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

Journal Article · 10 October 2024 · ACS Synthetic Biology

DOI: https://doi.org/10.1021/acssynbio.4c00405 · OSTI ID:2477599

Jilani, S. Bilal ^[1]; Alahuhta, Markus ^[2]; Bomble, Yannick J.  ^[2]; Olson, Daniel G.  ^[1]

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1. Dartmouth College, Hanover, NH (United States)
2. National Renewable Energy Laboratory (NREL), Golden, CO (United States)

Genetic approaches have been traditionally used to understand microbial metabolism, but this process can be slow in nonmodel organisms due to 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 we demonstrate a new approach that we call “cell-free systems biology”, where we use well-characterized enzymes and multienzyme cascades to serve as sources or sinks of intermediate metabolites. This allows us to isolate subnetworks within metabolism and study their systems-level properties. To demonstrate this, we worked with a threeenzyme 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. We showed that improved proton accounting allowed better prediction of pH changes and that active pH control allowed 2,3-butanediol titers of up to 2.1 M (189 g/L) from acetoin and 1.6 M (144 g/L) from pyruvate. The improved proton accounting provided a crucial insight that preventing the escape of CO₂ from the system largely eliminated the need for active pH control, dramatically simplifying our experimental setup. We then used this cascade reaction to understand limits to product formation in Clostridium thermocellum, an organism with potential applications for cellulosic biofuel production. We showed that the fate of pyruvate is largely controlled by electron availability and that reactions upstream of pyruvate limit overall product formation.

• Research Organization: Dartmouth College, Hanover, NH (United States)
• Sponsoring Organization: USDOE Office of Science (SC), Biological and Environmental Research (BER); USDOE Office of Science (SC), Basic Energy Sciences (BES)
• Grant/Contract Number: SC0022175
• OSTI ID: 2477599
• Journal Information: ACS Synthetic Biology, Journal Name: ACS Synthetic Biology Journal Issue: 11 Vol. 13; ISSN 2161-5063
• Publisher: American Chemical Society (ACS)Copyright Statement
• Country of Publication: United States
• Language: English