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Title: Clostridium thermocellum LL1210 pH homeostasis mechanisms informed by transcriptomics and metabolomics

Abstract

Background: Clostridium (Ruminiclostridium) thermocellum is a model fermentative anaerobic thermophile being studied and engineered for consolidated bioprocessing of lignocellulosic feedstocks into fuels and chemicals. Engineering efforts have resulted in significant improvements in ethanol yields and titers although further advances are required to make the bacterium industry-ready. For instance, fermentations at lower pH could enable co-culturing with microbes that have lower pH optima, augment productivity, and reduce buffering cost. C. thermocellum is typically grown at neutral pH, and little is known about its pH limits or pH homeostasis mechanisms. To better understand C. thermocellum pH homeostasis we grew strain LL1210 (C. thermocellum DSM1313 Δhpt ΔhydG Δldh Δpfl Δpta-ack), currently the highest ethanol producing strain of C. thermocellum, at different pH values in chemostat culture and applied systems biology tools.Results: Clostridium thermocellum LL1210 was found to be growth-limited below pH 6.24 at a dilution rate of 0.1 h -1. F1F0-ATPase gene expression was upregulated while many ATP-utilizing enzymes and pathways were downregulated at pH 6.24. These included most flagella biosynthesis genes, genes for chemotaxis, and other motility-related genes (> 50) as well as sulfate transport and reduction, nitrate transport and nitrogen fixation, and fatty acid biosynthesis genes. Clustering and enrichment of differentiallymore » expressed genes at pH values 6.48, pH 6.24 and pH 6.12 (washout conditions) compared to pH 6.98 showed inverse differential expression patterns between the F1F0-ATPase and genes for other ATP-utilizing enzymes. At and below pH 6.24, amino acids including glutamate and valine; long-chain fatty acids, their iso-counterparts and glycerol conjugates; glycolysis intermediates 3-phosphoglycerate, glucose 6-phosphate, and glucose accumulated intracellularly. Glutamate was 267 times more abundant in cells at pH 6.24 compared to pH 6.98, and intercellular concentration reached 1.8 μmol/g pellet at pH 5.80 (stopped flow).Conclusions: Clostridium thermocellum LL1210 can grow under slightly acidic conditions, similar to limits reported for other strains. This foundational study provides a detailed characterization of a relatively acid-intolerant bacterium and provides genetic targets for strain improvement. Future studies should examine adding gene functions used by more acid-tolerant bacteria for improved pH homeostasis at acidic pH values.« less

Authors:
; ; ; ; ; ; ; ; ;
Publication Date:
Research Org.:
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE
OSTI Identifier:
1618721
Alternate Identifier(s):
OSTI ID: 1435276
Grant/Contract Number:  
BESC; AC05-00OR22725
Resource Type:
Published Article
Journal Name:
Biotechnology for Biofuels
Additional Journal Information:
Journal Name: Biotechnology for Biofuels Journal Volume: 11 Journal Issue: 1; Journal ID: ISSN 1754-6834
Publisher:
Springer Science + Business Media
Country of Publication:
Netherlands
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Whitham, Jason M., Moon, Ji-Won, Rodriguez, Jr., Miguel, Engle, Nancy L., Klingeman, Dawn M., Rydzak, Thomas, Abel, Malaney M., Tschaplinski, Timothy J., Guss, Adam M., and Brown, Steven D. Clostridium thermocellum LL1210 pH homeostasis mechanisms informed by transcriptomics and metabolomics. Netherlands: N. p., 2018. Web. doi:10.1186/s13068-018-1095-y.
Whitham, Jason M., Moon, Ji-Won, Rodriguez, Jr., Miguel, Engle, Nancy L., Klingeman, Dawn M., Rydzak, Thomas, Abel, Malaney M., Tschaplinski, Timothy J., Guss, Adam M., & Brown, Steven D. Clostridium thermocellum LL1210 pH homeostasis mechanisms informed by transcriptomics and metabolomics. Netherlands. doi:10.1186/s13068-018-1095-y.
Whitham, Jason M., Moon, Ji-Won, Rodriguez, Jr., Miguel, Engle, Nancy L., Klingeman, Dawn M., Rydzak, Thomas, Abel, Malaney M., Tschaplinski, Timothy J., Guss, Adam M., and Brown, Steven D. Thu . "Clostridium thermocellum LL1210 pH homeostasis mechanisms informed by transcriptomics and metabolomics". Netherlands. doi:10.1186/s13068-018-1095-y.
@article{osti_1618721,
title = {Clostridium thermocellum LL1210 pH homeostasis mechanisms informed by transcriptomics and metabolomics},
author = {Whitham, Jason M. and Moon, Ji-Won and Rodriguez, Jr., Miguel and Engle, Nancy L. and Klingeman, Dawn M. and Rydzak, Thomas and Abel, Malaney M. and Tschaplinski, Timothy J. and Guss, Adam M. and Brown, Steven D.},
abstractNote = {Background: Clostridium (Ruminiclostridium) thermocellum is a model fermentative anaerobic thermophile being studied and engineered for consolidated bioprocessing of lignocellulosic feedstocks into fuels and chemicals. Engineering efforts have resulted in significant improvements in ethanol yields and titers although further advances are required to make the bacterium industry-ready. For instance, fermentations at lower pH could enable co-culturing with microbes that have lower pH optima, augment productivity, and reduce buffering cost. C. thermocellum is typically grown at neutral pH, and little is known about its pH limits or pH homeostasis mechanisms. To better understand C. thermocellum pH homeostasis we grew strain LL1210 (C. thermocellum DSM1313 Δhpt ΔhydG Δldh Δpfl Δpta-ack), currently the highest ethanol producing strain of C. thermocellum, at different pH values in chemostat culture and applied systems biology tools.Results: Clostridium thermocellum LL1210 was found to be growth-limited below pH 6.24 at a dilution rate of 0.1 h-1. F1F0-ATPase gene expression was upregulated while many ATP-utilizing enzymes and pathways were downregulated at pH 6.24. These included most flagella biosynthesis genes, genes for chemotaxis, and other motility-related genes (> 50) as well as sulfate transport and reduction, nitrate transport and nitrogen fixation, and fatty acid biosynthesis genes. Clustering and enrichment of differentially expressed genes at pH values 6.48, pH 6.24 and pH 6.12 (washout conditions) compared to pH 6.98 showed inverse differential expression patterns between the F1F0-ATPase and genes for other ATP-utilizing enzymes. At and below pH 6.24, amino acids including glutamate and valine; long-chain fatty acids, their iso-counterparts and glycerol conjugates; glycolysis intermediates 3-phosphoglycerate, glucose 6-phosphate, and glucose accumulated intracellularly. Glutamate was 267 times more abundant in cells at pH 6.24 compared to pH 6.98, and intercellular concentration reached 1.8 μmol/g pellet at pH 5.80 (stopped flow).Conclusions: Clostridium thermocellum LL1210 can grow under slightly acidic conditions, similar to limits reported for other strains. This foundational study provides a detailed characterization of a relatively acid-intolerant bacterium and provides genetic targets for strain improvement. Future studies should examine adding gene functions used by more acid-tolerant bacteria for improved pH homeostasis at acidic pH values.},
doi = {10.1186/s13068-018-1095-y},
journal = {Biotechnology for Biofuels},
number = 1,
volume = 11,
place = {Netherlands},
year = {2018},
month = {4}
}

Journal Article:
Free Publicly Available Full Text
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DOI: 10.1186/s13068-018-1095-y

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