| dc.description.abstract | Photoautotrophic metabolism represents the primary source of all food on earth as well as raw materials for bio-based production of fuels and chemicals. Therefore, improving photosynthetic efficiency is an important aim for metabolic engineering. There are few comprehensive methods that quantitatively describe photoautotrophic metabolism, though such information would be valuable for increasing photosynthetic capacity, enhancing biomass production, and rerouting carbon flux toward desirable end products. This dissertation presents the application of a novel metabolic flux analysis technique, isotopically nonstationary metabolic flux analysis (INST-MFA), to in vivo models of cyanobacterial and plant systems. First, we applied INST-MFA to map carbon fluxes in the cyanobacteria Synechococctus elongatus PCC 7942, which has been engineered to produce isobutyraldehyde. Flux analysis revealed a pathway bottleneck at the pyruvate node, which led to further rounds of strain engineering, resulting in significant increases in isobutyraldehyde productivity. Next, we applied INST-MFA to Arabidopsis thaliana rosettes to examine a more complex plant photosynthetic network. This was the first extension of INST-MFA to an in planta experimental system, taking into account compartmentalization and cellular heterogeneity, in order to quantify differences in carbon partitioning as a response to altered environmental light conditions. Finally, we extended the INST-MFA approach to identify metabolic pathway alterations in transgenic lines of Arabidopsis thaliana that had been engineered to express a bacterial carbonic anhydrase enzyme, which was the first step toward reconstituting an algal carbon concentrating mechanism in a C3 plant. Quantification of the global impact of this genetic perturbation on intermediary carbon fluxes revealed unexpected increases in photorespiratory activity that will guide further rounds of metabolic engineering. The application of INST-MFA toward improving photosynthetic capacity in both cyanobacterial and plant systems provides a scientific framework for similarly transformative steps in industrial photosynthetic microorganisms and crops that are important for biofuel and chemical feedstock production. | |
