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There is a fundamental lack of knowledge of the biosynthetic steps and the kinetic properties of the enzymes that catalyze the reactions that make tropane and granatane alkaloids. The synthetic biology and metabolic engineering toolbox for the development of these novel compounds is very limited. The main question guiding this research is: what molecular changes have resulted in the convergent evolution of tropane and granatane alkaloid metabolic systems? Tropane alkaloids and granatane alkaloids are structural homologs, with the only difference being one extra carbon in the granatane ring. Tropane and granatane alkaloids provide a way to reasonably study the rise of, and variation within, specialized metabolic pathways. An additional aim of this research is to use systems-based approaches to elucidate the biosynthetic pathway of granatane alkaloids in Punica granatum (pomegranate). Here we report the use of hairy root cultures for the production of granatane alkaloids in P. granatum. We established hairy root cultures using Agrobacterium rhizogenes harboring a pEGAD plasmid expressing enhanced green fluorescent protein (eGFP). The polyamines lysine, cadaverine, and methylcadaverine are hypothetical precursor compounds to granatane alkaloids. We report the quantities of these polyamines extracted from various tissues of the pomegranate plant. Further experimentation will also quantify the granatane alkaloids in the different pomegranate plant tissues as well. The first enzymatic step in the production of granatane alkaloids in P. granatum is hypothesized to be the decarboxylation of lysine. Here we report the experimentation of putative lysine decarboxylase proteins that were found using a pomegranate annotated genomic database. We also report the experimentation of putative S-adenosyl-L-methionine decarboxylase proteins for their possible substrate promiscuity for lysine.

Alkaloids are specialized metabolites produced by plants to protect themselves from biotic and abiotic forces. Tropane and granatane alkaloids are of special importance due to their vast pharmacological benefits. The evolutionary origin of the biosynthesis of these alkaloids is unknown. Understanding this aspect of tropane and granatane alkaloid biosynthesis will help us gain a greater understanding of what is necessary to produce these alkaloids using a systems biology approach. Tropane alkaloids are produced amongst seven different plant families; and granatane alkaloids are produced amongst four different families. The biosynthesis of tropane alkaloids is well studied in the Solanceae family. Here I report an alternate route to tropane alkaloid biosynthesis in the Erythroxylaceae, which utilizes a spermidine methyltransferase versus the well-studied putrescine methyltransferase found in the Solanaceae. I also provide evidence for a homologous step in granatane alkaloid biosynthesis using a novel N-methyltransferase called cadaverine methyltransferase. With the discovery and investigation of novel N-methyltransferases involved in both tropane and granatane alkaloid biosynthesis, we can utilize these enzymes to produce these compounds using a metabolic engineering platform in a microbial or plant system.