Molecular cloning of a cytochrome P450 taxane 10 beta-hydroxylase cDNA from Taxus and functional expression in yeast

The early steps in the biosynthesis of Taxol involve the cyclization of ger anylgeranyl diphosphate to taxa-4(5),11(12)-diene followed by cytochrome P4 50-mediated hydroxylation at C5, acetylation of this intermediate, and a se cond cytochrome P450-dependent hydroxylation at C10 to yield taxadien-5 alp ha -acetoxy-10 beta -ol. Subsequent steps of the pathway involve additional cytochrome P450 catalyzed oxygenations and CoA-dependent acylations, The l imited feasibility of reverse genetic cloning of cytochrome P450 oxygenases led to the use of Taxus cell cultures induced for Taxol production and the development of an approach based on differential display of mRNA-reverse t ranscription-PCR, which ultimately provided full-length forms of 13 unique but closely related cytochrome P450 sequences. Functional expression of the se enzymes in yeast was monitored by in situ spectrophotometry coupled to i n vivo screening of oxygenase activity by feeding taxoid substrates, This s trategy yielded a family of taxoid-metabolizing enzymes and revealed the ta xane 10 beta -hydroxylase as a 1494-bp cDNA that encodes a 498-residue cyto chrome P450 capable of transforming taxadienyl acetate to the 10 beta -hydr oxy derivative; the identity of this latter pathway intermediate was confir med by chromatographic and spectrometric means. The 10 beta -hydroxylase re presents the initial cytochrome P450 gene of Taxol biosynthesis to be isola ted by an approach that should provide access to the remaining oxygenases o f the pathway.