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

2024 Abstracts

Cell-Free Systems Biology: Characterizing Pyruvate Metabolism of Clostridium thermocellum with a Three-Enzyme Cascade Reaction


Daniel G. Olson* ([email protected]), S. Bilal Jilani


Dartmouth College


The overall goal of the project is to develop tools to improve the team’s systems-level understanding of metabolism in non-model organisms, such as Clostridium thermocellum, and use that understanding to increase product titer.


Genetic approaches have been traditionally used to understand microbial metabolism, but this process can be slow in non-model organisms with limited genetic tools. An alternative approach is to study metabolism directly in the cell lysate. This avoids the need for genetic tools, and is routinely used to study individual enzymatic reactions, but is not generally used to study systems-level properties of metabolism. Here the researchers demonstrate a new approach they call “cell-free systems biology” where they use well-characterized enzymes and multi-enzyme cascades to serve as sources or sinks of intermediate metabolites. This allows researchers to isolate subnetworks within metabolism and study their systems-level properties. To demonstrate this, the research team worked with a three-enzyme cascade reaction that converts pyruvate to 2,3-butanediol. Although it has been previously used in cell-free systems, its pH dependence was not well characterized, limiting its utility as a sink for pyruvate. The research team showed that improved proton accounting allowed better prediction of pH changes, and that active pH control allowed 2,3-butanediol titers of up to 1.1 M (189 grams per liter) from acetoin and 1.6 M (144 g/l) from pyruvate. The improved proton accounting provided a crucial insight that preventing the escape of carbon dioxide (CO2) from the system largely eliminated the need for active pH control, dramatically simplifying the team’s experimental setup. Researchers then used this cascade reaction to understand limits to product formation in C. thermocellum, an organism with potential applications for cellulosic biofuel production. This team showed that the fate of pyruvate is largely controlled by electron availability, and that reactions upstream of pyruvate limit overall product formation.

Funding Information

Funding for S. Bilal Jilani and Daniel Olson was provided by the U.S. DOE, Office of Science, BER program, GSP under Award Number DE-SC0022175.