Random sequencing of an induced Taxus cell cDNA library for identification of clones involved in Taxol biosynthesis

Abstract

Biosynthesis of the anticancer drug Taxol involves 19 enzymatic steps from the universal diterpenoid progenitor geranylgeranyl diphosphate derived by the plastidial methylerythritol phosphate pathway for isoprenoid precursor supply. 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, random sequencing of a cDNA library derived from Taxus cuspidata cells (induced for taxoid biosynthesis with methyl jasmonate) was undertaken. This effort revealed surprisingly high abundances for transcripts of several of the 12 defined genes of Taxol biosynthesis, yielded cDNAs encoding two previously uncharacterized cytochrome P450 taxoid hydroxylases, and provided candidate genes for all but one of the remaining seven steps of this extended sequence of reactions.paclitaxel ͉ Taxus cuspidata ͉ expressed sequence tags ͉ taxoid 10␤-hydroxylase

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).…”

“…1), with the terminal step of the reaction sequence being the N-benzoylation of 3′-N-debenzoyltaxol (6) to complete the pathway to Taxol (7) [14,16,17]. A cDNA encoding the presumptive N-benzoyl transferase was previously isolated by functional screening of a family of Taxus acyl/aroyl transferase clones [13], and the recombinant enzyme was characterized using the surrogate substrate 3′-N-debenzoyl-2′-deoxytaxol (5), not 3′-Ndebenzoyltaxol (6), because the former was more readily available [19]. With the recent synthetic preparation of N-debenzoyltaxol, described herein, it became possible to compare the presumed natural substrate of the reaction with the previously employed surrogate substrate, and to determine whether selectivity of this enzyme for the taxoid co-substrate is consistent with the proposed side chain assembly sequence (Fig.…”

“…The biosynthesis of Taxol requires nearly 20 steps beginning with the prenyl transferasemediated construction of the universal diterpenoid precursor geranylgeranyl diphosphate which is cyclized, in the committed step, to taxa-4(5),11(12)-diene [13]. 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.…”

“…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].…”