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Taxol Biosynthesis: Molecular Cloning of a Benzoyl- Coa:Taxane 2␣-O-Benzoyltransferase Cdna from Taxus and Functional Expression in Escherichia Coli

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Taxol Biosynthesis: Molecular Cloning of a Benzoyl- Coa:Taxane 2␣-O-Benzoyltransferase Cdna from Taxus and Functional Expression in Escherichia Coli Taxol biosynthesis: Molecular cloning of a benzoyl- CoA:taxane 2␣-O-benzoyltransferase cDNA from Taxus and functional expression in Escherichia coli Kevin Walker and Rodney Croteau* Institute of Biological Chemistry, Washington State University, Pullman, WA 99164-6340 Contributed by Rodney Croteau, October 16, 2000 A cDNA clone encoding a taxane 2␣-O-benzoyltransferase has metabolites are available to permit assay of 2-O-benzoyltrans- been isolated from Taxus cuspidata. The recombinant enzyme ferase activity with benzoyl-CoA as cosubstrate, the semisyn- catalyzes the conversion of 2-debenzoyl-7,13-diacetylbaccatin III, a thesis of 2-debenzoyl-7,13-diacetylbaccatin III did provide a semisynthetic substrate, to 7,13-diacetylbaccatin III, and thus ap- suitable taxoid substrate for screening the expressed set of T. pears to function in a late-stage acylation step of the Taxol cuspidata transacylase clones for 2␣-O-benzoyltransferase func- biosynthetic pathway. By employing a homology-based PCR clon- tion. Here, we describe the isolation and analysis of a cDNA ing strategy for generating acyltransferase oligodeoxynucleotide encoding the target taxane 2␣-O-benzoyltransferase (TBT), and probes, several gene fragments were amplified and used to screen we report on the properties of this recombinant enzyme of Taxol a cDNA library constructed from mRNA isolated from methyl biosynthesis. jasmonate-induced Taxus cells, from which several full-length acyltransferases were obtained and individually expressed in Esch- Materials and Methods erichia coli. The functionally expressed benzoyltransferase was Substrates. Authentic 10-deacetylbaccatin III was obtained from confirmed by radio-HPLC, 1H-NMR, and combined HPLC-MS verifi- Hauser Chemical Research (Boulder, CO). (2␣,5␣)-Dihydroxy- cation of the product, 7,13-diacetylbaccatin III, derived from 2-de- taxa-4(20),11(12)-diene was a gift from Robert Williams (Col- benzoyl-7,13-diacetylbaccatin III and benzoyl-CoA as cosubstrates orado State University, Boulder, CO). Benzoyl-CoA as the in the corresponding cell-free extract. The full-length cDNA has an sodium salt was purchased from Sigma and [7-14C]benzoic acid open reading frame of 1,320 base pairs and encodes a protein of was from NEN Life Sciences Products. All other reagents were 440 residues with a molecular weight of 50,089. The recombinant purchased from Aldrich, unless noted otherwise. benzoyltransferase has a pH optimum of 8.0, Km values of 0.64 mM and 0.30 mM for the taxoid substrate and benzoyl-CoA, respec- Synthesis of 7,13-Diacetylbaccatin III. To a stirred solution of ␣ ␮ tively, and is apparently regiospecific for acylation of the 2 - 10-deacetylbaccatin III (160 mg, 294 mol) in dry CH2Cl2 (5 ml) hydroxyl group of the functionalized taxane nucleus. This enzyme at 25°C under N2 were added acetic anhydride (20 eq), dimeth- may be used to improve the production yields of Taxol and for the ylaminopyridine (20 eq), and triethylamine (50 ␮l, 361 ␮mol). semisynthesis of drug analogs bearing modified aroyl groups at After 16 h, the reaction was diluted with EtOAc (50 ml) and the C2 position. quenched with water (10 ml). The mixture was stirred for 15 min, and the aqueous fraction was separated and extracted with paclitaxel ͉ 2-debenzoyl-7,13-diacetylbaccatin III EtOAc (twice with 25 ml). The combined organic fractions were washed with brine, 0.1 M HCl, and water, and dried over BIOCHEMISTRY he anticancer drug Taxol (generic name paclitaxel), pro- anhydrous MgSO4. The organic solvent was evaporated, and the Tduced by yew (Taxus) species, is one of the structurally more crude product was purified by silica gel flash column chroma- complex members of the taxoid family of diterpenoid natural tography (EtOAc͞hexane, 60:40, vol͞vol) to yield pure 7,13- products (1). Of the dozen enzymatic reactions involved in Taxol diacetylbaccatin III (see Fig. 2) (180 mg, 91% yield, 99% purity 1 1 ␦ biosynthesis (2, 3), there are five acyltransferase steps respon- by H NMR). H NMR (300-MHz, CDCl3) : 1.10 (s, CH3), 1.14 ϭ sible for the addition of five acyl groups present in the final highly (s, CH3), 1.74 (s, CH3), 1.77 (ddd, J 1.8, 10.8, and 14.7 Hz, ␤ ϭ functionalized product. The first and third acylation reactions of H-6 ), 1.90 (d, J 1.2 Hz, allylic-CH3) 1.97 (s, C(O)CH3), 2.12 ϭ the Taxol pathway appear to be catalyzed, respectively, by (s, C(O)CH3), 2.14 (s, C(O)CH3), 2.17 (d, J 8.7 Hz, H-14), 