| The application of plant cell culture for the commercial supply of valuable products is limited by low productivity. The goal of this thesis was to characterize Taxol metabolism in Taxus cell cultures, so that rate-limiting steps could be identified, and effective productivity enhancement strategies could be tested.; The kinetics of growth and Taxol production were characterized in T. canadensis, T. cuspidata, and T. baccata cell cultures. Maximum biomass yields reached 250-350 g/L with minimum doubling times of eight days, while Taxol accumulation rates reached a maximum of 1.7 mg/L-day, which is less than that necessary to make plant cell culture economically feasible for the supply of Taxol. Additionally, Taxus cultures lacked the ability to sustain high levels of Taxol accumulation over time, and there was significant variability between flasks.; Elicitation with methyl jasmonate was successful at enhancing Taxol yields to various extents in different Taxus cultures. Increases of 9-fold (C93AD-T. canadensis), 2.6-fold (PC2-T. baccata), and 1.1-fold (CO93P-T. canadensis) with corresponding maximum levels of 38 mg/L (360 {dollar}mu{dollar}g/flask), 2.0 mg/L (29 {dollar}mu{dollar}g/flask), and 31 mg/L (300 {dollar}mu{dollar}g/flask) were measured. Methyl jasmonate also stabilized Taxol accumulation, eliminating the loss of Taxol over time that was observed in unelicited cultures. However, other compounds similar in structure to methyl jasmonate that are linked to the lipoxygenase pathway were ineffective at stimulating Taxol production.; Methyl jasmonate enhanced Taxol release to the extracellular medium. The release of Taxol was characterized using transport inhibitors, transmission electron microscopy (TEM), and agents known to affect release. Release was shown to involve vesicular transport, and calcium was identified as a compound that strongly inhibits Taxol release. The use of cell wall-digesting enzymes was successful at stimulating Taxol release.; Taxol/taxanes were shown to be stable in acidified methanol and conditioned cell culture medium. However, when proteins were precipitated from the medium, Taxol levels decreased by more than half. Approximately 25% of Taxol was lost from cell cultures over time. This loss of Taxol correlated with an increase in the percentage of cell-associated Taxol.; The taxane biosynthetic pathway was studied using elicitation with methyl jasmonate, precursor feeding, and taxane spiking. Methyl jasmonate elicitation induced the accumulation of non-side chain containing taxanes, suggesting that the point of side chain addition is rate-limiting. Additionally, Taxol accounted for less than 20% of the total taxanes in cell culture, indicating that conversion of the non-side chain taxanes to Taxol could enhance yields even further. |
