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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.

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