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Title: Improved growth rate in Clostridium thermocellum hydrogenase mutant via perturbed sulfur metabolism

Abstract

Background: Metabolic engineering is a commonly used approach to develop organisms for an industrial function, but engineering aimed at improving one phenotype can negatively impact other phenotypes. This lack of robustness can prove problematic. Cellulolytic bacterium Clostridium thermocellum is able to rapidly ferment cellulose to ethanol and other products. Recently, genes involved in H 2 production, including the hydrogenase maturase hydG, were deleted from the chromosome of C. thermocellum. While ethanol yield increased, the growth rate decreased substantially compared to wild type. Results: Addition of 5 mM acetate to the growth medium improved the growth rate in C. thermocellum ΔhydG, whereas wild type remained unaffected. Transcriptomic analysis of the wild type showed essentially no response to the addition of acetate. However, in C. thermocellum ΔhydG, 204 and 56 genes were significantly differentially regulated relative to wild type in the absence and presence of acetate, respectively. Genes Clo1313_0108-0125, which are predicted to encode a sulfate transport system and sulfate assimilatory pathway, were drastically up-regulated in C. thermocellum ΔhydG in presence of added acetate. A similar pattern was seen with proteomics. Further physiological characterization demonstrated an increase in sulfide synthesis and elimination of cysteine consumption in C. thermocellum ΔhydG. In conclusion, sulfurmore » metabolism is perturbed in C. thermocellum ΔhydG, possibly to increase flux through sulfate reduction to act as an electron sink to balance redox reactions.« less

Authors:
 [1];  [2]; ORCiD logo [3]; ORCiD logo [2];  [2];  [2]; ORCiD logo [3]; ORCiD logo [2]; ORCiD logo [2]
  1. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center; Indian Inst. of Technology Delhi, Hauz Khas, New Delhi (India)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Biosciences Division; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center
  3. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). BioEnergy Science Center; Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States). Chemical Sciences Division
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:
1393809
Grant/Contract Number:
AC05-00OR22725
Resource Type:
Journal Article: Accepted Manuscript
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Volume: 10; Journal Issue: 1; Journal ID: ISSN 1754-6834
Publisher:
BioMed Central
Country of Publication:
United States
Language:
English
Subject:
09 BIOMASS FUELS; Cellulosic ethanol; Clostridium thermocellum; Redox balance; Metabolic engineering; Sulfate reduction

Citation Formats

Biswas, Ranjita, Wilson, Charlotte M., Giannone, Richard J., Klingeman, Dawn Marie, Rydzak, Thomas, Shah, Manesh B., Hettich, Robert L., Brown, Steven D., and Guss, Adam M.. Improved growth rate in Clostridium thermocellum hydrogenase mutant via perturbed sulfur metabolism. United States: N. p., 2017. Web. doi:10.1186/s13068-016-0684-x.
Biswas, Ranjita, Wilson, Charlotte M., Giannone, Richard J., Klingeman, Dawn Marie, Rydzak, Thomas, Shah, Manesh B., Hettich, Robert L., Brown, Steven D., & Guss, Adam M.. Improved growth rate in Clostridium thermocellum hydrogenase mutant via perturbed sulfur metabolism. United States. doi:10.1186/s13068-016-0684-x.
Biswas, Ranjita, Wilson, Charlotte M., Giannone, Richard J., Klingeman, Dawn Marie, Rydzak, Thomas, Shah, Manesh B., Hettich, Robert L., Brown, Steven D., and Guss, Adam M.. Tue . "Improved growth rate in Clostridium thermocellum hydrogenase mutant via perturbed sulfur metabolism". United States. doi:10.1186/s13068-016-0684-x. https://www.osti.gov/servlets/purl/1393809.
@article{osti_1393809,
title = {Improved growth rate in Clostridium thermocellum hydrogenase mutant via perturbed sulfur metabolism},
author = {Biswas, Ranjita and Wilson, Charlotte M. and Giannone, Richard J. and Klingeman, Dawn Marie and Rydzak, Thomas and Shah, Manesh B. and Hettich, Robert L. and Brown, Steven D. and Guss, Adam M.},
abstractNote = {Background: Metabolic engineering is a commonly used approach to develop organisms for an industrial function, but engineering aimed at improving one phenotype can negatively impact other phenotypes. This lack of robustness can prove problematic. Cellulolytic bacterium Clostridium thermocellum is able to rapidly ferment cellulose to ethanol and other products. Recently, genes involved in H2 production, including the hydrogenase maturase hydG, were deleted from the chromosome of C. thermocellum. While ethanol yield increased, the growth rate decreased substantially compared to wild type. Results: Addition of 5 mM acetate to the growth medium improved the growth rate in C. thermocellum ΔhydG, whereas wild type remained unaffected. Transcriptomic analysis of the wild type showed essentially no response to the addition of acetate. However, in C. thermocellum ΔhydG, 204 and 56 genes were significantly differentially regulated relative to wild type in the absence and presence of acetate, respectively. Genes Clo1313_0108-0125, which are predicted to encode a sulfate transport system and sulfate assimilatory pathway, were drastically up-regulated in C. thermocellum ΔhydG in presence of added acetate. A similar pattern was seen with proteomics. Further physiological characterization demonstrated an increase in sulfide synthesis and elimination of cysteine consumption in C. thermocellum ΔhydG. In conclusion, sulfur metabolism is perturbed in C. thermocellum ΔhydG, possibly to increase flux through sulfate reduction to act as an electron sink to balance redox reactions.},
doi = {10.1186/s13068-016-0684-x},
journal = {Biotechnology for Biofuels},
number = 1,
volume = 10,
place = {United States},
year = {Tue Jan 03 00:00:00 EST 2017},
month = {Tue Jan 03 00:00:00 EST 2017}
}

