Taxol biosynthesis: Identification and characterization of two acetyl CoA:taxoid-O-acetyl transferases that divert pathway flux away from Taxol production

Two cDNAs encoding taxoid-O-acetyl transferases (TAX 9 and TAX 14) were obtained from a previously isolated family of Taxus acyl/aroyl transferase cDNA clones. The recombinant enzymes catalyze the acetylation of taxadien-5α,13α-diacetoxy-9α,10β-diol to generate taxadien-5α,10β,13α-tri-acetoxy-9α-ol and taxadien-5α,9α,13α-triacetoxy-10β-ol, respectively, both of which then serve as substrates for a final acetylation step to yield taxusin, a prominent side-route metabolite of Taxus. Neither enzyme acetylate the 5α- or the 13α-hydroxyls of taxoid polyols, indicating that prior acylations is required for efficient peracetylation to taxusin. Both enzymes were kinetically characterized, and the regioselectivity of acetylation was shown to vary with pH. Sequence comparison with other taxoid acyl transferases confirmed that primary structure of this enzyme type reveals little about function in taxoid metabolism. Unlike previously identified acetyl transferases involved in Taxol production, these two enzymes appear to act exclusively on partially acetylated taxoid polyols to divert the Taxol pathway to side-route metabolites.     

Taxol biosynthesis: differential transformations of taxadien-5 alpha-ol and its acetate ester by cytochrome P450 hydroxylases from Taxus suspension cells

The biosynthesis of the diterpenoid antineoplastic drug Taxol in Taxus species involves the cyclization of the ubiquitous isoprenoid intermediate geranylgeranyl diphosphate to taxa-4(5),11(12)-diene followed by cytochrome P450-mediated hydroxylation (with allylic rearrangement) of this olefin precursor to taxa-4(20),11(12)-dien-5 alpha-ol, and further oxygenation and acylation reactions. Based on the abundances of naturally occurring taxoids, the subsequent order of oxygenation of the taxane core is considered to occur at C10, then C2 and C9, followed by C13, and finally C7 and C1. Circumstantial evidence suggests that the acetylation of taxadien-5 alpha-ol may constitute the third specific step of Taxol biosynthesis. To determine whether taxadienol or the corresponding acetate ester serves as the direct precursor of subsequent oxygenation reactions, microsomal preparations isolated from induced Taxus cells and optimized for cytochrome P450 catalysis were incubated with each potential substrate. Both taxadienol and taxadienyl acetate were oxygenated to the level of a diol and to higher polyols at comparable rates by cytochrome P450 enzymes of the microsomal preparation. Preparative-scale incubation allowed the isolation of sufficient quantities of the diol derived from taxadienol to permit the NMR-based structural elucidation of this metabolite as taxa-4(20),11(12)-dien-5 alpha,13 alpha-diol, which may represent an alternate route of taxoid metabolism in induced cells. GC-MS-based structural definition of the diol monoacetate derived in microsomes from taxadienyl acetate confirmed this metabolite as taxa-4(20),11(12)-dien-5 alpha-acetoxy-10 beta-ol, thereby indicating that acetylation at C5 of taxadienol precedes the cytochrome P450-mediated insertion of the C10-beta-hydroxyl group of Taxol.     

Taxoid metabolism: Taxoid 14beta-hydroxylase is a cytochrome P450-dependent monooxygenase

The production of the anticancer drug Taxol in Taxus (yew) cell cultures is often accompanied by the formation of side-route polyoxygenated taxoid metabolites bearing a 14beta-hydroxyl group. The recent acquisition of several new semisynthetic taxoid intermediates enabled the screening of a family of Taxus cytochrome P450 cDNA clones for the 14beta-hydroxylase and additional taxoid oxygenases. The candidate cytochrome P450 clones were functionally expressed in yeast and tested by in vivo feeding of radiolabeled 5alpha-acetoxy-10beta-hydroxy taxadiene and 5alpha,13alpha-dihydroxy taxadiene. One clone efficiently and specifically transformed the 5alpha-acetoxy-10beta-ol, but not the 5alpha,13alpha-diol, to a more polar product with the chromatographic properties of a taxoid triol monoacetate, and the identity of this product was confirmed by spectroscopic means as 5alpha-acetoxy-10beta,14beta-dihydroxy taxadiene. Microsome preparation from the transformed yeast allowed characterization of this new hydroxylase, which was shown to resemble other cytochrome P450 taxoid hydroxylases with pH optimum at 7.5 and a K(m) value for the taxoid substrate of about 50 microM. Because Taxol is unsubstituted at C14, the 14beta-hydroxylase cannot reside on the pathway to the target drug but rather appears to be responsible for diversion of the pathway to 14-hydroxy taxoids that are prominent metabolites of Taxus cell cultures. Manipulation of this hydroxylase gene could permit redirection of the pathway to increase flux toward Taxol and could allow the preparation of 13alpha,14beta-hydroxy taxoids as new therapeutic agents.     

