Taxadiene

The taxanes are a group of polycyclic diterpenes produced by various species of trees (such as yew) and are most commonly known as the potent anticancer drug paclitaxel (Taxol). The extraction process is costly. 

Transgenic tomato fruit expressing taxadiene synthase were able to reroute the production of taxadiene, allowing extraction of large amounts of highly purified taxadiene from freeze-dried tomato (Kovacs et al, 2007). 

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In plant plastids, GGPP is formed from products of glycolysis and is eight enzymatic steps away from central glucose metabolism. The MEP pathway (reviewed in recent literature - ) operates in plastids in plants and is a preferred source (non-mevalonate) of phosphate-activated prenyl units (IPPs) for plastid iso-prenoid accumulation, such as the phytol tail of chlorophyll, the backbones of carotenoids, and the cores of monoterpenes such as menthol, hnalool, and iridoids, diterpenes such as taxadiene, and the side chains of bioactive prenylated terpenophe-nolics such as humulone, lupulone, and xanthohumol. The mevalonic pathway to IPP that operates in the cytoplasm is the source of the carbon chains in isoprenes such as the polyisoprene, rubber, and the sesquiterpenes such as caryophyllene. 

Jennewein, S., Long, R.M., Williams, R.M. and Croteau, R. (2004) Cytochrome P450 taxadiene 5a-hydroxy-lase, a mechanistically unusual monooxygenase catalyzing the first oxygenation step of Taxol biosynthesis. 

Besumbes, O., Sauret-Giieto, S., Phillips, M.A. et al. (2004) Metabolic engineering of isoprenoidbiosynthesisin Arabidopsis for the production of taxadiene, the first committed precursor of Taxol. Biotechnology and Bioengineering, 88, 168-175. 

Kovacs, K., Zhang, L., Linforth, R.S.T. et al. (2007) Redirection of carotenoid metabolism for the efficient production of taxadiene [taxa-4(5),ll(12)-diene] in transgenic tomato fruit. Transgenic Research, 16,121-126. 

Huang, Q., Roessner, C.A., Croteau, R. and Scott, A.I. (2001) Engineering Escherichia coli for the synthesis of taxadiene, a key intermediate in the biosynthesis of Taxol. Bioorganic Medicinal Chemistry, 9, 2237-2242. 

By using an elegant 13C-labeling study that involved incubation of Phoma sp. with 1-13C and 1,2-13C acetate, Oikawa et al. [9], were able to isolate the proposed biosynthetic intermediate phomactatriene (or Sch 49026), with 13C incorporation from singly labeled acetate units as indicated by in Fig. 8.3. Phomactatriene is strikingly reminiscent of taxadiene, a biosynthetic intermediate for Taxol . The net biosynthesis for both involves geranylgeranyl diphosphate (GGDP) cyclization [9]. It is noteworthy that prior to isolation of phomactins, the only known related structure is cleomeolide, a diterpene from the herb Cleome viscosa [10] that remarkably resembles phomactin H. 

The metabolism of taxol by Eucalyptus perriniana cell suspension cultures has been recently reported to induce hydrolyses of ester bonds at C-13, C-10 and C-2 [222]. At this moment only very few data have been published about the microbial metabolism of taxoid compounds only site specific hydrolyses of acyl side-chains at C-13 or C-10 by extracellular and intracellular esterases of Nocar-dioides albus SC13,911 and N. luteus SC13,912, respectively, have been reported [223]. On the other hand, Hu et al. [224-226] have recently described some fungal biotransformations of related natural taxane diterpenes extracted from Chinese yews or their cell cultures, in order to obtain new active substances or precursors for hemisynthesis. The taxadiene 145, a 14 -acetylated derivative... 

Cyclization reactions of GGPP mediated by car-bocation formation, plus the potential for Wagner -Meerwein rearrangements, will allow many structural variants of diterpenoids to be produced. The toxic principle taxine from common yew (Taxus baccata Taxaceae) has been shown to be a mixture of at least eleven compounds based on the taxadiene skeleton which can be readily rationalized as in Figure 5.43, employing the same mechanistic principles as seen with mono- and sesqui-terpenes. 

