Metabolic flux analysis of photosynthetic systems

Avantika Anant Shastri, Purdue University

Abstract

Photosynthesis is the primary process on earth that fixes inorganic carbon dioxide into complex organic molecules. Potentially, the use of metabolic engineering will enable the production of many chemicals, including next generation fuels, by photosynthetic organisms. The knowledge of the interaction of carbon fixation pathways with other metabolic pathways, under different environmental conditions and mutations will increase fundamental biological understanding of photosynthetic metabolism as well as enable the modification of photosynthetic systems to achieve specific metabolic engineering goals. In this work, we study metabolic flux distributions in a model photosynthetic cyanobacterium, Synechocystis sp. PCC 6803 using two complementary approaches that are based on a stoichiometric model. We first reconstruct the biochemical reaction network of Synechocystis from its annotated genome and biochemical literature. We use the computational approach of flux balance analysis using linear optimization (FBA) on photoautotrophic metabolism for the first time, to evaluate maximum theoretical product yields and to obtain insights into the interactions between biochemical energy, carbon fixation and assimilation pathways. We predict and compare flux map topologies for the hetero-, auto- and mixotrophic modes of metabolism under conditions of optimal growth. We evaluate the effects of gene deletions or additions on theoretical biomass yield and metabolic flux distributions were and find a high degree of concurrence with experimental data found in literature. We also utilize the autotrophic flux distribution for technique development in the second part of this work. The second approach involves the development of a novel methodology for the experimental measurement of system-wide metabolic fluxes under autotrophic conditions using 13C labeling. Although steady state metabolic flux analysis using 13C labeling (steady state 13C MFA) is widely used for quantification of metabolic fluxes in hetero and mixotrophic metabolism, its application to autotrophic metabolism is prevented due to a unique problem associated with autotrophy on CO2. In this work, we identify and outline the first and only methodology that enables 13C MFA on autotrophic systems. The method involves estimation of metabolic fluxes from measurements of dynamic changes in 13C labeling patterns of intracellular metabolite pools in response to a step change in 13CO2 labeling (transient 13C MFA). We establish suitable rapid sampling, quenching, extraction, LC-MS/MS and GC-MS methods to quantify labeling patterns as well as concentrations of intracellular metabolites. We demonstrate the experimental methodology with a 13C labeling experiment under controlled bioreactor conditions. Important physiological findings include detection of photorespiration and evidence of glyoxylate shunt activity.

Degree

Ph.D.

Advisors

Morgan, Purdue University.

Subject Area

Biochemistry|Chemical engineering|Physiology

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