title: Nicotinamide cofactor ratios in engineered strains of Clostridium thermocellum and Thermoanaerobacterium saccharolyticum.
abstract: Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are bacteria under investigation for production of biofuels from plant biomass. Thermoanaerobacterium saccharolyticum has been engineered to produce ethanol at high yield (>90% of theoretical) and titer (>70 g/l). Efforts to engineer C. thermocellum have not, to date, been as successful, and efforts are underway to transfer the ethanol production pathway from T. saccharolyticum to C. thermocellum One potential challenge in transferring metabolic pathways is the possibility of incompatible levels of nicotinamide cofactors. These cofactors (NAD(+), NADH, NADP(+) and NADPH) and their oxidation state are important in the context of microbial redox metabolism. In this study we directly measured the concentrations and reduced oxidized ratios of these cofactors in a number of strains of C. thermocellum and T. saccharolyticum by using acid/base extraction and enzymatic assays. We found that cofactor ratios are maintained in a fairly narrow range, regardless of the metabolic network modifications considered. We have found that the ratios are similar in both organisms, which is a relevant observation in the context of transferring the T. saccharolyticum ethanol production pathway to C. thermocellum.
authors.affiliation: Thayer School of Engineering, Dartmouth College, 14 Engineering Drive, Hanover, NH 03755, USA BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37830, USA.
authors.forename: Dhananjay
authors.initials: D
authors.lastname: Beri
authors.affiliation: Thayer School of Engineering, Dartmouth College, 14 Engineering Drive, Hanover, NH 03755, USA BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37830, USA.
authors.forename: Daniel G
authors.initials: DG
authors.lastname: Olson
authors.affiliation: Thayer School of Engineering, Dartmouth College, 14 Engineering Drive, Hanover, NH 03755, USA BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37830, USA.
authors.forename: Evert K
authors.initials: EK
authors.lastname: Holwerda
authors.affiliation: Thayer School of Engineering, Dartmouth College, 14 Engineering Drive, Hanover, NH 03755, USA BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37830, USA Lee.R.Lynd@dartmouth.edu.
authors.forename: Lee R
authors.initials: LR
authors.lastname: Lynd
mesh_terms: D056804:Biofuels; D018533:Biomass; D048013:Acetivibrio thermocellus / Q000235:genetics / Q000378:metabolism*; D000431:Ethanol / Q000378:metabolism; D005285:Fermentation; D060847:Metabolic Engineering; D053858:Metabolic Networks and Pathways; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D009536:Niacinamide / Q000378:metabolism*; D010084:Oxidation-Reduction; D046950:Thermoanaerobacterium / Q000235:genetics / Q000378:metabolism*
chemical_list: D056804:Biofuels; D009243:NAD; D009536:Niacinamide; D000431:Ethanol; D009249:NADP
keywords: NADH; NADPH; acid/base extraction; cycling assay; metabolite extraction; nicotinamide cofactors