The
monoterpene indole alkaloids vindoline and
catharanthine, which are exclusively synthesized in the medicinal plant Catharanthus roseus, are the two important precursors for the production of pharmaceutically important anti-
cancer medicines
vinblastine and
vincristine. Hairy root culture is an ideal platform for
alkaloids production due to its industrial scalability, genetic and chemical stability, and availability of genetic engineering tools. However, C. roseus hairy roots do not produce
vindoline due to the lack of expression of the seven-step pathway from
tabersonine to
vindoline [Murata & De Luca (2015) Plant Journal, 44, 581-594]. The present study describes the genetic engineering of the first two genes
tabersonine 16-hydroxylase (T16H) and 16-O-methyl
transferase (16OMT) in the missing
vindoline pathway under the control of a
glucocorticoid-inducible promoter to direct
tabersonine toward
vindoline biosynthesis in C. roseus hairy roots. In two transgenic hairy roots, the induced overexpression of T16H and 16OMT resulted in the accumulation of
vindoline pathway metabolites 16-hydroxytabersonine and 16-methoxytabersonine. The levels of root-specific
alkaloids, including
lochnericine, 19-hydroxytabersonine and hörhammericine, significantly decreased in the induced hairy roots in comparison to the uninduced control lines. This suggests
tabersonine was successfully channeled to the
vindoline pathway away from the roots competing pathway based on the overexpression. Interestingly, another two new metabolites were detected in the induced hairy roots and proposed to be the epoxidized-16-hydroxytabersonine and lochnerinine. Thus, the introduction of
vindoline pathway genes in hairy roots can cause unexpected
terpenoid indole alkaloids (TIA) profile alterations. Furthermore, we observed complex transcriptional changes in TIA genes and regulators detected by RT-qPCR which highlight the tight regulation of the TIA pathway in response to T16H and 16OMT engineering in C. roseus hairy roots.