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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. And while recent metabolic engineering strategies have targeted eliminating canonical fermentation products (acetate, lactate, formate, and H 2), C. thermocellum also secretes amino acids, which has limited ethanol yields in engineered strains to approximately 70% of the theoretical maximum. To decrease amino acid secretion, we attempted to reduce ammonium assimilation by deleting the Type I glutamine synthetase (glnA) in 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 and α-ketoglutarate levels indicative of nitrogen-rich conditions. Here, we propose that deletion of glnA causes deregulation of nitrogen metabolism, leading to overexpression of nitrogen metabolism genes and, inmore » turn, elevated glutamine/α-ketoglutarate 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.« less

Authors:
 [1];  [1];  [2];  [1]; ORCiD logo [1];  [3];  [2]; ORCiD logo [1]; ORCiD logo [1]
  1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division, BioEnergy Science Center
  2. Univ. of Wisconsin, Madison, WI (United States). Dept. of Bacteriology
  3. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Dartmouth College, Hanover, NH (United States). Thayer School of Engineering
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Biological and Environmental Research (BER) (SC-23)
OSTI Identifier:
1393891
DOE Contract Number:  
AC05-00OR22725; AC02-05CH11231
Resource Type:
Journal Article
Resource Relation:
Journal Name: Metabolic Engineering; Journal Volume: 41; Journal Issue: C
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; cellulosic ethanol; clostridium thermocellum; glutamine synthetase; amino acid secretion

Citation Formats

Rydzak, Thomas, Garcia, David, Stevenson, David M., Sladek, Margaret, Klingeman, Dawn M., Holwerda, Evert K., Amador-Noguez, Daniel, Brown, Steven D., and Guss, Adam M. Deletion of Type I glutamine synthetase deregulates nitrogen metabolism and increases ethanol production in Clostridium thermocellum. United States: N. p., 2017. Web. doi:10.1016/j.ymben.2017.04.002.
Rydzak, Thomas, Garcia, David, Stevenson, David M., Sladek, Margaret, Klingeman, Dawn M., Holwerda, Evert K., Amador-Noguez, Daniel, Brown, Steven D., & Guss, Adam M. Deletion of Type I glutamine synthetase deregulates nitrogen metabolism and increases ethanol production in Clostridium thermocellum. United States. doi:10.1016/j.ymben.2017.04.002.
Rydzak, Thomas, Garcia, David, Stevenson, David M., Sladek, Margaret, Klingeman, Dawn M., Holwerda, Evert K., Amador-Noguez, Daniel, Brown, Steven D., and Guss, Adam M. Mon . "Deletion of Type I glutamine synthetase deregulates nitrogen metabolism and increases ethanol production in Clostridium thermocellum". United States. doi:10.1016/j.ymben.2017.04.002.
@article{osti_1393891,
title = {Deletion of Type I glutamine synthetase deregulates nitrogen metabolism and increases ethanol production in Clostridium thermocellum},
author = {Rydzak, Thomas and Garcia, David and Stevenson, David M. and Sladek, Margaret and Klingeman, Dawn M. and Holwerda, Evert K. and Amador-Noguez, Daniel and Brown, Steven D. and Guss, Adam M.},
abstractNote = {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. And 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 decrease amino acid secretion, we attempted to reduce ammonium assimilation by deleting the Type I glutamine synthetase (glnA) in 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 and α-ketoglutarate levels indicative of nitrogen-rich conditions. Here, we propose that deletion of glnA causes deregulation of nitrogen metabolism, leading to overexpression of nitrogen metabolism genes and, in turn, elevated glutamine/α-ketoglutarate 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.},
doi = {10.1016/j.ymben.2017.04.002},
journal = {Metabolic Engineering},
number = C,
volume = 41,
place = {United States},
year = {Mon May 01 00:00:00 EDT 2017},
month = {Mon May 01 00:00:00 EDT 2017}
}