title: Deletion of Type I glutamine synthetase deregulates nitrogen metabolism and increases ethanol production in Clostridium thermocellum.
abstract: Clostridium thermocellum rapidly deconstructs cellulose and ferments resulting hydrolysis products into ethanol and other products, and is thus a promising platform organism for the development of cellulosic biofuel production via consolidated bioprocessing. While recent metabolic engineering strategies have targeted eliminating canonical fermentation products (acetate, lactate, formate, and H2), C. thermocellum also secretes amino acids, which has limited ethanol yields in engineered strains to approximately 70% of the theoretical maximum. To investigate approaches to decrease amino acid secretion, we attempted to reduce ammonium assimilation by deleting the Type I glutamine synthetase (glnA) in an essentially wild type strain of C. thermocellum. Deletion of glnA reduced levels of secreted valine and total amino acids by 53% and 44% respectively, and increased ethanol yields by 53%. RNA-seq analysis revealed that genes encoding the RNF-complex were more highly expressed in ΔglnA and may have a role in improving NADH-availability for ethanol production. While a significant up-regulation of genes involved in nitrogen assimilation and urea uptake suggested that deletion of glnA induces a nitrogen starvation response, metabolomic analysis showed an increase in intracellular glutamine levels indicative of nitrogen-rich conditions. We propose that deletion of glnA causes deregulation of nitrogen metabolism, leading to overexpression of nitrogen metabolism genes and, in turn, elevated glutamine levels. Here we demonstrate that perturbation of nitrogen assimilation is a promising strategy to redirect flux from the production of nitrogenous compounds toward biofuels in C. thermocellum.
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, United States; BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN, United States.
authors.forename: Thomas
authors.initials: T
authors.lastname: Rydzak
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, United States; BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN, United States.
authors.forename: David
authors.initials: D
authors.lastname: Garcia
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, United States.
authors.forename: David M
authors.initials: DM
authors.lastname: Stevenson
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, United States; BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN, United States.
authors.forename: Margaret
authors.initials: M
authors.lastname: Sladek
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, United States; BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN, United States.
authors.forename: Dawn M
authors.initials: DM
authors.lastname: Klingeman
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, United States; Thayer School of Engineering at Dartmouth College, Hanover, NH, United States.
authors.forename: Evert K
authors.initials: EK
authors.lastname: Holwerda
authors.affiliation: Department of Bacteriology, University of Wisconsin-Madison, Madison, WI, United States.
authors.forename: Daniel
authors.initials: D
authors.lastname: Amador-Noguez
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, United States; BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN, United States.
authors.forename: Steven D
authors.initials: SD
authors.lastname: Brown
authors.affiliation: Biosciences Division, Oak Ridge National Laboratory, Oak Ridge, TN, United States; BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge, TN, United States. Electronic address: gussam@ornl.gov.
authors.forename: Adam M
authors.initials: AM
authors.lastname: Guss
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics*; D048013:Acetivibrio thermocellus / Q000235:genetics / Q000378:metabolism; D000431:Ethanol / Q000378:metabolism*; D017353:Gene Deletion; D005970:Glutamate Synthase / Q000235:genetics*; D009584:Nitrogen / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D000431:Ethanol; D005970:Glutamate Synthase; D009584:Nitrogen
keywords: Amino acid secretion; Cellulosic ethanol; Clostridium thermocellum; Glutamine synthetase