Characterization of Clostridium thermocellum strains with disrupted fermentation end-product pathways
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Clostridium thermocellum is a thermophilic, cellulolytic anaerobe that is a candidate microorganism for industrial biofuels production. Strains with mutations in genes associated with production of l-lactate (Δldh) and/or acetate (Δpta) were characterized to gain insight into the intracellular processes that convert cellobiose to ethanol and other fermentation end-products. Cellobiose-grown cultures of the Δldh strain had identical biomass accumulation, fermentation end-products, transcription profile, and intracellular metabolite concentrations compared to its parent strain (DSM1313 Δhpt Δspo0A). The Δpta-deficient strain grew slower and had 30 % lower final biomass concentration compared to the parent strain, yet produced 75 % more ethanol. A Δldh Δpta double-mutant strain evolved for faster growth had a growth rate and ethanol yield comparable to the parent strain, whereas its biomass accumulation was comparable to Δpta. Free amino acids were secreted by all examined strains, with both Δpta strains secreting higher amounts of alanine, valine, isoleucine, proline, glutamine, and threonine. Valine concentration for Δldh Δpta reached 5 mM by the end of growth, or 2.7 % of the substrate carbon utilized. These secreted amino acid concentrations correlate with increased intracellular pyruvate concentrations, up to sixfold in the Δpta and 16-fold in the Δldh Δpta strain. We hypothesize that the deletions in fermentation end-product pathways result in an intracellular redox imbalance, which the organism attempts to relieve, in part by recycling NADP+ through increased production of amino acids.
This work was supported by the Office of Biological and Environmental Research in the DOE Office of Science through the BioEnergy Science Center, a U.S. DOE Bioenergy Research Center. Oak Ridge National Laboratory is managed by UT-Batelle, LLC, for the U.S. Department of Energy under contract DE-AC05-00OR22725. We would like to thank Mascoma Corporation, Lebanon, NH, for the use of strains used in this study, Marybeth I. Maloney for performing DNA sequencing, and Kyle D. Hirst for performing the TOC/TN analysis.
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