Etoposide is an effective anticancer agent currently utilized in the clinic. The drug is semi-synthetically derived from podophyllotoxin, a lignan secondary metabolite isolated from Podophyllum rhizomes. However, the growing demand for the compound has critically endangered the traditional source, Podophyllum hexandrum. Though other podophyllotoxin-producing plants have been identified, it is desirable to relieve the severe pressure on natural sources. Current chemical synthetic routes, however, are not economically viable. Alternatively, we suggest metabolic engineering of a tractable host for overproduction, requiring the elucidation of the minimum set of genes sufficient for podophyllotoxin biosynthesis.
Herein, we present a platform for accelerated discovery in non-model plants. This involves the development of RNA-Sequencing conditions for the identification of potential gene candidates, and the screening of these candidates in a N. benthamiana system coupled with liquid chromatography-mass spectrometry metabolomics. We describe the usage of this platform for the identification of six new enzymes from Podophyllum hexandrum that complete the biosynthetic pathway to 4’-desmethyl-epipodophyllotoxin, a more direct precursor to Etoposide than podophyllotoxin. We also describe the full reconstitution of the pathway in N. benthamiana from the early lignan precursor, pinoresinol. The discovered genes enable future metabolic engineering efforts in Saccharomyces cerevisiae for industrial production of the anti-cancer agent.