Abstract
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 (Dldh) and/or acetate (Dpta) were characterized to gain insight into the intracellular processes that convert cellobiose to ethanol and other fermentation end-products. Cellobiose-grown cultures of the Dldh strain had identical biomass accumulation, fermentation end-products, transcription profile, and intracellular metabolite concentrations compared to its parent strain (DSM1313 Dhpt Dspo0A). The Dpta-deficient strain grew slower and had 30 % lower final biomass concentration compared to the parent strain, yet produced 75% more ethanol. A Dldh Dpta 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 Dpta. Free amino acids were secreted by all examined strains, with both Dpta strains secreting higher amounts of alanine, valine, isoleucine, proline, glutamine, and threonine. Valine concentration for Dldh Dpta 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 Dpta and 16-fold in the Dldh Dpta 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.
