| The plant, Podophyllum peltatum (American Mayapple) has been used for hundreds of years in folk medicine. The plant was uprooted, the root structure was boiled and the resulting extract was used for the removal of warts, treatment of parasitic worms, as a purgative agent and in larger doses to aid in suicide. The extraction was chemically analyzed to reveal the secondary metabolite podophyllotoxin (PPT).;The natural product PPT acts as a cytotoxic compound by inhibiting microtubule assembly, which lent itself to the development as a chemotherapeutic agent. PPT exhibited high levels of gastric toxicity, and was chemically modified to create the semi-synthetic drugs etoposide, teniposide, and etoposide phosphate which act as topoisomerase II inhibitors. These semi-synthetic chemotherapeutic agents are used for the treatment of many cancer types, including but not limited to: lung cancer, testicular cancer and, lymphoma and leukemia.;PPT is used as the precursor to these valuable chemotherapeutic agents making it in very high demand. However, the plant P. peltatum only grows in the wild and cannot not be cultivated as a renewable PPT resource. Furthermore, when this study began the biosynthetic pathway responsible for PPT production had yet to be discovered in any species. In an effort to discover the P. peltatum PPT biosynthetic genes, our laboratory considered next-generation genome sequencing. However, since no other related PPT-producing species had been sequenced, our sequencing effort would be a de novo assembly. To provide confidence in the eventual de novo assembly of the P. peltatum genome, genome size estimation of P. peltatum was performed using flow cytometry. P. peltatum was estimated to have a 2C genome size of 52.6327 +/- 0.5877 Gbp (53.82 pg DNA) (mean +/- SD, n=6). While this estimation contributes to the long-term goal of genomic sequencing of the plant, it also provided much insight to the difficulties researchers have faced since the beginning of P. peltatum research. The large genome can correlate to constraint on the distribution and abundance of the species. In addition, the copiousness of genetic material in P. peltatum can also account for seed germination difficulties our laboratory has faced since investigating this plant. The genome size estimation of the plant contributes to the sequencing effort, but also provides boundless insight on this challenging medicinal plant.;In an effort to discover a renewable PPT resource, our laboratory investigated the fungal endophytes of the P. peltatum. The P. peltatum endophyte 7 (PPE7), Phialocephala podophylli, is capable of PPT production. This fungus was further investigated using many molecular and bioinformatics techniques to elucidate the PPT pathway, revealing the complete sequence of the cinnamyl alcohol dehydrogenase (CAD) and secoisolariciresinol dehydrogenase (SD) genes that encode enzymes responsible for catalyzing upstream reactions in the PPE7 PPT biosynthetic pathway. To contribute to the development of a heterologous host producing a renewable source of PPT, our laboratory heterologously expressed the two genes in the host Pichia pastoris. The individual constitutive expression of PPE7 CAD and PPE7 SD, and the correlating HPLC analyses of the bioconversion reactions, provide evidence that the PPE7 genes are capable of conversion of their respective substrates to products. These novel enzymes can be further used to optimize the expression of the complete PPT pathway in P. pastoris, for the ultimate goal of a renewable resource of the valuable compound PPT. |
