A Taxus Paclitaxel Biosynthetic Genes Ii. Ginkgo Diterpene Cyclase Cloned

The novel diterpenoid Taxol (paclitaxel) first isolated from the bark of Pacific Yew (Taxus brevifolia) is one of the most powerful antitumor drugs. The first step in the Taxol biosynthesis from universal terpenoid pathway is the cyclization of geranylgeranyl diphosphate to taxa-4(5), 11(12)-diene by taxadiene synthase. The following step is a cytochrome P450-catalyzed hydroxylation of taxa-4(5), 11(12)-diene at C-5, with migration of the double-bond, to yield taxa-4(20), 11(12)-diene-5 α -ol. Subsequent reactions on the taxol pathway include additional oxygenations , hydroxy group acylations oxidation to a ketone, and generation of the oxytane ring system. Two cDNA fragments were cloned from a T. chinensis cDNA library in a previous work. The DNA sequencing showed that the ESTs obtained were 5'- and 3'-end fragments encoding taxadiene synthase. Due to a Pvu I site, two of which were also presented in the ampicillin resistance cassette region, a only restricted enzyme site could be used to ligate two fragments. A new vector pTTB has been constructed using a tetracycline resistance cassette instead of the ampicillin resistance cassette region in the cloning vector pThioHis B. Two cDNA fragments were ligated using PCR method followed by restricted enzyme digestion and ligation to generate pTTB2, which contained a 2,712 bp cDNA fragment with a 2, 586 bp open reading frame (ORF), encoding 862 amino acid residues including a presumptive plastidial peptide. The 3'-cDNA fragment and a truncated fragment were also subcloned by same method to give pTTB1 and pTTB3. The taxadiene synthase of T. chinensis most closely resembles the one from T. brevifolia ( 97% identity). The three...