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Abstract

Plant specialized metabolites have been historically used in traditional medicine, flavours and fragrances throughout centuries, and they still serve as a valuable source for new pharmaceutical and nutraceutical development. Montbretin A (MbA) is an acylated flavonol glycoside produced by the ornamental plant montbretia (Crocosmia x crocosmiiflora), and mainly accumulates in the underground storage organs, called corms. This unique metabolite is a highly specific inhibitor of the human pancreatic α-amylase (HPA), thus making it a promising candidate for drug development against type-2 diabetes. However, a production system for obtaining large quantities of MbA is currently unavailable. Metabolic engineering of MbA in alternative microbial or plant systems may lead to the large-scale production of MbA. The main goal of this thesis was to obtain foundational insight on MbA biosynthesis. I first examined the growth and development of the montbretia plant, and performed detailed metabolite analysis focusing on the corms. The results of the metabolite profiling revealed the spatiotemporal patterns of MbA biosynthesis. This provided a foundational knowledge for the development of the montbretia transcriptome by Irmisch et al. (submitted). Furthermore, based on the activity of corm protein extracts, I identified that a member of the BAHD-AT family is involved in the acylation step of MbA biosynthesis. The candidate BAHD-ATs were identified using the established montbretia transcriptome, and were cloned and heterologously expressed in Escherichia coli for functional characterization. Of the seven candidate ATs tested, two candidates, CcAT1 and CcAT2 catalyzed the formation of mini-MbA, the product of the third step of MbA biosynthesis. Additionally, CcAT1 and CcAT2 were transiently expressed in the leaves of Nicotiana benthamiana, which led to the formation of a surrogate mini-MbA compound. This provided preliminary insight towards the metabolic engineering of MbA in N. benthamiana. Furthermore, qRT-PCR analyses were performed to investigate the transcript abundance patterns of CcAT1 and CcAT2 during montbretia corm development. The transcript profiling of CcAT1 and CcAT2 further supported their in vivo roles of MbA biosynthesis. Overall, the results presented in this thesis provide new knowledge on specialized plant metabolism in a non-model plant species.