Taxol biosynthetic genes

The function and properties of heterologously expressed full-length cDNA clones, isolated from a Taxus cDNA library and specific to Taxol biosynthesis, are summarized. Recombinant enzymes are described that catalyze early steps of the pathway, including taxadiene synthase, taxadien-5alpha-ol-O-acetyltransferase and taxadien-5alpha-yl acetate 10beta-hydroxylase, and that catalyze late steps, including 10-deacetylbaccatin III-10beta-O-acetyltransferase and taxane 2alpha-O-benzoyltransferase. The properties of Taxus geranylgeranyl diphosphate synthase are also described; although this synthase does not mediate a committed step of Taxol biosynthesis, it does provide the universal plastidial diterpenoid precursor, geranylgeranyl diphosphate, for initiating Taxol biosynthesis.     

Molecular cloning and characterization of a cytochrome P450 taxoid 2alpha-hydroxylase involved in Taxol biosynthesis

The Taxol biosynthetic pathway, arising from the primary isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate in yew (Taxus), consists of approximately twenty steps, at least nine of which are thought to be cytochrome P450-mediated oxygenations. Several oxygenases involved in the early hydroxylation steps of the pathway have been identified and the corresponding genes have been cloned; however, defining the enzymes and their genes responsible for oxygenations in the central portion of the pathway is more difficult because neither the exact sequence of reactions nor the relevant intermediates are known. A surrogate substrate, (+)-taxusin (taxa-4(20),11(12)-dien-5alpha,9alpha,10beta,13alpha-tetraol tetraacetate), that was previously employed in the isolation of a taxoid 7beta-hydroxylase, was used here to functionally screen a family of cytochrome P450 oxygenases originating from a Taxus cell EST library. This in vivo screen in yeast led to the identification of a 1488bp cDNA clone (encoding a 495 residue protein) that was capable of producing 2alpha-hydroxytaxusin from taxusin with a K(m) value of 10.5 +/- 2.7 microM and k(cat) of about 0.05 s(-1) for the surrogate substrate. This structurally typical cytochrome P450 resembles most closely the previously isolated taxoid 7beta-hydroxylase, which also uses taxusin as a substrate, and both 2alpha- and 7beta-hydroxylases are capable of the reciprocal conversion of their respective pentaol tetraacetate products to the common hexaol tetraacetate. This C2-hydroxylase would appear to mediate the mid-pathway functionalization of the C2-position of the taxane core that ultimately bears a benzoyl group as an important Taxol pharmacophore. Overexpression of this cytochrome P450 taxoid 2alpha-hydroxylase in Taxus cells may improve Taxol yields and could prove useful in the production of other 2alpha-hydroxy taxoids as starting materials for subsequent acylation at this position.     

Taxus metabolomics: methyl jasmonate preferentially induces production of taxoids oxygenated at C-13 in Taxus x media cell cultures

Cells from suspension cultures of Taxus cuspidata were extracted with pentane as a source of relatively non-polar taxoids. Of the 13 taxoids identified in this fraction, eight were oxygenated at C-14 and two had not been previously described. These taxoids, along with existing taxoid standards, were employed to profile the metabolites of Taxus x media cv. Hicksii cell suspension cultures induced with methyl jasmonate to produce paclitaxel (Taxol). The majority of the taxoid metabolites produced in these induced cultures were oxygenated at C-13, and not C-14.     

Genetic engineering of taxol biosynthetic genes in Saccharomyces cerevisiae

Baccatin III, an intermediate of Taxol biosynthesis and a useful precursor for semisynthesis of the anti-cancer drug, is produced in yew (Taxus) species by a sequence of 15 enzymatic steps from primary metabolism. Ten genes encoding enzymes of this extended pathway have been described, thereby permitting a preliminary attempt to reconstruct early steps of taxane diterpenoid (taxoid) metabolism in Saccharomyces cerevisiae as a microbial production host. Eight of these taxoid biosynthetic genes were functionally expressed in yeast from episomal vectors containing one or more gene cassettes incorporating various epitope tags to permit protein surveillance and differentiation of those pathway enzymes of similar size. All eight recombinant proteins were readily detected by immunoblotting using specific monoclonal antibodies and each expressed protein was determined to be functional by in vitro enzyme assay, although activity levels differed considerably between enzyme types. Using three plasmids carrying different promoters and selection markers, genes encoding five sequential pathway steps leading from primary isoprenoid metabolism to the intermediate taxadien-5alpha- acetoxy-10beta-ol were installed in a single yeast host. Metabolite analysis showed that yeast isoprenoid precursors could be utilized in the reconstituted pathway because products accumulated from the first two engineered pathway steps (leading to the committed intermediate taxadiene); however, a pathway restriction was encountered at the first cytochrome P450 hydroxylation step. The means of overcoming this limitation are described in the context of further development of this novel approach for production of Taxol precursors and related taxoids in yeast.     

