Targeted redox and energy cofactor metabolomics in Clostridium thermocellum and Thermoanaerobacterium saccharolyticum Mike Himmel

Kyle Sander, Keiji G. Asano, Deepak Bhandari, Gary J. Van Berkel, Steven D. Brown, Brian Davison, Timothy J. Tschaplinski

Research output: Contribution to journalArticlepeer-review

6 Scopus citations

Abstract

Background: Clostridium thermocellum and Thermoanaerobacterium saccharolyticum are prominent candidate biocatalysts that, together, can enable the direct biotic conversion of lignocellulosic biomass to ethanol. The imbalance and suboptimal turnover rates of redox cofactors are currently hindering engineering efforts to achieve higher bioproductivity in both organisms. Measuring relevant intracellular cofactor concentrations will help understand redox state of these cofactors and help identify a strategy to overcome these limitations; however, metabolomic determinations of these labile metabolites have historically proved challenging. Results: Through our validations, we verified the handling and storage stability of these metabolites, and verified extraction matrices and extraction solvent were not suppressing mass spectrometry signals. We recovered adenylate energy charge ratios (a main quality indicator) above 0.82 for all extractions. NADH/NAD+ values of 0.26 and 0.04 for an adhE-deficient strain of C. thermocellum and its parent, respectively, reflect the expected shift to a more reduced redox potential when a species lacks the ability to re-oxidize NADH by synthesizing ethanol. This method failed to yield reliable results with C. bescii and poor-growing strains of T. saccharolyticum. Conclusions: Our validated protocols demonstrate and validate the extraction and analysis of selected redox and energy-related metabolites from two candidate consolidated bioprocessing biocatalysts, C. thermocellum and T. saccharolyticum. This development and validation highlights the important, but often neglected, need to optimize and validate metabolomic protocols when adapting them to new cell or tissue types.

Original languageEnglish
Article number270
JournalBiotechnology for Biofuels
Volume10
Issue number1
DOIs
StatePublished - Nov 30 2017

Funding

This work is supported by the BioEnergy Science Center (BESC), which is a U.S. Department of Energy Bioenergy Research Center supported by the Office of Biological and Environmental Research in the DOE Office of Science. The manuscript has been authored by UT-Battelle, LLC, under Contract No. DE-AC05-00OR22725 with the U.S. 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.

FundersFunder number
BioEnergy Science Center
U.S. Department of Energy Bioenergy Research Center
U.S. Department of Energy
Office of ScienceDE-AC05-00OR22725
Biological and Environmental Research

    Keywords

    • Adenylate energy charge
    • Clostridium thermocellum
    • Redox
    • Targeted metabolomics
    • Thermoanaerobacterium saccharolyticum

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