(357w) Metabolic Modeling and Systems Biology Characterization in the Green Alga Chromochloris Zofingiensis

Research Interests: Engineering organisms to improve the yields of valuable products presents a complex problem. Millions of years of evolution have allowed organisms to establish highly robust metabolic networks, and the perturbation of these networks to redirect carbon flux towards important products often requires a number of coordinated changes throughout the genome. In order to engineer cellular metabolism in a meaningful way, we must first understand it. In order to better understand the functioning and regulation of these networks, metabolic modeling of the fluxes through these networks is a useful practice that can be accomplished by employing chemical engineering toolsets. My research interests lie in this intersection of chemical engineering and biology, and in the use of chemical engineering strategies to solve problems in biological systems.

Chromochloris zofingiensis is a facultative heterotrophic green alga, and a high producer of lipids and astaxanthin, a naturally occurring pigment that has high value as an antioxidant. This organism is capable of a unique trophic switch, from autotrophy to purely heterotrophy in the presence of glucose, even while under continuous illumination. This switch is characterized by an accumulation of energy storage compounds, degradation of chlorophyll and restructuring of thylakoid membranes. To better understand the changes in metabolic flux distribution that accompany this trophic switch, we employed both flux balance analysis (FBA) and isotopically assisted metabolic flux analysis (MFA) on this organism.

I performed a detailed biomass composition analysis of this organism under three different media conditions that represented three different trophic modes for this organism, autotrophic growth, mixotrophic growth on acetate, and heterotrophic growth on glucose. In this analysis, the cellular content of lipids, proteins, carbohydrates, nucleic acids, chlorophyll, carotenoids, and starch was determined through various methodologies. In addition, I assessed the relative compositions of amino acids and fatty acids through derivatization and analysis via GC-MS. This biomass composition data was used to construct a biomass objective function which, along with experimentally determined growth rates and nutrient uptake rates, was used to conduct a flux balance analysis (FBA) of this organism. Through this analysis we were able to predict the formation of fermentation products in heterotrophic C. zofingiensis cultures, a prediction that was later confirmed through analysis of spent media samples.

Flux distributions determined in silico through FBA must also be measured in vivo, to evaluate the accuracy of computational findings and to highlight differences between physiological metabolism and optimized metabolism. To measure intracellular fluxes in vivo, I conducted an isotopically assisted metabolic flux analysis of heterotrophic C. zofingiensis. In this work, I created a central metabolic network by systematically pulling reactions from the genome scale metabolic model that was used in FBA. I conducted a series of tracer simulations to design a set of isotopically labeled glucose tracers that would enable the determination of intracellular fluxes. Cultures were grown on this isotopically labeled substrate until isotopic steady state had been achieved, at which point cellular biomass was quenched to rapidly halt metabolism, and metabolites were extracted for analysis. Metabolite isotopomer distributions were evaluated through mass spectrometry analysis, and these labeling patterns were used to calculate flux distributions within the cell.

Through my work with this organism, I have had to optimize other aspects of its cultivation. Flat panel photobioreactors are common in lab scale growth operations, intended to provide greater control of light intensity throughout the culture vessel. In these photobioreactors, gas is sparged from the bottom to provide mixing, however, during cultivation of C. zofingiensis in this reactor type it was found that this was not adequate to grow a homogenous culture. To overcome this lack of adequate mixing, I designed and 3D printed a novel paddlewheel to fix onto the sparge line within the culture vessel. This paddlewheel is bubble driven, and adds supplemental mixing to the culture. The inclusion of this paddlewheel improved culture performance and minimized culture settling within the vessel.

Through this work in my dissertation, I have become familiar with microbiology experiment design and cultivation, computational work in metabolic flux analysis, and a number of analytical chemistry techniques including the extraction and quantification of macromolecules, metabolomics, metabolite derivatization techniques and GC-MS analysis, LC-MS/MS analysis and analysis of mass spectrometry data. I have also developed skills in 3D printing and CAD design, and implemented these skills to optimize experimental design. As a chemical engineer with developed skills in microbiology and analytical chemistry, I aim to establish a career where I can employ these skills to solve engineering problems in complex biological systems.