Chinese Yew Taxane 13¦Á-hydroxylase Gene Cloning And Expression

The Taxol diterpene is an important drug for the treatment of a variety of cancers. The low yield of Taxol from natural sources and increasing applications of Taxol in chemotherapy have prompted intense efforts to improve biological production yield of the drug, that depends critically upon a detailed understanding of the biosynthetic pathways leading to Taxol and related taxoids and definition of the responsible enzymes and genes. The biosynthesis of Taxol from plant primary metabolism is a very complex process, including at least 20 steps to construct its tetracyclic skeleton and the addition of the various hydroxyl and acyl functional groups. Many results from Taxol biogenetic schemes show that hydroxylations and acylations are important for the bioactivity of Taxol.In this work, a new full-length hydroxylase gene was obtai- ned by homology-based PCR cloning strategy. Complete open reading frame was obtained by SOE-PCR amplifying. The ge- nomic DNA has an open-reading frame of 1,458 nucleotides, which encodes 486 amino acids with a calculated molecular weight of 54.7KDa and an estimated pI of 9.31. The deduced amino acid sequence resembles the sequences of other cloned hydroxylase (54-60% identity) involved directly in Taxol biosynthetic pathways. The analysis of the deduced amino acid sequence of 13 a -hydroxylase revealed some typical character- ristics of P450s and possessed several A-group P450 conserved domains. Through further trial-and-error experiments, a set of culture conditions that allowed production of a reasonable amount of soluble enzyme was established. SDS-PAGE result showed that a protein with an apparent molecular mass of 54 kDa appeared, supporting that the new hydroxylase gene could be normally translated into a protein as expected. The gene might give more information to Taxol biosynthesis and might be further investigated in plant metabolism engineering research. We finally aims at getting different precursors for semisynthesis, and our work is still going on .