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Expressing the

Shuen Hon1,2,3, Evert K Holwerda1,2,3, Robert S Worthen1,2,3

  • 11Thayer School of Engineering, Dartmouth College, 14 Engineering Drive, Hanover, NH 03755 USA.

Biotechnology for Biofuels
|September 12, 2018
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Summary
This summary is machine-generated.

Introducing pyruvate ferredoxin oxidoreductase A (pforA) from Thermoanaerobacterium saccharolyticum into Clostridium thermocellum significantly enhanced ethanol production. This metabolic engineering strategy improved both ethanol yield and titer, especially at higher substrate concentrations.

Keywords:
Clostridium thermocellumConsolidated bioprocessingEthanolIsobutanolPyruvate ferredoxin oxidoreductaseThermoanaerobacterium saccharolyticum

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Area of Science:

  • Microbiology
  • Metabolic Engineering
  • Synthetic Biology

Background:

  • Clostridium thermocellum is a target for metabolic engineering to enhance cellulose-to-ethanol fermentation.
  • The pyruvate ferredoxin oxidoreductase (PFOR) pathway is crucial for converting pyruvate to acetyl-CoA in C. thermocellum for ethanol production.

Purpose of the Study:

  • To improve ethanol yield and titer in Clostridium thermocellum by introducing the pyruvate ferredoxin oxidoreductase A (pforA) gene from Thermoanaerobacterium saccharolyticum.
  • To investigate the role of T. saccharolyticum pforA and ferredoxin in C. thermocellum's ethanol production pathway.

Main Methods:

  • Genetic engineering of Clostridium thermocellum to introduce the pforA gene and ferredoxin from Thermoanaerobacterium saccharolyticum.
  • Fermentation studies using cellobiose and Avicel as substrates to evaluate ethanol production metrics.
  • Gene deletion experiments to assess the impact of native C. thermocellum pfor genes.

Main Results:

  • Introduction of T. saccharolyticum pforA, along with four other T. saccharolyticum genes, significantly improved ethanol yield and titer in C. thermocellum.
  • The enhanced ethanol production was maintained even after deleting native C. thermocellum pfor genes.
  • Deletion of C. thermocellum pfor4 led to a notable decrease in isobutanol production.

Conclusions:

  • The T. saccharolyticum pforA gene successfully enhances ethanol production in C. thermocellum as part of the pyruvate-to-ethanol pathway.
  • High-yield ethanol production in the engineered strain is achievable with higher substrate concentrations (up to 50 g/L).
  • The introduction of pforA resulted in a 14% increase in maximum ethanol titer.