METABOLIC-INHIBITORS, ELICITORS, AND PRECURSORS AS TOOLS FOR PROBING YIELD LIMITATION IN TAXANE PRODUCTION BY TAXUS-CHINENSIS CELL-CULTURES

Inhibition of biosynthetic enzymes and translation and translocation p rocesses, elicitation, and precursor feeding were used to probe biosyn thetic pathway compartmentation, substrate-product relationships, and yield limitation of the diterpenoid taxanes in cell cultures of Taxus chinensis (PRO1-95). The results suggest the following: (i) the source of isopentenyl pyrophosphate in taxane production is likely plastidic rather than cytoplasmic; (ii) baccatin III may not be a direct precus or of Taxol (Taxol is a registered trademark of Bristol-Myers Squibb f or paclitaxel); (iii) baccatin III appears to have cytoplasmic and pla stidic biosynthetic components, while Taxol production is essentially plastidic; and (iv) arachidonic acid specifically stimulates Taxol pro duction but does not have a significant effect on baccatin III yield. Semiempirical mathematical models were used to describe these results and predict potential yield-limiting steps. Model simulations suggest that, under current operating conditions, Taxol production in Taxus ch inensis (PRO1-95) cultures is limited by the ability of the cells to c onvert phenylalanine to phenylisoserine rather than by the branch-poin t acyl transferase. This result is supported by the lack of improvemen t of Taxol yield by feeding phenylalanine or benzoylglycine. The metho ds described in this article, while specifically expanding our knowled ge of taxane production in PRO1-95 cultures, could be generally useful in investigating complex aspects of secondary metabolic pathways in p lant cell cultures, especially when details of the pathway and compart mentation are sparse.