Molecular cloning of a taxa-4(20),11(12)-dien-5 alpha-ol-O-acetyl transferase cDNA from Taxus and functional expression in Escherichia coli

The taxa-4(20),11(12)-dien-5 alpha-ol-O-acetyl transferase which catalyzes the third step of Taxol biosynthesis has been isolated from methyl jasmonat e-induced Taxus cells, and partially purified and characterized (K. Walker, R. E. B. Ketchum, M. Hezari, D. Gatfield, M. Golenowski, A. Barthol, and R . Croteau, Arch. Biochem. Biophys. 364, 273-279 1999). A revised purificati on method allowed internal amino acid microsequencing of the enzyme, from w hich primers were designed and employed to amplify a transacetylase gene-sp ecific fragment. This radiolabeled, 900-bp amplicon was used as a hybridiza tion probe to screen a cDNA library constructed from poly(A)(+) RNA isolate d from induced Taxus cells, from which a full-length transacetylase sequenc e was obtained, Expression of this clone from pCWori(+) in Escherichia coli JM109 cells yielded the functional enzyme, as determined by radiochemical assay and combined capillary gas chromatographic-mass spectrometric verific ation of the acetylated product. The full-length DNA has an open-reading fr ame of 1317 nucleotides corresponding to a deduced amino acid sequence of 4 39 residues that exhibits high sequence identity to the proteolytic fragmen ts of the native enzyme, which the recombinant transacetylase resembles in properties. Consistent with the size of the operationally soluble native en zyme, the DNA appears to encode a monomeric protein of molecular weight 49, 079 that bears no N-terminal organellar targeting information. Sequence com parison of the taxadien-5 alpha-ol-O-acetyl transferase with the few other known acyl transferases of plant origin indicates a significant degree of s imilarity between these enzymes (64-67%). The efficient conversion of taxad ien-5 alpha-yl acetate to further hydroxylated intermediates of the Taxol p athway confirms the significance of this acylation step and suggests this t axadienol transacetylase to be an important target for genetic manipulation to improve Taxol production. (C) 2000 Academic Press.