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Title: Cofactor Specificity of the Bifunctional Alcohol and Aldehyde Dehydrogenase (AdhE) in Wild-Type and Mutant Clostridium thermocellum and Thermoanaerobacterium saccharolyticum

Journal Article · · Journal of Bacteriology
DOI:· OSTI ID:1351568

Clostridium thermocellum and Thermoanaerobacterium saccharolyticumare thermophilic bacteria that have been engineered to produce ethanol from the cellulose and hemicellulose fractions of biomass, respectively. Although engineered strains of T. saccharolyticumproduce ethanol with a yield of 90% of the theoretical maximum, engineered strains ofC. thermocellumproduce ethanol at lower yields (~50% of the theoretical maximum). In the course of engineering these strains, a number of mutations have been discovered in theiradhEgenes, which encode both alcohol dehydrogenase (ADH) and aldehyde dehydrogenase (ALDH) enzymes. To understand the effects of these mutations, theadhEgenes from six strains ofC. thermocellumandT. saccharolyticumwere cloned and expressed inEscherichia coli, the enzymes produced were purified by affinity chromatography, and enzyme activity was measured. In wild-type strains of both organisms, NADH was the preferred cofactor for both ALDH and ADH activities. In high-ethanol-producing (ethanologen) strains ofT. saccharolyticum, both ALDH and ADH activities showed increased NADPH-linked activity. Interestingly, the AdhE protein of the ethanologenic strain ofC. thermocellumhas acquired high NADPH-linked ADH activity while maintaining NADH-linked ALDH and ADH activities at wild-type levels. When single amino acid mutations in AdhE that caused increased NADPH-linked ADH activity were introduced intoC. thermocellumandT. saccharolyticum, ethanol production increased in both organisms. Structural analysis of the wild-type and mutant AdhE proteins was performed to provide explanations for the cofactor specificity change on a molecular level. This work describes the characterization of the AdhE enzyme from different strains ofC. thermocellumandT. saccharolyticum.C. thermocellumandT. saccharolyticumare thermophilic anaerobes that have been engineered to make high yields of ethanol and can solubilize components of plant biomass and ferment the sugars to ethanol. In the course of engineering these strains, several mutations arose in the bifunctional ADH/ALDH protein AdhE, changing both enzyme activity and cofactor specificity. We show that changing AdhE cofactor specificity from mostly NADH linked to mostly NADPH linked resulted in higher ethanol production byC. thermocellumandT. saccharolyticum.

Research Organization:
National Renewable Energy Lab. (NREL), Golden, CO (United States)
Sponsoring Organization:
USDOE Office of Science (SC), Biological and Environmental Research (BER)
DOE Contract Number:
Report Number(s):
Journal Information:
Journal of Bacteriology, Vol. 197, Issue 15; ISSN 0021-9193
American Society for Microbiology
Country of Publication:
United States