Plants are exquisite biochemical factories responsible for synthesizing hundreds of thousands of diverse natural products. These compounds often have remarkable medicinal, pharmaceutical, and other human-health related benefits. Although plant natural products have had a great impact on disease treatment and are prime targets for drug development, full realization of their clinical potential has been hampered due to the target compounds being produced only in low amounts and in a small number of slow growing plant species. Efforts to engineer large-scale production in heterologous systems to combat the low abundance of these metabolites in nature, and to develop fermentation approaches to modify the pathways to produce novel pharmaceutical derivatives not found in nature have not met with great success. Furthermore, the known sources of most plant medicinal compounds have proven to be exceedingly difficult to approach biochemically and hence the genes encoding for the enzymes responsible for transformation of metabolites along the biosynthetic pathway remain elusive. The research presented in this dissertation will focus on the monoterpene indole alkaloid camptothecin that is currently used to produce semi-synthetic anti-tumor drugs.Three individual studies were performed to help elucidate the regulatory networks involved in the biosynthesis of camptothecin in Camptotheca acuminata. The second chapter in this dissertation focuses on whole-plant labeling of C. acuminata in a 13C-enriched atmosphere to generate stable isotope-labeled metabolites and intermediates to track transformation from one intermediate to the next. Liquid chromatography/mass spectrometry analysis (LC/MS) of leaf tissue extracts from labeled plants showed that the bottleneck in the production of camptothecin lies in the formation of downstream metabolites, but not in the formation of the iridoids in plants that are younger than 10 weeks old. The results showed that it is important to consider the developmental stage of the plant when performing metabolite studies in C. acuminata.The third chapter in this dissertation focuses on the metabolite profiling of wild-type and RNAisilenced C. acuminata. The LC/MS analysis of wild-type tissues showed that C. acuminata differs from other MIA-producing (monoterpene indole alkaloid) plants as it does not accumulate the well-known MIA precursor, strictosidine, and instead produces strictosidinic acid. Moreover, nearly all camptothecin pathway intermediates are present as multiple isomers, indicating an alternative seco-iridoid pathway. The analysis of root, stem, shoot apex, young and mature leaf tissues of transformed lines of C. acuminata demonstrated that the production of MIAs in tryptophan decarboxylase (TDC) silenced lines were deficient while the cyclase (CYC) silenced lines showed impaired ability to produce MIAs. These results indicate that the CYC knockout was effective in downregulating iridoid synthesis and the TDC knockout was able to successfully eliminate tryptamine synthesis.The fourth chapter in this dissertation presents the use of a F5 (pentafluorophenylpropyl)column for chromatographic separation for isolation and purification of strictosidinic acidisomers, and 1-D and 2-D NMR (nuclear magnetic resonance) spectroscopy methods for the structure elucidation of the isomers. The results from NMR analysis demonstrated that the two major strictosidinic acid forms are stereoisomers at their glucosylated C21 positions. The knowledge obtained from this study extends our understanding of the multiple isomers that are observed through the pathway.
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