Investigation into the biotechnological applications of Pantoea agglomerans for the production of high value terpenoids such as taxadiene

Williamson, J J (2017) Investigation into the biotechnological applications of Pantoea agglomerans for the production of high value terpenoids such as taxadiene. PhD thesis, University of Nottingham.

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Plant derived terpenoids represent a diverse class of chemicals with important roles such as fragrances, flavours and pharmaceuticals as well as high value medicinal compounds such as the antimalarial Artemisinin and the anti-cancer drug Paclitaxel (Taxol). High volume production of these products can be difficult due to reliance on plant production or economic difficulties in synthetic production. These high value terpenoids represent an interesting challenge to metabolic engineering and synthetic biology, not only the requirement to produce these chemicals at scale, but also because novel products can be developed via the intermediates of these pathways.

Research into methods of microbial production of Taxol and its derivatives have focused on the engineering of Escherichia coli and the precursor pathways leading to geranylgeranyl diphosphate (GGPP), since GGPP is the last non-dedicated precursor to Taxol. The work reported here has taken a different approach and has instead attempted to utilise natural diversity in search of a better host for production of these high value terpenoids, by selecting an organism that already produces a large amount of GGPP. As well as being a precursor to Taxol, GGPP can also be converted into carotenoid pigments such as lycopene, β-carotene and zeaxanthin. Therefore production of carotenoids can be used as a surrogate measure for predicting how well a species will perform in the production of heterologous isoprenoids. Genes for carotenoid biosynthesis from Pantoea species have been used in many heterologous expression experiments, however Pantoea have not been considered as a possible chassis for the production of isoprenoids. As a relatively close phylogenetic relative to E.coli, these Gram negative bacteria are easily transformable, genetically tractable and many tools which have been developed for E. coli function in Pantoea.

The work reported here addresses the developments of tools and techniques to assess the ability of Pantoea agglomerans to produce terpenoids including lycopene and taxadiene. The tools developed included: 1) a range of promoters, which were characterised in E. coli and P. agglomerans for their regulation of lycopene biosynthesis, 2) methods for the editing of the genome of P. agglomerans and 3) a range of vectors for the expression of the first 3 dedicated steps in Taxol biosynthesis, taxadiene synthase (txs), taxadiene 5α hydroxylase and taxadiene-5α-ol-O-acetyl transferase (5α) and taxane 10β-hydroxylase (10β). These genes initially could not be cloned in an operon together due to apparent toxicity. However by designing measures to minimise the burden imposed it was possible to express all four enzymes, of the three step process in P. agglomerans.

This work discusses the difficulties associated with working with a less well characterised organism (such as P. agglomerans), how we overcame these issues and the unexpected phenotypes exhibited by this genus.

Item Type: Thesis (University of Nottingham only) (PhD)
Supervisors: Hill, P.J.
Fray, R.G.
Seymour, G.
Subjects: Q Science > QP Physiology > QP501 Animal biochemistry
Faculties/Schools: UK Campuses > Faculty of Science > School of Biosciences
Item ID: 43555
Depositing User: Williamson, James
Date Deposited: 12 Oct 2017 12:23
Last Modified: 07 May 2020 12:32

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