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The limited bioavailability of plant-derived natural products with anticancer activity poses major challenges to the pharmaceutical industry. An example of this is camptothecin, a monoterpene indole alkaloid with potent anticancer activity that is extracted at very low concentrations from woody plants. Recently, camptothecin biosynthesis has been shown to become biotechnologically amenable in hairy-root systems of the natural producer  Ophiorrhiza pumila . Here, time-course expression and metabolite analyses were performed to identify novel transcriptional regulators of camptothecin biosynthesis in  O. pumila . It is shown here that camptothecin production increased over cultivation time and that the expression pattern of the WRKY transcription factor encoding gene  OpWRKY2  is closely correlated with camptothecin accumulation. Overexpression of  OpWRKY2  led to a more than three-fold increase in camptothecin levels. Accordingly, silencing of  OpWRKY2  correlated with decreased camptothecin levels in the plant. Further detailed molecular characterization by electrophoretic mobility shift, yeast one-hybrid and dual-luciferase assays showed that OpWRKY2 directly binds and activates the central camptothecin pathway gene  OpTDC . Taken together, the results of this study demonstrate that OpWRKY2 acts as a direct positive regulator of camptothecin biosynthesis. As such, a feasible strategy for the over-accumulation of camptothecin in a biotechnologically amenable system is presented.

The transcription factor OpWRKY2 positively regulates the biosynthesis of the anticancer drug camptothecin in Ophiorrhiza pumila