Figure 1 Scheme of the targets for genetic engineering of secondary metabolic pathway. Dark arrows, thickness related to flux; open arrows, increased activity; crosses through arrows, blocked steps, S, desired product.
screening and selection procedures (30-32). Therefore, the regulation of the biosynthesis of TIAs in cell cultures of C. roseus is being extensively studied in our laboratory in a collaborative project, the BSDL-project group Plant Cell Biotechnology, with the Institute of Molecular Plant Sciences Leiden, and the Department of Biochemical Engineering of the Delft University of Technology.
The TIA pathway is a complex metabolic network (Fig. 2). The bio-synthetic precursors for TIA biosynthesis are provided by the shikimate pathway and the terpenoid pathway (33-36). It was proved that the terpenoid moiety of the TIAs, secologanin, is not derived from the mevalonate pathway but instead from the MEP* pathway using a cell suspension culture of C. roseus (37). Because in the tryptophan branch of the shikimate pathway tryptophan decarboxylase (TDC, EC 22.214.171.124) operates at the interface between primary and secondary metabolism, it has been demonstrated to be a site for regulatory control of TIA biosynthesis (Fig. 3). TDC catalyzes the conversion of L-tryptophan to tryptamine, the indole moiety of the TIA. Strictosidine synthase (STR) catalyzes the condensation of tryptamine with secologanin by a reaction of the Pictet-Spengler type into 3a(S)-strictosi-
*In the 4th European Symposium on Plant Isoprenoids (April 21-23, 1999, Barcelona) it was agreed to use the term MEP (2-C-methyl-D-erythritol 4-phosphate) or Rohmer pathway.
^TDC geraniol tryptamine ^610H
loganin tryptamine sTR9ecologanin strictosidine «-
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