2.29 ϭ ␣ taxadien-5␣-ol O-acetyltransferase (TAT) (4), which converts (s, C(O)CH3), 2.53 (ddd, J 7.2, 9.6, and 14.4 Hz, H-6 ), the second specific pathway intermediate, taxa-4(20),11(12)- 3.89 (d, J ϭ 6.9 Hz, H-3), 4.09 (d, J ϭ 8.4 Hz, H-20␣), 4.24 dien-5␣-ol, to the acetate ester (5), and by 10-deacetylbaccatin (d, J ϭ 8.4 Hz, H-20␤), 4.91 (dd, J ϭ 1.8 and 9.6 Hz, H-5), 5.53 III 10-O-acetyltransferase (DAT), which converts 10-deacetyl- (dd, J ϭ 7.2 and 10.5 Hz, H-7), 5.60 (d, J ϭ 6.9 Hz, H-2), 6.10 baccatin III to baccatin III (6) as the last diterpenoid interme- (dt, J ϭ 1.2 and 8.7 Hz, H-13), 6.19 (s, H-10), 7.39–8.01 (aromatic diate before Taxol (3) (Fig. 1). cDNAs encoding both of these acetyltransferases have been isolated from an induced Taxus cuspidata cell library by a homology-based cloning strategy that Abbreviations: TBT, taxane 2␣-O-benzoyltransferase; APCI, atmospheric pressure chemical ionization; Mopso, 3-(N-morpholino)-2-hydroxypropanesulfonic acid. yielded a family of related acyltransferase sequences that Data deposition: The sequence reported in this paper has been deposited in the GenBank may contain all of the remaining transferases of Taxol biosyn- database (accession no. AF297618). thesis (6, 7). *To whom reprint requests should be addressed. E-mail: [email protected]. A survey of the 350 naturally occurring taxoid metabolites The publication costs of this article were defrayed in part by page charge payment. This characterized to date (1) suggests that the second acylation article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. reaction in the Taxol pathway is the benzoylation of the C2␣- §1734 solely to indicate this fact. hydroxyl group of an advanced taxane intermediate (2, 3) (Fig. Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.250491997. 1). Although no advanced, naturally occurring 2-deacyltaxoid Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.250491997 PNAS ͉ December 5, 2000 ͉ vol. 97 ͉ no. 25 ͉ 13591–13596 Downloaded by guest on September 28, 2021 Fig. 1. Outline of the Taxol biosynthetic pathway. The cyclization of geranylgeranyl diphosphate to taxadiene by taxadiene synthase and the hydroxylation to taxadien-5␣-ol by taxadiene 5␣-hydroxylase (a), the acetylation of taxadien-5␣-ol by taxa-4(20),11(12)-dien-5␣-ol O-acetyltransferase (TAT) (b), the conversion of a 2-debenzoyl ‘‘taxoid-type’’ intermediate to 10-deacetylbaccatin III by a taxane 2␣-O-benzoyltransferase (TBT) (c), the conversion of 10-deacetylbaccatin III to baccatin III by 10-deacetylbaccatin III 10-O-acetyltransferase (DBAT) (d), and the side-chain attachment to baccatin III to form Taxol (e) are illustrated. The broken arrow indicates several as-yet-undefined steps. protons). Atmospheric pressure chemical ionization (APCI) Synthesis of [7-14C]Benzoyl-CoA. The method was adapted from a MS: m͞z 671 (PHϩ). procedure for the synthesis of valproyl-CoA esters (8). To a solution of [7-14C]benzoic acid (3.3 mg, 27 ␮mol, specific activity ͞ ϭ ͞ Synthesis of 2-Debenzoyl-7,13-diacetylbaccatin III. To a stirred so- 18.5 Ci mol; 1 Ci 37 GBq) in CH2Cl2 tetrahydrofuran (5:2, ͞ lution of 7,13-diacetylbaccatin III (170 mg, 253 ␮mol) in dry vol vol, 1.4 ml) under N2 was added 1 M triethylamine in CH2Cl2 tetrahydrofuran (2 ml) at 0°C was added bis(2-methoxyeth- (3.0 ␮l, 30 ␮mol) in one portion, and the mixture was stirred for ␮ oxy)aluminum hydride (Ͼ65 wt % in toluene, 3 eq) dropwise. 10 min at room temperature. Ethyl chloroformate (2.57 l, 2.9 ␮ After stirring for 30 min at 0°C, the reaction was quenched by mg, 27 mol) was added in one portion and the reaction was dropwise addition of saturated NH4Cl, and the mixture was stirred for1hatroom temperature. The solvents were evapo- rated, and the residue was dissolved in 0.5 ml of t-butyl alcohol. stirred for 10 min, then warmed to room temperature and diluted ␮ with EtOAc (50 ml), followed by addition of water (10 ml). The CoA as the sodium salt (23 mg, 30 mol dissolved in 0.5 ml of aqueous phase was separated and extracted again with EtOAc 0.4 M NaHCO3) was added to the solution and the mixture was (twice with 25 ml). The combined organic fractions were washed stirred for 0.5 h at room temperature, then quenched with 1 M HCl (200 ␮l) and adjusted to pH 5 with 15 mM NaH PO (pH with brine and water, then dried over anhydrous MgSO . The 2 4 4 4.8). The solvents were evaporated under reduced pressure (5 h) solvent was evaporated and the crude product was purified by at room temperature. The residue was resuspended in 15 mM silica gel flash column chromatography (40–60% EtOAc gra- NaH2PO4 (pH 6.9, 7 ml). The crude product was purified by dient in hexane) to yield 2-debenzoyl-7,13-diacetylbaccatin III using a C Sep-Pak cartridge (500 mg C silica gel, Millipore) (see Fig.
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