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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}
}
  • Cited by 1
  • 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, we study a mutant strain of C. thermocellum that 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 cellulosemore » Avicel, effectively eliminating formate, acetate, and lactate production and reducing H 2 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 73.4 mM 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 CO 2 accumulation. In comparison, the maximum titer of wild type C. thermocellum was 14.1 mM ethanol on 10 g/L Avicel. In conclusion, 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.« less
  • The native ability of Clostridium thermocellum to rapidly consume cellulose and produce ethanol makes it a leading candidate for a consolidated bioprocessing (CBP) biofuel production strategy. C. thermocellum also synthesizes lactate, formate, acetate, H2, and amino acids that compete with ethanol production for carbon and electrons. Elimination of H2 production could redirect carbon flux towards ethanol production by making more electrons available for acetyl-CoA reduction to ethanol. C. thermocellum encodes four hydrogenases and rather than delete each individually, we targeted a hydrogenase maturase gene (hydG), involved in converting the three [FeFe] hydrogenase apoenzymes into holoenzymes. Further deletion of the [NiFe]more » hydrogenase (ech) resulted in a mutant that functionally lacks all four hydrogenases. H2 production in hydG ech was undetectable and ethanol yield increased nearly 2-fold compared to wild type. Interestingly, mutant growth improved upon the addition of acetate, which led to increased expression of genes related to sulfate metabolism, suggesting these mutants may use sulfate as a terminal electron acceptor to balance redox reactions. Genomic analysis of hydG revealed a mutation in adhE, resulting in a strain with both NADH- and NADPH-dependent alcohol dehydrogenase activities. While this same adhE mutation is found in ethanol tolerant C. thermocellum strain E50C, hydG and hydG ech are not more ethanol tolerant than wild type, illustrating the complicated interactions between redox balancing and ethanol tolerance in C. thermocellum. The dramatic increase in ethanol production here suggests that targeting protein post-translational modification is a promising new approach for inactivation of multiple enzymes simultaneously for metabolic engineering.« less
  • Background: The native ability of Clostridium thermocellum to rapidly consume cellulose and produce ethanol makes it a leading candidate for a consolidated bioprocessing (CBP) biofuel production strategy. C. thermocellum also synthesizes lactate, formate, acetate, H-2, and amino acids that compete with ethanol production for carbon and electrons. Elimination of H-2 production could redirect carbon flux towards ethanol production by making more electrons available for acetyl coenzyme A reduction to ethanol. Results: H-2 production in C. thermocellum is encoded by four hydrogenases. Rather than delete each individually, we targeted hydrogenase maturase gene hydG, involved in converting the three [FeFe] hydrogenase apoenzymesmore » into holoenzymes. Further deletion of the [NiFe] hydrogenase ( ech) resulted in a mutant that functionally lacks all four hydrogenases. H-2 production in ΔhydGΔech was undetectable, and the ethanol yield nearly doubled to 64% of the maximum theoretical yield. Genomic analysis of ΔhydG revealed a mutation in adhE, resulting in a strain with both NADH- and NADPH-dependent alcohol dehydrogenase activities. While this same adhE mutation was found in ethanol-tolerant C. thermocellum strain E50C, ΔhydG and ΔhydGΔech are not more ethanol tolerant than the wild type, illustrating the complicated interactions between redox balancing and ethanol tolerance in C. thermocellum. Conclusions: Finally, the dramatic increase in ethanol production suggests that targeting protein post-translational modification is a promising new approach for simultaneous inactivation of multiple enzymes.« less
  • Clostridium thermocellum ferments cellulose, is a promising candidate for ethanol production from cellulosic biomass, and has been the focus of studies aimed at improving ethanol yield. Thermoanaerobacterium saccharolyticum ferments hemicellulose, but not cellulose, and has been engineered to produce ethanol at high yield and titer. Recent research has led to the identification of four genes in T. saccharolyticum involved in ethanol production: adhE, nfnA, nfnB and adhA. We introduced these genes into C. thermocellum and observed significant improvements to ethanol yield, titer, and productivity. The four genes alone, however, were insufficient to achieve in C. thermocellum the ethanol yields andmore » titers observed in engineered T. saccharolyticum strains, even when combined with gene deletions targeting hydrogen production. Here, this suggests that other parts of T. saccharolyticum metabolism may also be necessary to reproduce the high ethanol yield and titer phenotype in C. thermocellum.« less
    Cited by 4