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|Title:||Quantitative Mass Spectrometry for Comparative Analysis of Yeast Proteome Composition and Dynamics|
|Abstract:||Metabolic engineering of microbes to overproduce chemical building blocks and biofuels is an attractive option for replacing existing expensive and/or environmentally harmful syntheses. Rational metabolic engineering requires a detailed model of metabolic regulation in the organism in question, one that captures the interdependencies between metabolite concentrations, protein copy numbers, and reaction fluxes. Deducing these dependencies requires high-throughput quantitation of the relevant molecules on a global scale. Thus, the main purpose of this thesis is to quantify relative and absolute protein expression levels for four metabolically diverse yeast species of engineering interest – Saccharomyces cerevisiae, Issatchenkia orientalis, Yarrowia lipolytica, and Rhodosporidium toruloides – under various growth conditions, complementing fluxes and metabolite abundances collected for identical samples by the Rabinowitz Lab. Using established proteomics methods for absolute protein abundance estimation, I showed that protein abundances are highly correlated between these four species. To further confirm this finding, I devised methods to accurately measure protein abundances between species via quantification of a small subset of conserved tryptic peptides, demonstrating that the median change in protein expression level between S. cerevisiae and I. orientalis is < 10%. Furthermore, I found that proteome-scale expression responses to different growth conditions are largely identical across the species investigated, despite drastic metabolome differences preliminarily reported by the Rabinowitz Lab. Together, these results support the hypothesis that metabolic fluxes are predominantly regulated via allosteric inhibition and activation of enzymes whose copy numbers remain mostly unchanged.|
|Type of Material:||Princeton University Senior Theses|
|Appears in Collections:||Chemistry, 1926-2019|
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