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Narraidoo, Nathalie (2011) Investigation of Taxol biosynthetic genes for the production of novel taxanes in heterologous plant systems. PhD thesis, University of Nottingham.

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Abstract

The diterpenoid paclitaxel (TaxolTM) is one of the most effective anticancer drugs, used against a wide range of cancers. It is produced as a secondary metabolite in the vascular cambial region of the bark of Taxus brevifolia from which it was first extracted in 1971. Taxol also accumulates in low concentrations in several other Taxus species. As the demands for Taxol greatly exceeded its supply, alternative routes for producing the drug and its related taxanes were developed. Taxol is presently manufactured by semisynthesis from its precursors baccatin III and 10-deacetylbaccatin III found in Taxus needles. The biosynthesis of Taxol mostly occurs via the 2-C-methyl D-erythritol 4- phosphate pathway and requires at least 19 enzymatic steps from the precursor geranylgeranyl diphosphate. This study set out to heterologously express the early genes of the Taxol biosynthetic pathway in Nicotiana tabacum for the subsequent redirection of this precursor for the synthesis of novel taxanes.

The first five genes of the Taxol biosynthetic pathway, namely taxadien-5-α-hydroxylase, taxadien-5α-acetyltransferase taxoids 10β, 13α- and 7β-hydroxylase, were isolated from Taxus baccata mRNA. Individual transgenic tobacco lines were generated expressing each of the first three enzymes of the biosynthetic pathway. These lines were crossed with each other in order to obtain all three transgenes expressed together in individual transgenic lines. Progenies from the crosses, expressing the first three transgenes were analysed, however, GS-MS analysis failed to detect the compound taxadiene-5α-ol and its acetylated compound taxadiene-5α-yl acetate. The expression of the Taxol biosynthetic genes in transgenic tobacco plants were accompanied by phenotypic effects, including dwarfism and low fertility of the transgenic plants. To circumvent these sterility issues which made crossing of the plants difficult, a construct was prepared carrying the first two genes of the Taxol biosynthetic pathway, to be transformed in yellow flesh tomato mutant at a later stage.

The localisation of taxadiene synthase, 5α-hydroxylase and taxadien-5α acetyltransferase was investigated by making translational fusions to fluorescent protein tags. Confocal microscopy was used to detect the fluorescent proteins GFP, YFP and CFP in Arabiodopsis thaliana roots and tobacco leaf and root cells. Taxadiene synthase was found to be localised to the plastids, taxadien-5-α-hydroxylase spatially positioned on the plastid envelope and the endoplasmic reticulum membrane and taxadien-5α acetyltransferase was localised to the endoplasmic reticulum.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Fray, R.G. Seymour, G.
Subjects:	R Medicine > RC Internal medicine > RC 254 Neoplasms. Tumors. Oncology (including Cancer) R Medicine > RS Pharmacy and materia medica
Faculties/Schools:	UK Campuses > Faculty of Science > School of Biosciences
Item ID:	13269
Depositing User:	EP, Services
Date Deposited:	13 May 2013 13:49
Last Modified:	13 Oct 2017 19:19
URI:	https://eprints.nottingham.ac.uk/id/eprint/13269

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