Redirection of carotenoid metabolism for the efficient production of taxadiene [taxa-4(5),11(12)-diene] in transgenic tomato fruit

Abstract

The taxanes are a group of polycyclic diterpenes produced by various species of yew. The potent anticancer drug paclitaxel (marketed as Taxol) is the commercially most important taxane with annual sales in 2000 exceeding 1.6 billion dollars. Paclitaxel is currently obtained either by direct extraction from yew trees or by the extraction of the precursor 10-deactilbaccatin III, which is then converted to paclitaxel by semi-synthesis. Apart from cost, one of the main draw backs to taxol in cancer treatment is the development of resistance by tumours, commonly due to the expression of ABC transporter efflux pumps which remove the drug from the target cell. A number of natural taxanes and semisynthetic derivates, have recently been shown to act as potent inhibitors of ABC transport proteins. These compounds have no effect upon microtubule polymerization (the normal target of paclitaxel), but have the ability to restore drug sensitivity when given in combination with paclitaxel to resistant cell lines. In work to be described elsewhere, we sort to carry out a structure function analysis of the ability of novel oxidised taxanes to act as ABC transporter inhibitors. For this study 100 mg or more of taxadiene [taxa-4(5),11(12)-diene], the first taxane in the paclitaxel pathway, was required as starting material from which to synthesize these compounds. Taxadiene is synthesised directly from geranylgeranyl diphosphate (GGPP), which is found in most plant tissues where it serves as a common precursor for many metabolites. The synthesis and use of GGDP are tightly regulated in most vegetative organs, however, in tomato fruit it is used almost exclusively for the production of coloured carotenoids which accumulate to high levels in the plastid as lycopene crystals. Expressing taxadiene synthase in a yellow-fruited tomato line that lacks the ability to utilise GGPP for carotenoid synthesis allowed GGPP normally utilised for making carotenoids to be re-routed for the production of taxadiene, allowing the facile extraction of 160 mg of highly pure taxadiene from 1 kg of freeze dried fruit.

Production of taxadiene from cultured ginseng roots transformed with taxadiene synthase gene

“…In contrast to the reduced growth shown in the TS-transformed Arabidopsis and tomato (29,31), no phenotypic or growth differences were observed between the cultured wild-type and the transgenic ginseng roots. Presumably, the newly introduced TS gene in Arabidopsis and tomato might convert GGPP to taxadiene, and therefore lower the amount of endogenous gibberellin (31).…”

“…The presence and expression of the TS gene in the transgenic cultured ginseng root and the de novo formation of a compound with the same fragmentation pattern as taxadiene confirmed the successful transformation of cultured ginseng roots to produce taxadiene. The taxadiene yield from the transformed ginseng roots with the TS gene was 9.10 μg/g dry weight, which was higher than the 5 μg/g dry weight produced in moss (30), but lower than the 20 μg/g dry weight produced in transgenic tomato fruits (29).…”

“…Crude hexane extracts from cultured wild-type ginseng or taxadiene synthase transformed ginseng roots were analyzed. (A) The peak at 13.16 min in the gas chromatogram had the mass spectra profile matched exactly with the taxadiene mass spectra profile of Kovacs et al (2007). (B) Taxadiene levels in the taxadiene synthase gene transformed ginseng roots.…”

“…Several studies have indicated methods to taxadiene in tomato and Arabidopsis using metabolic engineering due to the high price (29)(30)(31). The presence and expression of the TS gene in the transgenic cultured ginseng root and the de novo formation of a compound with the same fragmentation pattern as taxadiene confirmed the successful transformation of cultured ginseng roots to produce taxadiene.…”

“…In the initial attempt, the taxadiene existence was identified by a comparison of the peaks of the MS profile as was reported by Kovacs et al (29). The control wild-type cultured ginseng plants displayed no peak for taxadiene.…”