Catharanthus roseus, a tropical plant, produces the valuable anti-cancer compounds, vincristine and vinblastine in extremely low amounts. My research objectives were to examine the response of plant secondary metabolism to various metabolic perturbations, and indicate limitations in the reaction network.
In hairy roots, tabersonine is an important intermediate in the synthesis of vindoline, a monomer in the formation of the anti-cancer compounds that are generally absent from cell and hairy root cultures. To understand how much metabolic flux is directed toward the tabersonine branchpoint, transient profiles of lochnericine and horhammericine in relation to tabersonine in both dark and light-adapted cultures were quantified. The results demonstrated that the accumulation of lochnericine was growth related, similar to tabersonine, and that light repressed the formation of all three alkaloids. Using enzyme inhibitors, the involvement of separate P-450 mononoxygenase dependent enzymes in the biosynthesis of horhammericine and lochnericine was demonstrated. Furthermore, horhammericine and lochnericine were observed to be turned over.
A search for rate limiting regions of the pathway was accomplished through precursor feeding studies. By identifying precursors, which after feeding, significantly enhance the production of alkaloids, the specific precursor branch that is limiting flux to alkaloids can be elucidated. Precursors fed from the terpenoid portion of the pathway at 21 days in the culture cycle were found to enhance the specific yield of tabersonine. This result suggests that during the early stationary phase period (21--24 days) flux limitations may occur upstream of geraniol. On the other hand, the rate-limiting pathway could not be identified during the late growth phase (17--21 days). In part, this was due to the fact that tryptophan served as a precursor of indole acetic acid (IAA), a plant growth regulator. Therefore, changes in indole alkaloid accumulation and root growth due to tryptophan feeding were similar to those induced by exogenously added IAA. Since feeding tryptamine or terpenoid precursors did not significantly enhance indole alkaloid accumulation the rate-limitation may be downstream of loganin.
The metabolic flux distribution between separate alkaloid branches was quantified by monitoring the transient profiles of multiple alkaloids. The flux towards the Iboga alkaloids decreased while the flux to the Aspidosperma alkaloids increased between 12 and 26 days. In contrast, the flux distribution between the Corynanthe branch and the sum of the Iboga and Aspidosperma branches remained unchanged during this period. Despite significant changes in extracellular nutrient levels during this period, the total flux to the alkaloids remained constant between 12 and 26 days.
Through the precursor feeding and biogenetic flux analysis studies, flux limitations to alkaloids likely exist in the terpenoid pathway leading to secologanin. With the tools developed to quantify metabolic flux through a simple model, metabolic flux in metabolically engineered plant cell and tissue cultures can be routinely analyzed.
| Identifer | oai:union.ndltd.org:RICE/oai:scholarship.rice.edu:1911/19420 |
| Date | January 1999 |
| Creators | Morgan, John Allen |
| Contributors | Shanks, Jacqueline V. |
| Source Sets | Rice University |
| Language | English |
| Detected Language | English |
| Type | Thesis, Text |
| Format | 138 p., application/pdf |
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