Investigation of the secoisolariciresinol dehydrogenase gene in the podophyllotoxin biosynthetic pathway of PPE7

Podophyllotoxin (PPT) is a plant natural product that serves as a precursor for the synthesis of many well-known chemotherapeutic drugs. Since the source plants for PPT are limited in availability, we have turned our attention to another viable source of this compound. Using the endophytic fungus PPE7 isolated from the rhizomes of Podophyllum peltatum, our ultimate goal is to engineer the PPT biosynthetic pathway into a suitable heterologous host to lead to the sustainable, commercial production of PPT. This research is centered on the secoisolariciresinol dehydrogenase (SD) gene encoding the enzyme that facilitates the dehydrogenation of secoisolariciresinol to form matairesinol, a mid-pathway intermediate product in PPT biosynthesis. We have successfully confirmed the full SD gene sequence in PPE7 and created a synthetic construct of the PPE7 SD gene sequence that lacks intronic sequences and is codon-optimized for protein expression in P. pastoris. Additionally, western analysis confirmed that the protein was successfully expressed and the synthetic gene is integrated into the P. pastoris genome. The next step in determining if the recombinant PPE7 SD heterologous protein is functional is to perform a bioconversion assay. The cells expressing this protein will be tested for in vivo and ex vivo production of the target compound matairesinol and will be compared to control cells containing the expression vector that lacks the synthetic SD gene. The product that results from this reaction will be analyzed by LC-MS. This enzyme is a key step in the production of PPT and the other bio-active metabolites from this pathway