Fig. 2 Biosynthetic route to terpenes. Geranyl diphosphate (5) famesyl diphosphate (6) gera-nylgeranyl diphosphate (7) (-)-limonene (8) (-)-camphene (9) taxadiene (10) casbene (II) capsidiol (12). IPP = isopentenyl diphosphate, DMAPP = dimethylallyl diphosphate...

Hu, Sun and Scott described recently an efficient synthesis of some taxadiene derivatives266. In their approach, alcohol (66) is treated with carbon disulphide followed by methyl iodide under standard conditions for the formation of xanthates, to afford the dithiocarbonate (67) in a process that presumably involved a Claisen rearrangement (Scheme 9). Interestingly, 67 undergoes smooth reaction with B SnH to provide the required products (68, 69)266. 

HUANG, E. X., HUANG, Q. L WILDUNG, M. R CROTEAU, R SCOTT, A. I. Overproduction, in Escherichia coli, of soluble taxadiene synthase, a key enzyme in the taxol biosynthetic pathway, Protein Expression Purification, 1998,13,90-%. 

WILLIAMS, D. C., WILDUNG, M. R., JIN, A. Q. W., DALAL, D., OLIVER, J. S., COATES, R. M., CROTEAU, R., Heterologous expression and characterization of a "pseudomature" form of taxadiene synthase involved in paclitaxel (Taxol) biosynthesis and evaluation of a potential intermediate and inhibitors of the multistep diterpene cyclization reaction, Arch. Biochem. Biophys., 2000, 379, 137-146. 

The genes encoding several of the hydroxylation and acylation steps have been cloned and functionally expressed in E. coli (for a summary, see Marasco and Schmidt-Dannert, 2007). Whereas it was possible to produce the main skeleton, taxadiene, in recombinant bacteria and yeast in good yield, the later steps of taxol biosynthesis appear to be more difficult to achieve in recombinant systems at present (Jennewein et al., 2005 Dejong et al. 2006). 

Wildung, M.R. and Croteau, R. (1996) A cDNA clone for taxadiene synthase, the diterpene cyclase that catalyzes the committed step of taxol biosynthesis. /. Biol. Chem., 271, 9201-4. 

To date over 30 plant terpenoid synthases have been cloned as cDNAs, and many of these were found to encode enzymes of secondary metabolism (43). Isolation and analysis of six genomic clones encoding monoterpene ((—)-pinene and (—)-limonene), sesquiterpene ((E)-a-bisabolene and S-selinene) and diterpene (abietadiene) synthases from Abies grandis, and a diterpene (taxadiene) synthase from Taxus brevifolia have been reported (44). Overexpression of a cotton farnesyl diphosphate synthase (EPPS) in transgenic Artemesia annua has resulted in 3- to 4-fold increase in the yield of the sesquiterpenoid anti-malarial drug, artemisinin, in hairy roots (45). 

Prolonged (ca 3 h) irradiation of Winterstein esters leds to the formation of the corresponding phenylpropionates [41] (Scheme 4) The reaction is typical of Winterstein s acid derivatives and does not involve antenna-like effects from chromophores on the terpenoid moiety. Similar results were obtained in fact with simple aliphatic esters of this P-aminoacid [41]. Under these conditions (irradiation for several hours), alkaloids of the lS-oxo.A4 20 11-taxadiene-type (taxine B and comptonine-type) undergo photocyclization to 3,11-cyclotaxanes [40], The photocyclization of this type of alkaloids could be accomplished without reductive deamination using shorter (17 min) irradiation times [S3]. 

Taxadiene undergoes an extended series of oxygenation and acylation reactions to yield taxol. So far, 8 out of 20 enzymatic steps have been in vitro catalyzed through cloned cDNAs [53]. Several regioselective CoA thioester-dependent acyltransferases, including a C13-side-chain N-benzoyltransferase which predominantly forms one 3 -epimer of 2 -deoxytaxol, are involved in the synthesis. This indicates that there is still a long way to go before taxol could be produced in microorganisms from a reconstituted biosynthesis. 

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