Clostridium thermocellum LL1210 pH homeostasis mechanisms informed by transcriptomics and metabolomics

Jason M. Whitham, Ji Won Moon, Miguel Rodriguez, Nancy L. Engle, Dawn M. Klingeman, Thomas Rydzak, Malaney M. Abel, Timothy J. Tschaplinski, Adam M. Guss, Steven D. Brown

Research output: Contribution to journalArticlepeer-review

17 Scopus citations

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 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.

Original languageEnglish
Article number98
JournalBiotechnology for Biofuels
Volume11
Issue number1
DOIs
StatePublished - Apr 5 2018

Funding

This work is supported by the BioEnergy Science Center (BESC), which is a US Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. ORNL is managed by UT‑Battelle, LLC, Oak Ridge, TN, USA, for the DOE under contract DE‑AC05‑00OR22725. RNA‑Seq data were generated by the US Department of Energy Joint Genome Institute, a DOE Office of Science User Facility, is supported by the Office of Science of the US Department of Energy under Contract No. DE‑AC02‑05CH11231. The manuscript has been authored by UT‑Battelle, LLC, under Contract No. DE‑AC05‑00OR22725 with the US Department of Energy. The funders had no role in study design, data collection and interpretation, preparation of the manuscript, or the decision to submit the work for publication. The publisher, by accepting the article for publication, acknowledges that the United States Government retains a non‑exclusive, paid‑up, irrevocable, world‑wide license to publish or reproduce the published form of this manuscript, or allow others to do so, for United States Government purposes. The Department of Energy will provide public access to these results of federally sponsored research in accordance with the DOE Public Access Plan (http://energy.gov/downloads/ doe‑public‑access‑plan). This work is supported by the BioEnergy Science Center (BESC), which is a US Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. ORNL is managed by UT‑Battelle, LLC, Oak Ridge, TN, USA, for the DOE under contract DE‑AC05‑00OR22725.

FundersFunder number
BioEnergy Science Center
US Department of Energy Joint Genome Institute
U.S. Department of EnergyDE‑AC02‑05CH11231, DE‑AC05‑00OR22725
Office of Science
Biological and Environmental Research

    Keywords

    • Clostridium thermocellum
    • FF-ATPase
    • GOGAT
    • Glutamate decarboxylase
    • Glutamate dehydrogenase
    • Glutamine synthetase
    • Proton pumping
    • Urease
    • pH homeostasis

    Fingerprint

    Dive into the research topics of 'Clostridium thermocellum LL1210 pH homeostasis mechanisms informed by transcriptomics and metabolomics'. Together they form a unique fingerprint.

    Cite this