Influence of Precursor Availability on Alkaloid Accumulation by Transgenic Cell Line of Catharanthus roseus

Abstract

We have used a transgenic cell line of Catharanthus roseus (L.) G. Don to study the relative importance of the supply of biosynthetic precursors for the synthesis of terpenoid indole alkaloids. Line S10 carries a recombinant, constitutively overexpressed version of the endogenous strictosidine synthase (Str) gene. Various concentrations and combinations of the substrate tryptamine and of loganin, the immediate precursor of secologanin, were added to suspension cultures of S10. Our results indicate that high rates of tryptamine synthesis can take place under conditions of low tryptophan decarboxylase activity, and that high rates of strictosidine synthesis are possible in the presence of a small tryptamine pool. It appears that the utilization of tryptamine for alkaloid biosynthesis enhances metabolic flux through the indole pathway. However, a deficiency in the supply of either the iridoid or the indole precursor can limit flux through the step catalyzed by strictosidine synthase. Precursor utilization for the synthesis of strictosidine depends on the availability of the cosubstrate; the relative abundance of these precursors is a cell-line-specific trait that reflects the metabolic status of the cultures.

Figure 1

Biosynthesis of strictosidine in C. roseus.

Figure 2

Effect of feeding loganin on TIA accumulation under conditions of low tryptamine availability. White bars represent cultures treated on d 0 and sampled on d 3; shaded bars represent cultures treated on d 11 and harvested on d 14. The concentration of exogenously added precursors is expressed in micromoles. L+T indicates that both loganin and tryptamine were added. Triplicate cultures were pooled upon harvesting and analyzed as single samples.

Figure 3

Time course of alkaloid production (A) and TDC activity (B) under normal conditions (○) and in the presence of 200 μm exogenous loganin (•). Δ, Daily difference in alkaloid content between treated and control cultures. Duplicate or triplicate cultures were pooled upon harvesting and analyzed as single samples.