The purpose of this research was to study the factors that affecting the growth and Taxol production by Taxus species. Taxol, a complex diterpene alkaloid, is approved by the FDA for the treatment of ovarian and breast cancer. It was originally isolated from the bark of Taxus brevifolia. However, the bark contains very low concentrations of Taxol. Currently, Taxol is manufactured via a semi-synthetic method, but the production is limited and Taxol is stilI an expensive drug. Plant cell culture method has been recognized as a promising alternative for Taxol production. Nevertheless, the low or unstable productivity has become an obstacle to attain high yield of Taxol. This research focused on the experimental and computational modelling aspects of Taxol production in Taxus cell cultures. Callus and cell suspension cultures were successfully initiated from the seedlings of T. baccata. The addition of 1.5 % (w/v) insoluble polyvinylpolypyrrolidone (PVPP) and use of half strength of picloram in callus maintenance medium reduced the problem of cell darkening considerably. In suspension cultures, the non-ionic XAD-4 adsorbent was added to overcome the same problem. Fructose was the best carbon source compared to sucrose and glucose. The addition of fructose (10 gIL) on day 8 and methyl jasmonate (lOO IlM) on day 10 increased Taxol production to 17 mgIL from the suspension cultures initiated from needle explants of the seedlings. The experimental data obtained from this study were used in the development of the computer models: the kinetic model, fundamental and integrated dynamic metabolic flux analysis models. The in silico Taxus metabolism was reconstructed and computational metabolic flux balancing method was used in order to obtain fluxes of all the metabolic reactions with linear programming and optimisation in GAMS environment (General Algebraic Modeling System). The objective function of optimisation was either the maximisation of the specific growth rate or the maximisation of the specific Taxol production rate. Experimental values of nutrient uptake rates such as glucose, fructose and oxygen during the course of the batch culture were used as constraints to obtain different sets of optimised flux distributions for different periods of the batch culture. The computational results indicated that the transhydrogenase reactions were important to balance the need of NADPH for biosynthetic reactions in Taxus metabolism. The variation in biomass composition, inclusion of starch biosynthesis and degradation reactions, and Taxol precursors did not affect the growth of cells significantly. When the secondary cell wall biosynthesis was incorporated in the model, both growth and Taxol production rates were reduced. An increase in in silico Taxol production was observed when methyl jasmonate elicitation was combined with phenylalanine addition. An integrated dynamic model was constructed in order to combine the abilities of Excel- VBA and GAMS to optimise the objective function, handle mathematical kinetic expressions and visualise the outputs. As an example of its application, Taxol concentrations were automatically computed after the optimisation during the time course of the batch culture. These models can be developed further and used in future in order to define some strategies such as media formulation, precursor addition and genetic engineering targets in silico in order to manipulate the metabolism and increase the Taxol yield.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:595834 |
| Date | January 2007 |
| Creators | Karim, Khairiah Abd |
| Publisher | University of Manchester |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
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