Deletion of hydrogen loss increased ethanol yield in Clostridium thermocellum

A major focus of biofuel research is to engineer a microorganism that can efficiently solubilize and ferment cellulosic biomass to liquid fuels by one-step consolidated bioprocessing (CBP). CBP is a potentially advantageous approach for the production of biofuels, but requires an organism capable of hydrolyzing biomass to sugars and fermenting the sugars to ethanol at commercially viable titers and yields. Clostridium thermocellum, a thermophilic anaerobe, can ferment cellulosic biomass to ethanol, organic acids and H₂. Deletion of the pathways leading to these byproducts can potentially redirect carbon flux towards ethanol pathway. H₂ production was blocked by deletion of a hydrogenase maturase gene (hydG), which is involved in converting Fe-Fe hydrogenase apoenzymes into holoenzymes by assembling the active site. This functionally inactivated all the Fe-Fe hydrogenases simultaneously. In the DhydG mutant, the Ni-Fe hydrogenase-encoding ech was also deleted to obtain a mutant that functionally lacks all hydrogenase. The ethanol production increased by ~2-fold in DhydGDech compared to wild type, and H₂ production was completely eliminated. The DhydG and DhydGDech strains exhibited improved growth and ethanol productivity in the presence of acetate in the medium. The effect of acetate on growth of C. thermocellum and its mutants was studied using transcriptomics, revealing that 94 genes were up-regulated and 154 down-regulated in mutants in comparison to the wild type DSM 1313. The major up-regulated genes were related to sulfur metabolism and electron transport, suggesting possible mechanisms for improved redox balancing in hydrogenase deletion mutants.