Cloning and expression of geranylgeranyl diphosphate synthase and isolation and characterization of taxadiene -5 -hydroxylase: Two enzymes involved in the early steps of taxol biosynthesis.

摘要

Taxol is a diterpenoid natural product of great interest and importance to both the basic research and biomedical communities. The exact reactions and order of biosynthetic steps required for the formation of this compound have not yet been fully elucidated. All of the information we have gathered regarding the biosynthesis of taxol is from studies which focus on either the late or early enzymatic reactions in this pathway. This dissertation contains reports of novel discoveries related to the early steps of taxol biosynthesis.;In the initial phase of this project, the enzyme geranylgeranyl diphosphate synthase, which is responsible for the formation of geranylgeranyl diphosphate, the twenty carbon precursor of taxol, was cloned from T. canadensis and overexpressed in yeast.;As this work progressed, the enzyme taxadiene-5-hydroxylase, which is responsible for the first oxygenation step in taxol biosynthesis, was isolated and biochemically characterized as a cytochrome P450 monooxygenase. Furthermore, the product of this reaction was identified as taxa-4(20),11(12)-dien-5alpha-ol. Given the difficulties with the isolation of cytochrome P450 enzymes from woody plant tissues, methods were developed for the recovery of functional taxadiene-5-hydroxylase. These methods can be used for the recovery of many such enzymes from woody tissues.;Finally, a degenerate primer-based PCR strategy was used to obtain probes for many different cytochrome P450 genes from T. canadensis in an effort to obtain a sequence for taxadiene-5-hydroxylase as well as any other P450s involved in taxol biosynthesis. Nine full length cytochrome P450 clones were obtained and expressed in yeast. One half of the active expression constructs were screened for taxadiene-5-hydroxylase and taxa-4(20),11(12)-dien-5alpha-ol hydroxylase activities. Unfortunately, none of the recombinant enzymes tested could catalyze either reaction. It is, however, highly likely that one or more of the uncharacterized P450s involved in taxol biosynthesis are represented in this bank of clones.