(340a) Quantifying Transient Metabolic Fluxes Using Stable Isotopes, Mass Spectrometry, and Computational Modeling

The desire to apply engineering principles to biological systems has been a primary research objective for thousands of applications across a multitude of disciplines: from genetic engineering for the manufacture and production of biofuels, pharmaceuticals, and specialty chemicals, to systems biology approaches for characterizing and understanding health, disease, and other biological phenomena. As we strive for a more sophisticated understanding and subsequent utilization of these biological systems, it is imperative that equally complex tools for probing cellular metabolism be established simultaneously. My dissertation work focuses first on the development of experimental, analytical techniques to inform fundamental models that quantify reaction-level metabolism of non-model organisms and then apply these techniques to address real-world systems biology and metabolic engineering challenges.

Metabolic engineering of cyanobacteria for biofuel production. Cyanobacteria are a potential platform for the sustainable production of liquid fuels due to their ability to synthesize energy-dense compounds directly from atmospheric carbon dioxide. Recent research forays into this application have been encumbered by poor yields of desired fuel compounds due, in part, to a limited understanding of cyanobacterial metabolism. To enable the goal of logically engineering a commercially competitive cyanobacterial strain, I used isotope assisted metabolic flux analysis to identify reaction pathways for engineered strain production to improve titers of the target product. Specifically, I probed the metabolism of the fast-growing cyanobacterium, Synechococcus sp. PCC 7002 (WT), and a genetically engineered Synechoccous strain (LS) developed in our laboratory to produce limonene, a drop-in biofuel precursor to jet fuel. A major challenge to implementing this work centered on measuring and modeling metabolic pathways in a non-model photosynthetic system, which requires transient isotope labeling as a consequence of the single-carbon isotope label source (CO₂). Additionally, cyanobacteria display properties of both gram-negative and gram-positive bacteria, which necessitated surveying a variety of experimental quenching and extraction techniques to ensure optimal, rapid halting of metabolism and sufficient metabolite recovery.

Quantifying clotting and sex hormone effects on human blood platelet metabolism. The introduction of Exogenous female sex hormones to the human body, such as estrogen in the form of oral contraception, increases the risk of developing blood clots (venous thromboembolism) by ~3-4 fold. While this risk factor has been evident for over 50 years, the relationship between female sex hormones and the function of platelets, the specialized blood cells vital to clot initiation and propagation, is still unclear. As an initial, novel approach to elucidating the mechanisms by which estrogen influences platelet function, my work characterizes platelet metabolism during exposure to estrogen and thrombin, a potent platelet agonist. Ours is the first study to apply metabolic flux analysis techniques in platelet cells to directly quantify platelet metabolism and identified a metabolic shift that causes activated platelets to switch from anaerobic to oxidative processing of glucose. This required overcoming the additional challenges of performing metabolic flux analysis in an anuclear cell, which neither grows nor divides, and cannot be cultured in traditional laboratory monoculture but rather must be isolated from whole blood.

My dissertation work has provided me with extensive experience in metabolomics, metabolic flux analysis, mass spectrometry (LC-MS/MS), microbiology, technical design and optimizing experimental workflows, computational and statistical analysis, and writing and publishing scientific manuscripts. I look forward to a career in which I can apply my skills to systems biology challenges to produce a more sustainable, medically equipped, productive world.