Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

2023 Abstracts

The Use of Deuterated Water as a Substrate-Agnostic and Cost-Effective Isotope Tracer for Investigating Reversibility and Thermodynamics of Reactions in Central Carbon Metabolism


Melanie M. Callaghan1,2, Eashant Thusoo1,2, Bishal D. Sharma2,4, David M. Stevenson1,2, Daniel Olson2,4, Lee R. Lynd2,4, and Daniel Amador-Noguez1,2,3* ([email protected])


1University of Wisconsin–Madison; 2Center for Bioenergy Innovation (CBI); 3Great Lakes Bioenergy Research Center; and 4Dartmouth College



Integrate advanced mass spectrometry, computational modeling, and metabolic engineering to develop an experimental-computational approach for the in vivo genome-scale determination of Gibbs free energies (ΔG) in metabolic networks suitable for high-throughput thermodynamic profiling of engineered organisms and emerging model systems.


Successful manipulation of microbial systems for biotechnology applications requires a quantitative understanding of their metabolism. Stable isotope tracers (e.g., 13C, 15N, 18O, and 2H tracers), in combination with mass-spectrometry–based metabolomics, have become a widely used tool for the quantitative analysis of metabolism. Steady-state and dynamic isotope tracer experiments can provide information on metabolic network structure, metabolic fluxes, and thermodynamics of metabolic reactions and pathways.

The use of isotope tracers to estimate ΔG of reactions in central carbon metabolism constitutes a recent development. Rather than relying on measurements of product and reactant concentrations, this approach estimates ΔG from forward (J+) and backward (J) reaction fluxes via the relation ΔG = −RT ln(J+/J). The measurement of forward-to-backward J+/J ratios can be performed using 2H-labeled and 13C-labeled tracers and relies on the generation of distinctive metabolite labeling patterns by reversible reactions within a pathway. Both the measurement of metabolic fluxes and the estimation of in vivo ΔG of reactions have been previously accomplished by placing the heavy isotopes—most commonly 13C, 15N, or 2H—into nutrient substrates such as 13C or 2H-labeled sugars, 2H-labeled fatty acids, 15N-labeled amino acids, 15N-labeled ammonia, 13C-labeled CO2 or formic acid, and many others.

For some applications, the use of isotopically labeled nutrient substrates may not be feasible due to availability and/or high cost. One salient example of this is metabolic flux analysis in industrially relevant cellulolytic microbes, such as Clostridium thermocellum, that metabolize complex substrates (i.e., lignocellulosic biomass). In this work, the use of deuterated water (also named as “heavy water,” 2H2O, or D2O) is explored as a non-nutrient, substrate-agnostic, cost-effective isotope tracer for investigating reversibility of reactions in central carbon metabolism. Researchers reasoned that the use of 2H2O as a tracer (i.e., by growing bacteria in culture media containing a defined amount of 2H2O) can provide information on reversibility of dehydration/hydration reactions, isomerization reactions, aldol reactions, and transamination reactions that result in the incorporation of protons from water into C-H bonds, and thus allow characterization of pathway thermodynamics. The team reports the successful use of deuterated water to investigate the reversibility of glycolytic reactions on three bacterial species of industrial interest: the model bacterium Escherichia coli, the cellulolytic and ethanologenic bacterium C. thermocellum, and the ethanologenic bacterium Zymomonas mobilis, each harboring distinct versions of glycolysis. This work will aid in the construction of accurate metabolic models that incorporate thermodynamic constraints and guide fast rational engineering of microbial networks.

Funding Information

This material is based upon work supported by the U.S. Department of Energy, Office of Science, Biological and Environmental Research (BER) Program, Early Career Research Program under Award Number DE-SC0018998. This work was also supported by the Center for Bioenergy Innovation (CBI), from the U.S. Department of Energy Bioenergy Research Centers supported by the BER Program in the DOE Office of Science.