Administering cultured Taxus cells with early precursors reveals bifurcations in the taxoid biosynthetic pathway

Administering Taxus suspension cells with labeled 5alpha-hydroxytaxadiene and 5alpha,10beta-dihydroxytaxadiene, and the corresponding 5alpha-acetate esters, demonstrated that acetylation at C5 of the monool precursor promotes the formation of 14beta-hydroxy taxoids, such as taxuyunnanine C, at the expense of 13alpha-hydroxy taxoids, including Taxol and its congeners, but that the major bifurcation in taxoid biosynthesis, toward 13alpha- or 14beta-hydroxy taxoids, occurs after 10beta-hydroxylation of the taxane core.     

联合调控对中国红豆杉细胞关键酶基因表达的影响

红豆杉悬浮培养细胞可以持续提供抗癌药物紫杉醇及一些紫杉烷类。在中国红豆杉悬浮培养细胞中,云南紫杉烷C(Tc)是主要的紫杉烷。为了更理性地调控紫杉醇或有用紫杉烷的生产,有必要深入了解其生物合成过程。采用实时定量PCR(Real-time Quantitative PCR,即RQPCR)技术考察经调控后紫杉醇及紫杉烷代谢中关键酶基因—TASY,T5αH,TDAT,T10βH,TαH,T14βH表达水平的变化。在细胞培养的第7天和12天,分别以100μmol/L2,3-二羟丙基茉莉酸(DHPJA)诱导,同时在细胞培养第7天进行20g/L蔗糖饲喂、100g/LXAD-7HP的原位吸附。该联合调控处理使得细胞培养第30天时,Tc产量高达1517±37mg/L,是对照处理的11.1倍,是DHPJA重复诱导联合蔗糖饲喂处理的1.7倍。RQ-PCR结果显示:DHPJA的加入可使6个基因表达水平显著提高,但在12小时后快速下降,需补充DHPJA以再次提高基因表达水平。吸附剂同时引入会延缓基因表达水平的提高速度,但却能维持基因表达处于一个较高的水平,表现为在细胞培养中后期,基因表达水平将显著高于无吸附剂的调控体系。与13α-羟化相对应的TαH基因有所不同,吸附剂的存在更显著地抑制其表达,但仍有维持表达的功能。     

Preliminary assessment of the C13-side chain 2'-hydroxylase involved in taxol biosynthesis

The biosynthesis of the anticancer drug Taxol in yew (Taxus) species is thought to involve the preliminary formation of the advanced taxane diterpenoid intermediate baccatin III upon which the functionally important N-benzoyl phenylisoserinoyl side chain is subsequently assembled at the C13-O-position. In vivo feeding studies with Taxus tissues and characterization of the two transferases responsible for C13-side chain construction have suggested a sequential process in which an aminomutase converts alpha-phenylalanine to beta-phenylalanine which is then activated to the corresponding CoA ester and transferred to baccatin III to yield beta-phenylalanoyl baccatin III (i.e., N-debenzoyl-2'-deoxytaxol) that undergoes subsequent 2'-hydroxylation and N-benzoylation to afford Taxol. However, because the side chain transferase can utilize both beta-phenylalanoyl CoA and phenylisoserinoyl CoA in the C13-O-esterification of baccatin III, ambiguity remained as to whether the 2'-hydroxylation step occurs before or after transfer of the amino phenylpropanoyl moiety. Using cell-free enzyme systems from Taxus suspension cells, no evidence was found for the direct hydroxylation of beta-phenylalanine to phenylisoserine; however, microsomal preparations from this tissue appeared capable of the cytochrome P450-mediated hydroxylation of beta-phenylalanoyl baccatin III to phenylisoserinoyl baccatin III (i.e., N-debenzoyltaxol) as the penultimate step in the formation of Taxol and related N-substituted taxoids. These preliminary results, which are consistent with the proposed side chain assembly process, have clarified an important step of Taxol biosynthesis and set the foundation for cloning the responsible cytochrome P450 hydroxylase gene.     

