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Title: Cellulosic ethanol production via consolidated bioprocessing at 75 °C by engineered Caldicellulosiruptor bescii

In this paper, we report that the C. bescii genome does not encode an acetaldehyde/alcohol dehydrogenase or an acetaldehyde dehydrogenase and no ethanol production is detected in this strain. The recent introduction of an NADH-dependent AdhE from C. thermocellum (Fig. 1a) in an ldh mutant of this strain resulted in production of ethanol from un-pretreated switchgrass, but the thermolability of the C. thermocellum AdhE at the optimum growth temperature of C. bescii (78 °C) meant that ethanol was not produced above 65 °C. The adhB and adhE genes from Thermoanaerobacter pseudethanolicus 39E, an anaerobic thermophile that produces ethanol as a major fermentation product at 70 °C, were cloned and expressed in an ldh deletion mutant of C. bescii. The engineered strains produced ethanol at 75 °C, near the ethanol boiling point. The AdhB expressing strain produced ethanol (1.4 mM on Avicel, 0.4 mM on switchgrass) as well as acetate (13.0 mM on Avicel, 15.7 mM on switchgrass). The AdhE expressing strain produced more ethanol (2.3 mM on Avicel, 1.6 mM on switchgrass) and reduced levels of acetate (12.3 mM on Avicel, 15.1 mM on switchgrass). These engineered strains produce cellulosic ethanol at the highest temperature of any microorganism to date.more » In addition, the addition of 40 mM MOPS to the growth medium increased the maximal growth yield of C. bescii by approximately twofold. In conclusion, the utilization of thermostable enzymes will be critical to achieving high temperature CBP in bacteria such as C. bescii. The ability to produce ethanol at 75 °C, near its boiling point, raises the possibility that process optimization could allow in situ product removal of this end product to mitigate ethanol toxicity.« less
 [1] ;  [1] ;  [1] ;  [2] ;  [2] ;  [1]
  1. Univ. of Georgia, Athens, GA (United States); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center (BESC)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center (BESC); Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division
Publication Date:
OSTI Identifier:
Grant/Contract Number:
Accepted Manuscript
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Volume: 8; Journal Issue: 1; Journal ID: ISSN 1754-6834
BioMed Central
Research Org:
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
Sponsoring Org:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
Country of Publication:
United States
59 BASIC BIOLOGICAL SCIENCES; 09 BIOMASS FUELS Cellulosic ethanol; Metabolic engineering; Caldicellulosiruptor bescii; Alcohol dehydrogenase; Thermoanaerobacter pseudethanolicus 39E