Six enzymes from mayapple that complete the biosynthetic pathway to the etoposide aglycone

Abstract

Podophyllotoxin is the natural product precursor of the chemotherapeutic etoposide, yet only part of its biosynthetic pathway is known. We used transcriptome mining in Podophyllum hexandrum (mayapple) to identify biosynthetic genes in the podophyllotoxin pathway. We selected 29 candidate genes to combinatorially express in Nicotiana benthamiana (tobacco) and identified six pathway enzymes, including an oxoglutarate-dependent dioxygenase that closes the core cyclohexane ring of the aryltetralin scaffold. By coexpressing 10 genes in tobacco-these 6 plus 4 previously discovered-we reconstitute the pathway to (-)-4'-desmethylepipodophyllotoxin (the etoposide aglycone), a naturally occurring lignan that is the immediate precursor of etoposide and, unlike podophyllotoxin, a potent topoisomerase inhibitor. Our results enable production of the etoposide aglycone in tobacco and circumvent the need for cultivation of mayapple and semisynthetic epimerization and demethylation of podophyllotoxin.

Fig. 1.. Biosynthetic pathway of (−)-podophyllotoxin in P. hexandrum.

Uncharacterized steps are indicated by dashed lines.

Fig. 2.. Expression analysis to identify candidate genes.

(A) qRT-PCR analysis of podophyllotoxin biosynthetic genes after P. hexandrum leaf wounding (at t = 0 hour). Relative expression levels were normalized to t = 0 hour. Data are average values (three technical replicates) ± one SD. (B) Hierarchical clustering of RNA-Seq expression data after filtering by enzyme family and expression level. Heat map depicts the expression levels from a single node from the resulting cluster. Color key: Known biosynthetic genes (black), candidate genes (red), genes identified in this report (red with black arrows).

Fig. 3.. Six genes identified for the biosynthesis of the etoposide aglycone.

(A) Average LC-MS ion abundance ± one SD (three biological replicates) are shown for (−)-podophyllotoxin intermediates and derivatives produced in tobacco after expression of indicated enzymes and (−)-matairesinol infiltration. (B) Extracted ion chromatograms (EIC) for the etoposide aglycone, (−)-4′-desmethylepipodophyllotoxin (m/z = 401) in tobacco leaves expressing GFP or DIR, PLR, SDH, CYP719A23, and the six genes identified in this report with and without the infiltration of (+)-pinoresinol. Arrows indicate glycosylated derivatives of (−)-4′-desmethylepipodophyllotoxin. (C) Average amounts of (−)-4′-desmethylepipodophyllotoxin detected in tobacco ± one SD (three biological replicates).