Transgenic hairy roots of Catharanthus roseus were established with glucocorticoid inducible tryptophan decarboxylase (TDC) expression alone or in combination with inducible expression of a feedback-resistant anthranilate synthase alpha subunit (ASα) from Arabidopsis. Northern blot analysis confirmed transcription of the anthranilate synthase gene upon induction in the double line (TDC+ASα) and in vitro enzyme assays confirmed increased resistance to feedback inhibition by tryptophan. In TDC enzyme assays, increases of 48% and 87% in the TDC and double lines, respectively, were noted. Although the TDC line showed no significant increase in tryptamine levels on induction, induction of the double line resulted in increases in tryptamine levels of as much as six-fold for a 3 day late exponential induction. Downstream effects on alkaloids were noted in the TDC line where serpentine specific yields increased as much as 129% on induction. No effects on measured alkaloids were noted in the double line, but the two clones have very different basal alkaloid biosynthetic capacities. Within this study, the engineering of the indole pathway in C. roseus hairy roots is reported, and the role of the indole pathway in alkaloid biosynthesis explored.
Bibliographical noteFunding Information:
The authors would like to thank Dr. Nam-Hai Chua at the Rockefeller University for providing the inducible promoter plasmid (pTA7002), Dr. Eugene Nester at the University of Washington for providing the A. rhizogenes 15834 strain used, and Dr. Gerald Fink at Whitehead Institute for Biomedical Research for providing the Arabidopsis ASA1 clone. This work was supported by National Science Foundation Grants BES-00037300 and BES-0224593 (S.I.G. and K.-Y. S.) and BES-9906978 and BES-0224600 (J.V.S.). Erik Hughes was partially supported by a training grant from the National Institutes of Health (T32-GM08362).