Taxol: biosynthesis, molecular genetics, and biotechnological applications

Over the past decade, Taxol and its closely related structural analogue Taxotere have emerged as very important antitumor agents. Their widespread use in the treatment of a variety of cancer types, their likely approval for the treatment of additional forms of cancer, and their use at earlier stages of intervention will lead to increased demand for these drugs in the future. Because of yield considerations, Taxol and Taxotere are currently derived via semisynthesis from the advanced taxoid 10-deacetylbaccatin III, which must be isolated from yew (Taxus) trees. Thus, efforts are underway to produce Taxol (and other advanced taxoids for use in semisynthesis) by alternate, biotechnological means. This article provides a current overview of research on taxoid biosynthesis and an assessment of bioengineering applications for taxoid production in yew cell culture.     

Taxane diterpenoids from Taxus yunnanensis and Taxus cuspidata

Chemical examination of the seeds of the Chinese yew, Taxus runnanensis Cheng et L. K. Fu and the Japanese yew, Taxus cuspidata Sieb et Zucc, resulted in the isolation of four taxane diterpenoids. The structures of these taxoids were established as (12alpha)-2alpha-acetoxy-5alpha,9alpha, 10beta-trihydroxy-3,11-cyclotax-4(20)-en-13-one; 2alpha,7beta,13alpha-triacetoxy-5alpha, 9alpha-dihydroxy-2(3-->20)abeotaxa-4(20),11-dien-10-one; 9alpha,10beta-diacetoxy-5alpha-cinnamoyloxytaxa- 4(20),11-dien-13alpha-ol and the known 2alpha,7beta,9alpha,10beta,13-pentaacetoxytax a-4(20),12-diene-5alpha,11beta-diol on the basis of spectral analysis.     

Partial purification and characterization of acetyl coenzyme A: taxa-4(20),11(12)-dien-5alpha-ol O-acetyl transferase that catalyzes the first acylation step of taxol biosynthesis

The acetylation of taxa-4(20),11(12)-dien-5alpha-ol is considered to be the third specific step of Taxol biosynthesis that precedes further hydroxylation of the taxane nucleus. An operationally soluble acetyl CoA:taxadienol-O-acetyl transferase was demonstrated in extracts of Taxus canadensis and Taxus cuspidata cells induced with methyl jasmonate to produce Taxol. The reaction was dependent on both cosubstrates and active enzyme, and the product of this acetyl transferase was identified by radiochromatographic and GC-MS analysis. Following determination of the time course of acetyl transferase appearance in induced cell cultures, the operationally soluble enzyme was partially purified by a combination of anion exchange, hydrophobic interaction, and affinity chromatography on immobilized coenzyme A resin. This acetyl transferase has a pI and pH optimum of 4.7 and 9.0, respectively, and a molecular weight of about 50,000 as determined by gel permeation chromatography. The enzyme shows high selectivity and high affinity for both cosubstrates, with Km values of 4.2 and 5.5 microM for taxadienol and acetyl CoA, respectively. The enzyme does not acetylate the more advanced Taxol precursors, 10-deacetylbaccatin III or baccatin III. This acetyl transferase is insensitive to monovalent and divalent metal ions, is only weakly inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide, and coenzyme A, and resembles in general properties the few other O-acetyl transferases of higher plant origin that have been examined.     

Regio- and stereoselective hydroxylation of taxoids by filamentous fungi

Paclitaxel (Taxol), is one of the most promising chemotherapeutic agents developed for cancer treatment in past two decades. Microorganisms such as filamentous fungi are known to perform regio- and stereoselective hydroxylation of taxoids. Herein, we describe highly regio- and stereoselective hydroxylation at the 1beta and 9alpha positions of the taxane skeleton. Such hydroxylation reactions proceed readily for the taxadienes as substrates rather than taxoids having an oxetane ring. The presence of different oxygen substituents on the taxane nucleus, such as 5-acetoxy, has a significant effect on the selectivity and yield of the hydroxylation catalyzed by the microbial oxidases.     

