Effects of terpenoid precursor feeding on Catharanthus roseus hairy roots over‐expressing the alpha or the alpha and beta subunits of anthranilate synthase

Abstract

Among the pharmacologically important terpenoid indole alkaloids produced by Catharanthus roseus are the anti-cancer drugs vinblastine and vincristine. These two drugs are produced in small yields within the plant, which makes them expensive to produce commercially. Metabolic engineering has focused on increasing flux through this pathway by various means such as elicitation, precursor feeding, and introduction of genes encoding specific metabolic enzymes into the plant. Recently in our lab, a feedback-resistant anthranilate synthase alpha subunit was over-expressed in C. roseus hairy roots under the control of a glucocorticoid inducible promoter system. Upon induction we observed a large increase in the indole precursors, tryptophan, and tryptamine. The current work explores the effects of over-expressing the anthranilate synthase alpha or alpha and beta subunits in combination with feeding with the terpenoid precursors 1-deoxy-D-xylulose, loganin, and secologanin. In feeding 1-deoxy-D-xylulose to the hairy root line expressing the anthranilate synthase alpha subunit, we observed an increase of 125% in hörhammericine levels in the induced samples, while loganin feeding increased catharanthine by 45% in the induced samples. Loganin feeding to the hairy root line expressing anthranilate synthase alpha and beta subunits increases catharanthine by 26%, ajmalicine by 84%, lochnericine by 119%, and tabersonine by 225% in the induced samples. These results suggest that the terpenoid precursors to the terpenoid indole alkaloids are important factors in terpenoid indole alkaloid production.

Five year maintenance of the inducible expression of anthranilate synthase in Catharanthus roseus hairy roots

“…The parallel cultivation of the ASab-1 line starting at 2.25 years after liquid adaptation and the stabilized metabolite profile from 2-5 years provides additional evidence that the line has become stable and is insensitive to differences in sub-culturing techniques. The increase in enzyme activity reported at Peebles et al, 2005Peebles et al, , 2006 5 years may explain why there is an observed change in metabolite levels over the first 2 years (Figs. 3 and 4).…”

“…At this point, a mixture of hairy roots containing different backgrounds may still exist. Over time as the five best growing hairy root tips are chosen for Peebles et al, 2005Peebles et al, , 2006 sub-culturing, we maybe selecting for a faster growing, more homogeneous culture. We are currently using a modified procedure in which each selection is based on a single root tip from the infected plant.…”

The Leaf Epidermome ofCatharanthus roseusReveals Its Biochemical Specialization

“…The high K m values for both loganic acid and AdoMet suggest that the catalytic activity of LAMT may be limiting the production of terpenoid component of MIA biosynthesis in C. roseus (Peebles et al, 2006).…”

Manipulating indole alkaloid production by Catharanthus roseus cell cultures in bioreactors: from biochemical processing to metabolic engineering

“…The induced TDC line showed no significant increase in tryptamine level but 129% increase in serpentine production, whereas TDC-ASa line showed 6-fold increase in tryptamine level but no increase in serpentine production (Hughes et al 2004b). Secologanin biosynthesis limitation in such transgenic hairy roots was confirmed (Peebles et al 2006). In feeding 1-deoxy-D-xylulose to ASa-overexpressing hairy root line, an increase of 125% in hoerhammericine was observed, while loganin feeding increased catharanthine by 45%.…”