(746g) Towards the Elucidation and Metabolic Engineering of the Podophyllotoxin Biosynthetic Pathway

Towards the elucidation and metabolic engineering of the podophyllotoxin biosynthetic pathway

Warren Lau¹, Elizabeth Sattely¹
(1) Department of Chemical Engineering, Stanford University, 381 North-South Mall,
Stanford, CA, 94305-5025

The lignan compound, podophyllotoxin, is an isolated secondary metabolite used for the semi-synthesis of successfully utilized anti-tumor drugs etoposide, etopophos and teniposide. The growing demand for podophyllotoxin as a precursor for anti-cancer drugs has endangered the traditional source, Podophyllum emodi, which provides 0.1-1% by mass of the biologically active compound. Though other Podophyllum sources have been identified, there is an urgency to relieve the severe pressure on natural sources. Chemical synthesis is one possible solution, but current synthetic routes are not economically feasible for large scale production. Another possible alternative is the overproduction of the molecule in a tractable host organism by pathway engineering. However, there are currently an estimated number of five unknown genes missing for the complete biosynthesis of podophyllotoxin. Also, traditional hosts, such as E. coli, do not carry the complex machinery necessary to produce many plant proteins in their soluble and functional form. Herein, we present the development of an in planta screening platform that combines rapid, transient expression in the model plant, Arabidopsis thaliana (Arabidopsis) with liquid chromatography-mass spectrometry (LC-MS) metabolomics that will enable the functional characterization of candidate genes that are potentially involved in podophyllotoxin biosynthesis. Additionally, we will discuss the usage of this platform as an effective means of evaluating expression constructs for metabolic engineering efforts with a specific focus on building the initial pathway towards podophyllotoxin in Arabidopsis.