MS/MS profiling of taxoids from the needles of Taxus wallichiana

Ammonium cationisation has been used for taxoid profiling of partially purified methanolic extracts of needles of Taxus wallichiana growing in different regions of the Himalayas (Kashmir, Himachal Pradesh, UP Hills, Darjeeling, Sikkim and Arunachal Pradesh) by electrospray ionisation tandem mass spectrometry (MS/MS). The MS/MS spectra of the [M + NH4]+ or [M + H]+ ions gave structurally diagnostic fragment ions which revealed information about the taxane skeleton as well as the number and nature of the substituents. The rearranged 11(15-->1)-abeo-taxanes showed a characteristic elimination of the hydroxyisopropyl group with an acetoxy/benzoyloxy group from C-9. The identification of the taxoids was achieved by comparison of the MS/MS spectra with those of authentic taxoids or was based on biogenetic grounds. The results were corroborated by liquid chromatography-MS analysis. Out of the 50 taxoids identified, 21 belonged to the rearranged class. The presence of paclitaxel in the samples from four regions was confirmed: the study also revealed the occurrence of several basic taxoids in these samples. MS/MS profiling by electrospray ionisation was shown to be a fast and reliable technique for the analysis of taxoid samples.     

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

The taxa-4(20),11(12)-dien-5alpha-ol-O-acetyl transferase which catalyzes the third step of Taxol biosynthesis has been isolated from methyl jasmonate-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 purification method allowed internal amino acid microsequencing of the enzyme, from which primers were designed and employed to amplify a transacetylase gene-specific fragment. This radiolabeled, 900-bp amplicon was used as a hybridization probe to screen a cDNA library constructed from poly(A)(+) RNA isolated from induced Taxus cells, from which a full-length transacetylase sequence 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 verification of the acetylated product. The full-length DNA has an open-reading frame of 1317 nucleotides corresponding to a deduced amino acid sequence of 439 residues that exhibits high sequence identity to the proteolytic fragments of the native enzyme, which the recombinant transacetylase resembles in properties. Consistent with the size of the operationally soluble native enzyme, the DNA appears to encode a monomeric protein of molecular weight 49,079 that bears no N-terminal organellar targeting information. Sequence comparison of the taxadien-5alpha-ol-O-acetyl transferase with the few other known acyl transferases of plant origin indicates a significant degree of similarity between these enzymes (64-67%). The efficient conversion of taxadien-5alpha-yl acetate to further hydroxylated intermediates of the Taxol pathway confirms the significance of this acylation step and suggests this taxadienol transacetylase to be an important target for genetic manipulation to improve Taxol production.     

利用RNAi抑制曼地亚红豆杉细胞紫杉烷14β-羟基化酶基因的表达

目的：利用RNA干扰技术抑制紫杉烷14β-羟基化酶(简称14OH)基因的表达。方法：以曼地亚红豆杉(Taxus×media)为材料,通过农杆菌介导的双元载体转化系统将针对14OH基因构建的RNA干扰载体导入该植物细胞中。应用PCR-Southern技术对转基因细胞进行了鉴定。另外,利用RT-PCR技术检测14OH基因mRNA表达水平,并采用HPLC技术对转基因细胞系中三种C-14氧取代的紫杉烷成分进行测定。结果：Southern检测证实转基因实验获得成功,RT-PCR结果表明转基因细胞系中14OH基因mRNA表达水平与对照组相比显著下降,HPLC分析显示C-14氧取代的紫杉烷含量也有明显降低。结论：该RNA干扰技术有效地抑制了曼地亚红豆杉细胞内14OH基因的表达,有望为提高紫杉醇的生物合成产量提供一条新途径。     

The kinetics of taxoid accumulation in cell suspension cultures of Taxus following elicitation with methyl jasmonate

Cell suspension cultures of Taxus canadensis and Taxus cuspidata rapidly produced paclitaxel (Taxol) and other taxoids in response to elicitation with methyl jasmonate. By optimizing the concentration of the elicitor, and the timing of elicitation, we have achieved the most rapid accumulation of paclitaxel in a plant cell culture, yet reported. The greatest accumulation of paclitaxel occurred when methyl jasmonate was added to cultures at a final concentration of 200 microM on day 7 of the culture cycle. The concentration of paclitaxel increased in the extracellular (cell-free) medium to 117 mg/day within 5 days following elicitation, equivalent to a rate of 23.4 mg/L per day. Paclitaxel was only one of many taxoids whose concentrations increased significantly in response to elicitation. Despite the rapid accumulation and high concentration of paclitaxel, its concentration never exceeded 20% of the total taxoids produced in the elicited culture. Two other taxoids, 13-acetyl-9-dihydrobaccatin III and baccatin VI, accounted for 39% to 62% of the total taxoids in elicited cultures. The accumulation of baccatin III did not parallel the pattern of accumulation for paclitaxel. Baccatin III continued to accumulate until the end of the culture cycle, at which point most of the cells in the culture were dead, implying a possible role as a degradation product of taxoid biosynthesis, rather than as a precursor.