title: Elimination of hydrogenase active site assembly blocks H2 production and increases ethanol yield in Clostridium thermocellum.
abstract: BACKGROUND
  The native ability of Clostridium thermocellum to rapidly consume cellulose and produce ethanol makes it a leading candidate for a consolidated bioprocessing (CBP) biofuel production strategy. C. thermocellum also synthesizes lactate, formate, acetate, H2, and amino acids that compete with ethanol production for carbon and electrons. Elimination of H2 production could redirect carbon flux towards ethanol production by making more electrons available for acetyl coenzyme A reduction to ethanol.
  
  
  RESULTS
  H2 production in C. thermocellum is encoded by four hydrogenases. Rather than delete each individually, we targeted hydrogenase maturase gene hydG, involved in converting the three [FeFe] hydrogenase apoenzymes into holoenzymes. Further deletion of the [NiFe] hydrogenase (ech) resulted in a mutant that functionally lacks all four hydrogenases. H2 production in ∆hydG∆ech was undetectable, and the ethanol yield nearly doubled to 64% of the maximum theoretical yield. Genomic analysis of ∆hydG revealed a mutation in adhE, resulting in a strain with both NADH- and NADPH-dependent alcohol dehydrogenase activities. While this same adhE mutation was found in ethanol-tolerant C. thermocellum strain E50C, ∆hydG and ∆hydG∆ech are not more ethanol tolerant than the wild type, illustrating the complicated interactions between redox balancing and ethanol tolerance in C. thermocellum.
  
  
  CONCLUSIONS
  The dramatic increase in ethanol production suggests that targeting protein post-translational modification is a promising new approach for simultaneous inactivation of multiple enzymes.
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA ; BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA ; Current address: Centre for Rural Development and Technology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi, 110016 India.
authors.forename: Ranjita
authors.initials: R
authors.lastname: Biswas
authors.affiliation: BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA ; Thayer School of Engineering at Dartmouth College, Hanover, NH 03755 USA.
authors.forename: Tianyong
authors.initials: T
authors.lastname: Zheng
authors.affiliation: BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA ; Thayer School of Engineering at Dartmouth College, Hanover, NH 03755 USA.
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Olson
authors.affiliation: BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA ; Thayer School of Engineering at Dartmouth College, Hanover, NH 03755 USA.
authors.forename: Lee R
authors.initials: LR
authors.lastname: Lynd
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA ; BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA ; One Bethel Valley Road, Oak Ridge, TN 37831-6038 USA.
authors.forename: Adam M
authors.initials: AM
authors.lastname: Guss
keywords: Cellulosic ethanol; Clostridium thermocellum; Hydrogenase maturation; Metabolic engineering