Characterization of Clostridium thermocellum strains with disrupted fermentation end-product pathways

摘要

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 (Delta ldh) and/or acetate (Delta pta) were characterized to gain insight into the intracellular processes that convert cellobiose to ethanol and other fermentation end-products. Cellobiose-grown cultures of the Delta ldh strain had identical biomass accumulation, fermentation end-products, transcription profile, and intracellular metabolite concentrations compared to its parent strain (DSM1313 Delta hpt Delta spo0A). The Delta pta-deficient strain grew slower and had 30 % lower final biomass concentration compared to the parent strain, yet produced 75 % more ethanol. A Delta ldh Delta pta 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 Delta pta. Free amino acids were secreted by all examined strains, with both Delta pta strains secreting higher amounts of alanine, valine, isoleucine, proline, glutamine, and threonine. Valine concentration for Delta ldh Delta pta 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 Delta pta and 16-fold in the Delta ldh Delta pta 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.