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

S. Bilal Jilani, Markus Alahuhta, Yannick Bomble, Daniel Olson

Research output: Contribution to journalArticlepeer-review

Abstract

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 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. 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.
Original languageAmerican English
Pages (from-to)3587-3599
Number of pages13
JournalACS Synthetic Biology
Volume13
Issue number11
DOIs
StatePublished - 2024

NREL Publication Number

  • NREL/JA-2700-92039

Keywords

  • 2 3-butanediol
  • 2 3-butanediol dehydrogenase
  • acetivibrio thermocellus
  • acetolactate decarboxylase
  • acetolactate synthase
  • formate dehydrogenase
  • hungateiclostridium
  • Ruminiclostridium

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