title: Elimination of metabolic pathways to all traditional fermentation products increases ethanol yields in Clostridium thermocellum.
abstract: Clostridium thermocellum has the natural ability to convert cellulose to ethanol, making it a promising candidate for consolidated bioprocessing (CBP) of cellulosic biomass to biofuels. To further improve its CBP capabilities, a mutant strain of C. thermocellum was constructed (strain AG553; C. thermocellum Δhpt ΔhydG Δldh Δpfl Δpta-ack) to increase flux to ethanol by removing side product formation. Strain AG553 showed a two- to threefold increase in ethanol yield relative to the wild type on all substrates tested. On defined medium, strain AG553 exceeded 70% of theoretical ethanol yield on lower loadings of the model crystalline cellulose Avicel, effectively eliminating formate, acetate, and lactate production and reducing H2 production by fivefold. On 5 g/L Avicel, strain AG553 reached an ethanol yield of 63.5% of the theoretical maximum compared with 19.9% by the wild type, and it showed similar yields on pretreated switchgrass and poplar. The elimination of organic acid production suggested that the strain might be capable of growth under higher substrate loadings in the absence of pH control. Final ethanol titer peaked at 73.4mM in mutant AG553 on 20 g/L Avicel, at which point the pH decreased to a level that does not allow growth of C. thermocellum, likely due to CO2 accumulation. In comparison, the maximum titer of wild type C. thermocellum was 14.1mM ethanol on 10 g/L Avicel. With the elimination of the metabolic pathways to all traditional fermentation products other than ethanol, AG553 is the best ethanol-yielding CBP strain to date and will serve as a platform strain for further metabolic engineering for the bioconversion of lignocellulosic biomass.
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830, United States; BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37830, United States; Bredesen Center for Interdisciplinary Research and Graduate Education, University of Tennessee - Knoxville, Knoxville, TN 37996, United States.
authors.forename: Beth
authors.initials: B
authors.lastname: Papanek
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830, United States; BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37830, United States.
authors.forename: Ranjita
authors.initials: R
authors.lastname: Biswas
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830, United States; BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37830, United States.
authors.forename: Thomas
authors.initials: T
authors.lastname: Rydzak
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN 37830, United States; BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN 37830, United States; Bredesen Center for Interdisciplinary Research and Graduate Education, University of Tennessee - Knoxville, Knoxville, TN 37996, United States. Electronic address: gussam@ornl.gov.
authors.forename: Adam M
authors.initials: AM
authors.lastname: Guss
mesh_terms: D018533:Biomass; D002482:Cellulose / Q000378:metabolism; D048013:Acetivibrio thermocellus / Q000235:genetics* / Q000378:metabolism*; D003470:Culture Media; D000431:Ethanol / Q000378:metabolism*; D005285:Fermentation; D006863:Hydrogen-Ion Concentration; D060847:Metabolic Engineering; D053858:Metabolic Networks and Pathways / Q000235:genetics; D009154:Mutation / Q000235:genetics; D008897:Panicum / Q000378:metabolism; D010957:Plasmids; D032107:Populus / Q000378:metabolism
chemical_list: D003470:Culture Media; D000431:Ethanol; D002482:Cellulose
keywords: Biofuels; Clostridium thermocellum; Consolidated bioprocessing; Lignocellulose