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  • Large-scale production of lignocellulosic biofuel is a potential solution to sustainably meet global energy needs. One-step consolidated bioprocessing (CBP) is a potentially advantageous approach for the production of biofuels, but requires an organism capable of hydrolyzing biomass to sugars and fermenting the sugars to ethanol at commercially viable titers and yields. Clostridium thermocellum, a thermophilic anaerobe, can ferment cellulosic biomass to ethanol and organic acids, but low yield, low titer, and ethanol sensitivity remain barriers to industrial production. Here, we deleted the hypoxanthine phosphoribosyltransferase gene in ethanol tolerant strain of C. thermocellum adhE*(EA) in order to allow use of previouslymore » developed gene deletion tools, then deleted lactate dehydrogenase (ldh) to redirect carbon flux towards ethanol. Upon deletion of ldh, the adhE*(EA) ldh strain produced 30% more ethanol than wild type on minimal medium. The adhE*(EA) ldh strain retained tolerance to 5% v/v ethanol, resulting in an ethanol tolerant platform strain of C. thermocellum for future metabolic engineering efforts.« less
  • Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical assaysmore » confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.« less
  • Clostridium thermocellum is a thermophilic, obligately anaerobic, Gram-positive bacterium that is a candidate microorganism for converting cellulosic biomass into ethanol through consolidated bioprocessing. Ethanol intolerance is an important metric in terms of process economics, and tolerance has often been described as a complex and likely multigenic trait for which complex gene interactions come into play. Here, we resequence the genome of an ethanol-tolerant mutant, show that the tolerant phenotype is primarily due to a mutated bifunctional acetaldehyde-CoA/alcohol dehydrogenase gene (adhE), hypothesize based on structural analysis that cofactor specificity may be affected, and confirm this hypothesis using enzyme assays. Biochemical assaysmore » confirm a complete loss of NADH-dependent activity with concomitant acquisition of NADPH-dependent activity, which likely affects electron flow in the mutant. The simplicity of the genetic basis for the ethanol-tolerant phenotype observed here informs rational engineering of mutant microbial strains for cellulosic ethanol production.« less
  • The antibiotic protein synthesis inhibitor chloramphenicol specifically blocked the incorporation of (35 S) sulfate into the residue protein of two marine bacteria, Pseudomonas halodurans and Alteromonas luteo-violaceus. Simultaneous inhibition of total protein synthesis occurred, but incorporation of 35 S into low-molecular-weight organic compounds continued. A. luteo-violaceus rapidly autolyzed, with similar reduction in cell counts, total culture protein and cellular sulfur, whereas P. halodurans remained viable. Treatment with chloramphenicol, growth during nitrogen and carbon limitation, and the carbon and energy sources used for growth did not alter the sulfur content of P. halodurans protein. The mean value (1.09%, by weight), representingmore » a wide variety of environmentally relevant growth conditions, was in agreement with model protein composition. The variability of cellular composition of P. halodurnas and A. luteo-violaceus is discussed with respect to the measurement of bacterial growth in natural environments. Total carbon and nitrogen per cell varied greatly (coefficient of variation, ca. 100%) depending on growth conditions. Variation in total sulfur and protein per cell was much less (coefficient of variation, less than 50%), but the least variation was found for sulfate incorporation into residue protein (coefficient of variation, ca. 15%). Thus, sulfate incorporation into residue protein can be used as an accurate measurement of de novo protein synthesis in these bacteria. (Refs. 26).« less