title: Coexpression in yeast of Taxus cytochrome P450 reductase with cytochrome P450 oxygenases involved in Taxol biosynthesis.
abstract: To maximize redox coupling efficiency with recombinant cytochrome P450 hydroxylases from yew (Taxus) species installed in yeast for the production of the anticancer drug Taxol, a cDNA encoding NADPH:cytochrome P450 reductase from T. cuspidata was isolated. This single-copy gene (2,154 bp encoding a protein of 717 amino acids) resembles more closely other reductases from gymnosperms (approximately 90% similarity) than those from angiosperms (<80% similarity). The recombinant reductase was characterized and compared to other reductases by heterologous expression in insect cells and was shown to support reconstituted taxoid 10beta-hydroxylase activity with an efficiency comparable to that of other plant-derived reductases. Coexpression in yeast of the reductase along with T. cuspidata taxoid 10beta-hydroxylase, which catalyzes an early step of taxoid biosynthesis, demonstrated significant enhancement of hydroxylase activity compared to that supported by the endogenous yeast reductase alone. Functional transgenic coupling of the Taxus reductase with a homologous cytochrome P450 taxoid hydroxylase represents an important initial step in reconstructing Taxol biosynthesis in a microbial host.
authors.affiliation: Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164-6340, USA.
authors.forename: Stefan
authors.initials: S
authors.lastname: Jennewein
authors.forename: Hangil
authors.initials: H
authors.lastname: Park
authors.forename: Jinghong M
authors.initials: JM
authors.lastname: DeJong
authors.forename: Robert M
authors.initials: RM
authors.lastname: Long
authors.forename: Arthur P
authors.initials: AP
authors.lastname: Bollon
authors.forename: Rodney B
authors.initials: RB
authors.lastname: Croteau
mesh_terms: D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000378:metabolism*; D017239:Paclitaxel / Q000096:biosynthesis*; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism*; D020946:Taxus / Q000201:enzymology*
chemical_list: D003577:Cytochrome P-450 Enzyme System; D017239:Paclitaxel