title: Cellulosic ethanol production via consolidated bioprocessing at 75 °C by engineered Caldicellulosiruptor bescii.
abstract: BACKGROUND
  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.Fig. 1Proposed scheme for the pyruvate to ethanol pathway in C. thermocellum and T. pseudethanolicus 39E. a The C. thermocellum ethanol pathway. The red colored AdhE (Cthe_0423) is already expressed and tested in C. bescii [26]. b The T. pseudethanolicus 39E ethanol pathway. The green colored AdhE (Teth39_0206) and blue colored AdhB (Teth39_0218) are expressed and tested in C. bescii in this study.
  
  
  RESULTS
  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. In addition, the addition of 40 mM MOPS to the growth medium increased the maximal growth yield of C. bescii by approximately twofold.
  
  
  CONCLUSIONS
  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.
authors.affiliation: Department of Genetics, University of Georgia, Athens, GA USA.|The BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN USA.
authors.forename: Daehwan
authors.initials: D
authors.lastname: Chung
authors.affiliation: Department of Genetics, University of Georgia, Athens, GA USA.|The BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN USA.
authors.forename: Minseok
authors.initials: M
authors.lastname: Cha
authors.affiliation: Department of Genetics, University of Georgia, Athens, GA USA.|The BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN USA.
authors.forename: Elise N
authors.initials: EN
authors.lastname: Snyder
authors.affiliation: The BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN USA.|Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN USA.
authors.forename: James G
authors.initials: JG
authors.lastname: Elkins
authors.affiliation: The BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN USA.|Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN USA.
authors.forename: Adam M
authors.initials: AM
authors.lastname: Guss
authors.affiliation: Department of Genetics, University of Georgia, Athens, GA USA.|The BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN USA.
authors.forename: Janet
authors.initials: J
authors.lastname: Westpheling
keywords: Alcohol dehydrogenase; Caldicellulosiruptor bescii; Cellulosic ethanol; Metabolic engineering; Thermoanaerobacter pseudethanolicus 39E