Flux analysis in central carbon metabolism in plants: 13C NMR experiments and analysis

Sriram, Ganesh
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Metabolic flux analysis is crucial in metabolic engineering. This research concentrated on improvements in 13C labeling-based flux analysis, a powerful flux quantification method, particularly oriented toward application to plants. Furthermore, systemic 13C flux analyses were performed on two model plant systems: Glycine max (soybean) embryos, and Catharanthus roseus hairy roots.;The concepts 'bond integrity', 'bondomer' and the algorithm 'Boolean function mapping' were introduced, to facilitate efficient flux evaluation from carbon bond labeling experiments, and easier flux identifiability analysis.;13C labeling experiments were performed on developing soybean (Glycine max) embryos and C. roseus hairy roots. A computer program, NMR2Flux, was developed to automatically calculate fluxes from the labeling data. This program accepts a user-defined metabolic network model, and incorporates recent mathematical advances toward accurate and efficient evaluation of fluxes and their standard deviations. Several physiological insights were obtained from the flux results. For instance, in soybean embryos, the reductive pentose phosphate pathway was active in the plastid and negligible in the cytosol. Also, unknown fluxes (such as plastidic fructose-1,6-bisphosphatase) could be identified and quantified. To the best of the author's knowledge, this is the most comprehensive flux analysis of a plant system to date.;Investigations on flux identifiability were carried out for the soybean embryo system. Using these, optimal labeling experiments were designed, that utilize judicious combinations of labeled varieties of two substrates (sucrose and glutamine), to maximize the statistical quality of the evaluated fluxes.;The identity of four intense peaks observed in the 2-D [13C, 1H] spectra of protein isolated from soybean embryos, was investigated. These peaks were identified as levulinic acid and 5-hydroxymethyl furfural, and were degradation products of glycosylating sugars associated with soybean embryo protein. A 2-D NMR study was conducted on them, and it was shown that the metabolic information in the degradation products can be used toward metabolic flux or pathway analysis.;In addition, the elemental make-up and composition of the biomass of C. roseus hairy roots (crucial toward flux analysis) is reported. 89.2% (+/-9.7%) of the biomass was accounted for.*;*This dissertation is a compound document (contains both a paper copy and a CD as part of the dissertation). The CD requires the following system requirements: Adobe Acrobat.

Chemical engineering