Taxol Biosynthesis and Molecular Genetics

Abstract

Biosynthesis of the anticancer drug Taxol in Taxus (yew) species involves 19 steps from the universal diterpenoid progenitor geranylgeranyl diphosphate derived by the plastidial methyl erythritol phosphate pathway for isoprenoid precursor supply. Following the committed cyclization to the taxane skeleton, eight cytochrome P450-mediated oxygenations, three CoA-dependent acyl/aroyl transfers, an oxidation at C9, and oxetane (D-ring) formation yield the intermediate baccatin III, to which the functionally important C13-side chain is appended in five additional steps. To gain further insight about Taxol biosynthesis relevant to the improved production of this drug, and to draw inferences about the organization, regulation, and origins of this complex natural product pathway, Taxus suspension cells (induced for taxoid biosynthesis by methyl jasmonate) were used for feeding studies, as the foundation for cell-free enzymology and as the source of transcripts for cDNA library construction and a variety of cloning strategies. This approach has led to the elucidation of early and late pathway segments, the isolation and characterization of over half of the pathway enzymes and their corresponding genes, and the identification of candidate cDNAs for the remaining pathway steps, and it has provided many promising targets for genetically engineering more efficient biosynthetic production of Taxol and its precursors.

Specificity of the N-benzoyl transferase responsible for the last step of Taxol biosynthesis

“…Benzoyl-CoA, and [7][8][9][10][11][12][13][14] C]benzoyl-CoA (1.1 mCi/mol) employed for preliminary kinetic assay development, were prepared as previously described, as were tigloyl-CoA (from tiglic acid) and N-debenzoyl-2′-deoxytaxol (5) [17,19].…”

“…A 100-µL aliquot of protein solution (up to 100 µM TAX10 protein) in the above noted buffer was combined with up to 500 µM of either N-debenzoyl-2′-deoxytaxol (5) or N-debenzoyltaxol (6) and up to 500 µM [7][8][9][10][11][12][13][14] C]benzoyl-CoA in the same buffer, and incubated at 31°C for up to 1 h. Assay mixtures were then extracted with two 0.1-mL portions of CH 2 Cl 2 , and the organic extracts were pooled and reduced to dryness under a stream of purified N 2 . The residue was dissolved in 50 µL of CH 3 CN, filtered through a 0.2-µm nylon syringe filter (Millex-GN, Millipore, Bedford, MA) into a sample vial, and analyzed by HPLC with UV-and radiomonitoring (Agilent 1100 Series with G1311A quaternary pump, and coupled to a G1314A UV-detector and Packard Series A-100 radio-detector).…”

“…This parental olefin is then functionalized by a series of oxygenations, acylations, and other reactions to yield the late stage intermediate baccatin III (4, Fig. 1) [14], upon which the functionally significant Nbenzoyl phenylisoserine side chain [15] is then assembled at the C13-O-position to complete the pathway to Taxol (7).…”

“…Feeding studies with Taxus tissues, using both amino acid precursors and advanced taxoid intermediates, metabolite profiling of Taxus cells in culture, and demonstration of relevant activities in cell free enzyme systems (see [14,16,17] for reviews of this work) have indicated that side chain construction requires five steps ( Fig. 1) involving aminomutase-catalyzed conversion of (S)-α-phenylalanine (1) to (R)-β-phenylalanine (2), ligation to the corresponding CoA ester (3), transfer of the aminoacyl group to the C13-hydroxyl of baccatin III (4) to yield N-debenzoyl-2′-deoxytaxol (i.e., 13-O-β-phenylalanoyl baccatin III, 5), hydroxylation at the 2′-position in the side chain to afford N-debenzoyltaxol (i.e., 13-O-phenylisoserinoyl baccatin III, 6), and N-benzoylation as the final side chain modification.…”

“…In the case of the transfer reactions, functional screening of a family of previously acquired Taxus acyl/aroyl transferase cDNAs [13] yielded the C13 phenylpropanoid side chain-CoA acyl transferase that mediates the 13-O-esterification of baccatin III [20], and the benzoyl CoA-dependent transferase responsible for the final benzamidation step of the Taxol pathway [19]. The lack of absolute substrate specificity by the recombinant transferases led to some ambiguities with regard to the precise timing of hydroxylation and acyl transfer steps in side chain assembly [14] that were only recently resolved by demonstration that the cytochrome P450 2′-hydroxylase operates only on Ndebenzoyl-2′-deoxytaxol (5), thus implicating the construction sequence of O-esterification, followed by 2′-hydroxylation, followed by N-benzamidation [17]. In this context, it is important to note that the recombinant terminal N-transferase was characterized using the more readily available N-debenzoyl-2′-deoxytaxol (5) as a surrogate taxoid co-substrate [19].…”