EFFECTS OF OVER-EXPRESSION OF STRICTOSIDINE SYNTHASE AND TRYPTOPHAN DECARBOXYLASE ON ALKALOID PRODUCTION BY CELL-CULTURES OF CATHARANTHUS-ROSEUS

Cells of Catharanthus roseus (L.) G. Don were genetically engineered t o over-express the enzymes strictosidine synthase (STR; EC 4.3.3.2) an d tryptophan decarboxylase (TDC; EC 4.1.1.28), which catalyze key step s in the biosynthesis of terpenoid indole alkaloids (TIAs). The cultur es established after Agrobacterium-mediated transformation showed wide phenotypic diversity, reflecting the complexity of the biosynthetic p athway. Cultures transgenic for Str consistently showed tenfold higher STR activity than wild-type cultures, which favored biosynthetic acti vity through the pathway. Two such lines accumulated over 200 mg.L-1 o f the glucoalkaloid strictosidine and/or strictosidine-derived TIAs, i ncluding ajmalicine, catharanthine, serpentine, and tabersonine, while maintaining wild-type levels of TDC activity. Alkaloid accumulation b y highly productive transgenic lines showed considerable instability a nd was strongly influenced by culture conditions, such as the hormonal composition of the medium and the availability of precursors. High tr ansgene-encoded TDC activity was not only unnecessary for increased pr oductivity, but also detrimental to the normal growth of the cultures. In contrast, high STR activity was tolerated by the cultures and appe ared to be necessary, albeit not sufficient, to sustain high rates of alkaloid biosynthesis. We conclude that constitutive over-expression o f Str is highly desirable for increased TIA production. However, given its complexity, limited intervention in the TIA pathway will yield po sitive results only in the presence of a favorable epigenetic environm ent.