediate. The work presented highlights the inherent plasticity and potential for evolution of alternative product profiles of these monoterpene synthases of conifer defense against pests.
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