(provide by applicant) Taxol, a highly functionalized diterpenoid, is an important anticancer drug isolated from yew (Taxus) species. Total synthesis of taxol is not practical and, for the foreseeable future, the supply of this drug, and its precursors for semisynthesis, must rely on biological methods of production. The development of improved biological processes must be based upon a detailed understanding of the pathway for taxol biosynthesis, the enzymes which catalyze the sequence of reactions, and the genes encoding these enzymes, especially those responsible for slow steps, since the molecular genetic manipulation of the pathway can be expected to lead to the production of the drug in larger quantities at reasonable cost. The long-term goal of improving taxol production is being reached by determining the number, types and sequence of enzymatic steps in the transformation of the isoprenoid branch-point intermediate geranylgeranyl diphosphate to the diterpenoid natural product, and by assessing the contribution of each step to pathway flux in order to evaluate importance as a cDNA cloning (and gene overexpression) target. Defining this multi-step pathway is being 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 the first four specific steps of the taxol pathway and several downstream' transformations, and has provided the tools for cDNA isolation with which seven pathway genes have been obtained.
The specific aims of this ongoing project are: 1. to define the early oxygenation steps leading from the olefin precursor of taxol, taxadiene, to the level of a pentaol, to characterize the responsible cytochrome P450 enzymes, and to acquire the corresponding genes by a homology-based cloning strategy; 2. to define the late-stage oxygenation steps that complete the modification of the taxoid core, to characterize the responsible cytochrome P450 enzymes, and to acquire the corresponding genes by a similar cloning strategy; 3. to complete comparative studies on the three recombinant acyltransferases now in hand (for acylation at C2, C5 and C 10), and to define the acyltransferases involved in C13 side-chain assembly and to isolate the corresponding cDNAs; 4. to elucidate the remaining modifications to the taxane core (oxidation at C9 and oxetane D-ring formation) and the origin of the 3-phenylisoserine side chain, and to devise suitable cDNA cloning strategies; and 6. to engineer Taxus cells for overexpression of slow pathway steps to increase taxoid production yields, and to exploit both 'sense' and 'antisense' technology to assist in defining pathway sequence and flux controls.
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