BIOSYNTHESIS of Taxol

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Total synthesis of the anticancer agent Taxol is not practical and, for the foreseeable future, the supply of this diterpenoid drug, and its precursors for semisynthesis, must continue to rely on biological processes involving the isolation from yew (Taxus) species or cell cultures derived therefrom. Improvement of these biological methods of production must be based upon a detailed understanding of the complex pathway for Taxol biosynthesis, the enzymes which catalyze the sequence of reactions, and the genes for these enzymes, especially those responsible for slow steps, since the molecular genetic manipulation of the pathway can be expected to lead the production of the drug in high yield at more reasonable cost. The goal of improving Taxol production will be reached by determining the types and order of enzymatic steps from the diterpenoid branch-point intermediate geranylgeranyl diphosphate to the end- product, by cDNA cloning of the responsible genes, and by assessing the flux contribution of each step (and of diversionary side routes) by gene overexpression and suppression. Defining this multistep pathway is accomplished through the use of cell-free enzyme systems from induced yew (Taxus) cultured cells, combined with in vivo feeding studies, to determine the progression from simple to complex metabolites. This systematic approach has identified several early, intermediate and late steps of the Taxol pathway, and provided the tools for cDNA isolation with which thirteen pathway genes have been obtained and characterized by a broad range of cloning and expression strategies. The specific aims of this project are: 1. to clone and characterize the remaining five genes corresponding to intermediate enzymatic steps that complete modification of the taxoid core (Clp-hydroxylase, C9a-hydroxylase and C9 oxidase, C4,C20-epoxidase and oxomutase catalyzing formation the oxetane ring); 2. to clone and characterize the remaining two genes corresponding to the aroyl CoA ligase and C2'-hydroxylase needed to complete assembly of the C13 Af-benzoyl phenylisoserine side-chain; 3. to clone the corresponding genes and characterize the taxoid C7-0-, C9-O-, and C13-0-acetyltransferases which, along with taxoid 14p-hydroxylase, constitute major diversions of intermediate taxoids away from Taxol; and 4. to engineer Taxus cells for overexpression and suppression of each pathway (and side-route) gene, and to assess the influence on metabolic profile and yield as a means of ordering the sequence of reactions and defining flux controls at each step. Completion of these objectives will provide an understanding of Taxol biosynthesis and the foundation for multigene transgenic approaches to improve theproduction of Taxol, and of related intermediates for semisynthesis of Taxol and second generation taxoid drugs.