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 Llactate (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 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 6-fold 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.
