Taxadiene synthase, a diterpene cyclase, catalyzes the conversion of geranylgeranyl diphosphate (GGPP) to taxadiene, a key intermediate in Taxol biosynthesis in yew. A 2 151 bp cDNA fragment encoding taxadiene synthas...Taxadiene synthase, a diterpene cyclase, catalyzes the conversion of geranylgeranyl diphosphate (GGPP) to taxadiene, a key intermediate in Taxol biosynthesis in yew. A 2 151 bp cDNA fragment encoding taxadiene synthase of Taxus chinensis (Pilg.) Rehd. was cloned by homology-based PCR and cDNA library screening. The 5′-terminal 611 bp cDNA fragment of taxadiene synthase was isolated by PCR. The two fragments were ligated together and gave a 2*!712 bp cDNA fragment with a 2*!586 bp open reading frame (ORF), encoding 862 amino acid residues including a presumptive plastidial transit peptide. The taxadiene synthase of T. chinensis most closely resembles the one from T. brevifolia (97% identity). Heterologous overexpression of 2.5 kb cDNA fragment from T. chinensis was obtained using a fusion expression vector pET-32a and the Escherichia coli strain BL21trxB. The expressed proteins from E. coli BL21trxB were present as inclusion bodies. After the inclusion bodies were denatured, renatured and refolded, the recombinant enzyme was purified by a single step with a His-binding metal affinity column. The catalytic product of taxadiene synthase of T. chinensis was detected by capillary gas chromatography-mass spectrometry (GC-MS) and identified as taxa-4(5),11(12)-diene.展开更多
文摘Taxadiene synthase, a diterpene cyclase, catalyzes the conversion of geranylgeranyl diphosphate (GGPP) to taxadiene, a key intermediate in Taxol biosynthesis in yew. A 2 151 bp cDNA fragment encoding taxadiene synthase of Taxus chinensis (Pilg.) Rehd. was cloned by homology-based PCR and cDNA library screening. The 5′-terminal 611 bp cDNA fragment of taxadiene synthase was isolated by PCR. The two fragments were ligated together and gave a 2*!712 bp cDNA fragment with a 2*!586 bp open reading frame (ORF), encoding 862 amino acid residues including a presumptive plastidial transit peptide. The taxadiene synthase of T. chinensis most closely resembles the one from T. brevifolia (97% identity). Heterologous overexpression of 2.5 kb cDNA fragment from T. chinensis was obtained using a fusion expression vector pET-32a and the Escherichia coli strain BL21trxB. The expressed proteins from E. coli BL21trxB were present as inclusion bodies. After the inclusion bodies were denatured, renatured and refolded, the recombinant enzyme was purified by a single step with a His-binding metal affinity column. The catalytic product of taxadiene synthase of T. chinensis was detected by capillary gas chromatography-mass spectrometry (GC-MS) and identified as taxa-4(5),11(12)-diene.
