Ethanol Tolerance in Engineered Strains of Clostridium thermocellum: Article No. 137

Daniel Olson, Marybeth Maloney, Anthony Lanahan, Nicholas Cervenka, Ying Xia, Angel Pech-Canul, Shuen Hon, Samantha Ziegler, Yannick Bomble, Lee Lynd

Research output: Contribution to journalArticlepeer-review

2 Scopus Citations


Clostridium thermocellum is a natively cellulolytic bacterium that is promising candidate for cellulosic biofuel production, and can produce ethanol at high yields (75-80% of theoretical) but the ethanol titers produced thus far are too low for commercial application. In several strains of C. thermocellum engineered for increased ethanol yield, ethanol titer seems to be limited by ethanol tolerance. Previous work to improve ethanol tolerance has focused on the WT organism. In this work, we focused on understanding ethanol tolerance in several engineered strains of C. thermocellum. We observed a tradeoff between ethanol tolerance and production. Adaptation for increased ethanol tolerance decreases ethanol production. Second, we observed a consistent genetic response to ethanol stress involving mutations at the AdhE locus. These mutations typically reduced NADH-linked ADH activity. About half of the ethanol tolerance phenotype could be attributed to the elimination of NADH-linked activity based on a targeted deletion of adhE. Finally, we observed that rich growth medium increases ethanol tolerance, but this effect is eliminated in an adhE deletion strain. Together, these suggest that ethanol inhibits growth and metabolism via a redox-imbalance mechanism. The improved understanding of mechanisms of ethanol tolerance described here lays a foundation for developing strains of C. thermocellum with improved ethanol production.
Original languageAmerican English
Number of pages15
JournalBiotechnology for Biofuels and Bioproducts
StatePublished - 2023

NREL Publication Number

  • NREL/JA-2700-87818


  • acetaldehyde dehydrogenase
  • Acetivibrio thermocellus
  • AdhE
  • alcohol dehydrogenase
  • biofuel
  • Hungateiclostridium thermocellum
  • next-generation sequencing
  • Ruminiclostridium thermocellum
  • tolerance
  • whole genome sequencing


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