Suppression of camptothecin biosynthetic genes results in metabolic modification of secondary products in hairy roots of Ophiorrhiza pumila

Secondary metabolic flux was modified in the hairy roots of Ophiorrhiza pumila using RNAi technology to produce camptothecin, an anti-neoplastic monoterpenoid alkaloid, and related alkaloids. [Display omitted] ► Hairy root of Ophiorrhiza pumila is a feasible system for camptothecin production. ► Camptothecin related alkaloids were reduced in the RNAi hairy root of O. pumila. ► Analysis of RNAi hairy roots is a potential method to identify intermediates. Camptothecin is a monoterpenoid indole alkaloid that exhibits anti-tumor activity. In Ophiorrhiza pumila, production of camptothecin and its related alkaloids was high in the hairy roots, but not in the cell suspension culture derived from hairy roots. To identify the intermediates in camptothecin biosynthesis, expression of genes encoding tryptophan decarboxylase (TDC) and secologanin synthase (SLS), the two enzymes catalyzing the early steps in camptothecin biosynthesis, were suppressed in the hairy roots of O. pumila by RNA interference (RNAi), and metabolite changes were investigated. In most TDC- and SLS-suppressed lines, accumulation of camptothecin and related alkaloids, strictosidine, strictosamide, pumiloside, and deoxypumiloside was reduced. The accumulation levels of secologanin exhibited a strong negative correlation with the expression level of TDC, and that of loganin exhibited a negative correlation with the expression level of SLS. Some hairy root-specific chromatographic peaks detected by liquid chromatography Fourier transform ion cyclotron resonance mass spectrometry (LC-FTICR-MS) exhibited positive or negative correlation with TDC expression, suggesting their possible involvement in camptothecin biosynthesis.