Final Technical Report--Quantitative analysis of metabolic regulation by integration of metabolomics, proteomics, and fluxomics
This project used an -omics driven systems biology approach to investigate metabolic regulation in a systematic, large-scale, and quantitative manner. Efforts involved two microorganisms, the biofuel producing yeast Saccharomyces cerevisiae and the cellulose degrading bacterium Clostridium celluloyticum. In yeast, we used a combination of metabolomics, proteomics, and fluxomics to measure enzyme concentrations, metabolite concentrations, and metabolic fluxes across 25 steady-state yeast cultures. We then assessed the extent to which flux can be explained by a Michaelis-Menten relationship between enzyme, substrate, product, and potential regulator concentrations. This revealed three novel instances of cross-pathway metabolic regulation which we biochemically verified. Overall, substrate concentrations were the strongest driver of the net rates of cellular metabolic reactions, with metabolite concentrations collectively having more than double the physiological impact of enzymes. Thus, our studies in yeast argued for metabolism being substantially cell-regulating. In the cellulolytic bacterium, we developed and employed new tracer strategies to measure glycolytic reaction reversibility and thereby thermodynamics. This resulted in the striking observation that C. cellulolyticum glycolysis is nearly fully thermodynamically reversible, with no strongly forward driven step. The phosphofructokinase step, which is strongly forward driven in most species, is modified to use inorganic pyrophosphate as opposed to ATP as the phosphate donor. This increases the ATP yield of glycolysis in exchange for its becoming a slow, reversible pathway. Thus, our studies in cellulolytic bacteria argued for their having evolved a uniquely energy-efficient form of glycolysis. Collectively, these findings highlight the importance of metabolite concentrations and enzyme cofactor choices in controlling pathway fluxes.
- Research Organization:
- Princeton Univ., NJ (United States)
- Sponsoring Organization:
- USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division
- DOE Contract Number:
- SC0012461
- OSTI ID:
- 1487155
- Report Number(s):
- DOE-Princeton-12461
- Country of Publication:
- United States
- Language:
- English
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