title: Improved growth rate inClostridium thermocellumhydrogenase mutant via perturbed sulfur metabolism.
abstract: BACKGROUND
  Metabolic engineering is a commonly used approach to develop organisms for an industrial function, but engineering aimed at improving one phenotype can negatively impact other phenotypes. This lack of robustness can prove problematic. Cellulolytic bacteriumClostridium thermocellumis able to rapidly ferment cellulose to ethanol and other products. Recently, genes involved in H2production, including the hydrogenase maturasehydGand NiFe hydrogenaseech, were deleted from the chromosome ofC. thermocellum. While ethanol yield increased, the growth rate of ΔhydGdecreased substantially compared to wild type.
  
  
  RESULTS
  Addition of 5 mM acetate to the growth medium improved the growth rate inC. thermocellum ∆hydG, whereas wild type remained unaffected. Transcriptomic analysis of the wild type showed essentially no response to the addition of acetate. However, inC. thermocellum ΔhydG, 204 and 56 genes were significantly differentially regulated relative to wild type in the absence and presence of acetate, respectively. Genes, Clo1313_0108-0125, which are predicted to encode a sulfate transport system and sulfate assimilatory pathway, were drastically upregulated inC. thermocellum ΔhydGin the presence of added acetate. A similar pattern was seen with proteomics. Further physiological characterization demonstrated an increase in sulfide synthesis and elimination of cysteine consumption inC. thermocellum ΔhydG.Clostridium thermocellum ΔhydGΔechhad a higher growth rate thanΔhydGin the absence of added acetate, and a similar but less pronounced transcriptional and physiological effect was seen in this strain upon addition of acetate.
  
  
  CONCLUSIONS
  Sulfur metabolism is perturbed inC. thermocellum ΔhydGstrains, likely to increase flux through sulfate reduction to act either as an electron sink to balance redox reactions or to offset an unknown deficiency in sulfur assimilation.
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 ; 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: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA ; BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA.
authors.forename: Charlotte M
authors.initials: CM
authors.lastname: Wilson
authors.affiliation: BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA ; Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA.
authors.forename: Richard J
authors.initials: RJ
authors.lastname: Giannone
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.
authors.forename: Dawn M
authors.initials: DM
authors.lastname: Klingeman
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.
authors.forename: Thomas
authors.initials: T
authors.lastname: Rydzak
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.
authors.forename: Manesh B
authors.initials: MB
authors.lastname: Shah
authors.affiliation: BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA ; Chemical Sciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830 USA.
authors.forename: Robert L
authors.initials: RL
authors.lastname: Hettich
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.
authors.forename: Steven D
authors.initials: SD
authors.lastname: Brown
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; Metabolic engineering; Redox balance; Sulfate reduction