Expression of a feedback-resistant anthranilate synthase in Catharanthus roseus hairy roots provides evidence for tight regulation of terpenoid indole alkaloid levels.

Abstract

Different plant species produce a variety of terpenoid indole alkaloids, which are of interest as plant defensive secondary metabolites and as valuable pharmaceuticals. Although significant progress has been made, the mechanisms regulating the levels of this important class of compounds require continued elucidation. Previous precursor feeding studies have indicated that alkaloid accumulation can be improved during the exponential growth phase of hairy root cultures through enhanced tryptophan availability. To test this relationship, transgenic hairy root cultures of Catharanthus roseus were established with a glucocorticoid-inducible promoter controlling the expression of an Arabidopsis feedback-resistant anthranilate synthase alpha subunit. Enzyme assays demonstrated that the Arabidopsis alpha subunit is compatible with the native beta subunit and that anthranilate synthase activity is more resistant to tryptophan inhibition in induced than in uninduced extracts. The metabolic effects of expressing the feedback-resistant anthranilate synthase alpha subunit were also dramatic. Over a 6-day induction period during the late exponential growth phase, tryptophan and tryptamine specific yields increased from almost undetectable levels to 2.5 mg/g dry weight and from 25 microg/g to 267 microg/g dry weight, respectively. The greater than 300-fold increase in tryptophan levels observed in these studies under certain induction conditions compares favorably with the fold increases obtained in previous constitutive expression studies. Despite the large increases in tryptophan and tryptamine, the levels of most terpenoid indole alkaloids were not significantly altered, with the exception of lochnericine, which increased 81% after a 3-day induction period. These results suggest that terpenoid indole alkaloid levels are tightly controlled.