Submitted to: Archives of Biochemistry and Biophysics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 3/30/1997
Publication Date: N/A
Interpretive Summary: Taxol, a novel anticancer drug originally isolated from the Pacific yew, is currently in clinical use, but the increasing demand for this drug still requires intensive work on alternative approaches to its long-term supply. One of the alternatives to solving the long-term supply issue for taxol is taxol production via plant cell culture. Over the past several years, we have developed elite cell lines from yew species that produce taxol and related taxanes. In order to increase yields in this system, it is important to understand the steps in the biosynthetic pathway which may be limiting production. This paper describes the detection of taxadiene synthase in cell cultures; this enzyme is responsible for the first committed step in taxol biosynthesis. The levels and timing of the appearance of the enzyme relative to taxol production suggest that this enzyme is not limiting the formation of taxol in this system.
Technical Abstract: 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 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 and electrophoretically 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 culutres of T. canadensis indicate that rate-limiting transformations lay farther down the pathway than the cyclization step in this system.