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Title: Final Technical Report--Quantitative analysis of metabolic regulation by integration of metabolomics, proteomics, and fluxomics

Abstract

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 themore » 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.« less

Authors:
ORCiD logo [1]
  1. Princeton Univ., NJ (United States)
Publication Date:
Research Org.:
Princeton Univ., NJ (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER). Biological Systems Science Division
OSTI Identifier:
1487155
Report Number(s):
DOE-Princeton-12461
DOE Contract Number:  
SC0012461
Resource Type:
Technical Report
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CHEMISTRY; 97 MATHEMATICS AND COMPUTING; metabolomics; isotope tracing; biofuels; ethanol; bayesian analysis; optimiziation; thermodynamics; yeast; bacteria; cellulose degradation; metabolic flux

Citation Formats

Rabinowitz, Joshua D. Final Technical Report--Quantitative analysis of metabolic regulation by integration of metabolomics, proteomics, and fluxomics. United States: N. p., 2018. Web. doi:10.2172/1487155.
Rabinowitz, Joshua D. Final Technical Report--Quantitative analysis of metabolic regulation by integration of metabolomics, proteomics, and fluxomics. United States. https://doi.org/10.2172/1487155
Rabinowitz, Joshua D. 2018. "Final Technical Report--Quantitative analysis of metabolic regulation by integration of metabolomics, proteomics, and fluxomics". United States. https://doi.org/10.2172/1487155. https://www.osti.gov/servlets/purl/1487155.
@article{osti_1487155,
title = {Final Technical Report--Quantitative analysis of metabolic regulation by integration of metabolomics, proteomics, and fluxomics},
author = {Rabinowitz, Joshua D.},
abstractNote = {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.},
doi = {10.2172/1487155},
url = {https://www.osti.gov/biblio/1487155}, journal = {},
number = ,
volume = ,
place = {United States},
year = {2018},
month = {12}
}

Works referenced in this record:

Characterizing the in vivo role of trehalose in Saccharomyces cerevisiae using the AGT1 transporter
journal, April 2015


Common and divergent features of galactose-1-phosphate and fructose-1-phosphate toxicity in yeast
journal, April 2018


Bisphosphoglycerate mutase controls serine pathway flux via 3-phosphoglycerate
journal, August 2017


Systems-level analysis of mechanisms regulating yeast metabolic flux
journal, October 2016


Metabolite Spectral Accuracy on Orbitraps
journal, May 2017


Reversal of Cytosolic One-Carbon Flux Compensates for Loss of the Mitochondrial Folate Pathway
journal, June 2016


Chemical Basis for Deuterium Labeling of Fat and NADPH
journal, October 2017