Taxol production and taxadiene synthase activity in Taxus canadensis cell suspension cultures

The cyclization of geranylgeranyl diphosphate to taxa-4(5),11(12)-diene represents the first committed, and a slow, step in the complex biosynthetic pathway leading to the anticancer drug Taxol. The cyclization enzyme, taxadiene synthase, has been previously purified from Pacific yew (Taxus brevifolia) stem and characterized, and the corresponding cDNA has been isolated. To better assess the role of taxadiene synthase in the control of pathway flux in Canadian yew (T. canadensis) cells, a reliable system for production of Taxol in suspension culture, the enzyme from this source was isolated and shown to be chromatographically, electrophoretically, and kinetically identical to that of T. brevifolia stem. Results from the analysis of enzyme activity levels during the time course of Taxol accumulation in developing cell cultures of T. canadensis indicate that rate-limiting transformations lay farther down the pathway than the cyclization step in this system. (C) 1997 Academic Press.