Engineering Clostridium thermocellum for hydrogen production

Clostridium thermocellum is a gram-positive, thermophilic (55 – 60^oC) and anaerobic bacterium displaying one of the highest growth rates on cellulose. It is an attractive candidate of consolidated bioprocessing (CBP), a process configuration which directly converts plant biomass into downstream metabolic products without separate additions of cellulase enzymes.   During fermentation, C. thermocellum secretes an array of products branching downstream of pyruvate including H₂, formate, lactate, acetate, ethanol, and amino acids.  To improve the H₂ molar yield (mol H₂/mol hexose), our goals include optimizing cellulose fermentation in bioreactors, and genetically modify the strain by eliminating pathways competing for the electron for H₂ production. Hydrogen production in C. thermocellum is primarily catalyzed by hydrogenases which accept electrons from a low potential carrier such as reduced ferredoxin to reduce H⁺.  One major reaction to produce reduced ferredoxin is catalyzed by pyruvate:ferredoxin oxidoreductase (PFOR) using pyruvate and oxidized ferredoxin as substrates and produces acetyl-CoA.  We have shown that re-directing the carbon flux towards pyruvate to acetyl-CoA via PFOR by knocking out pathways branching away from pyruvate improved the rate of H2 production. We have also used ¹³C-tracer experiments to assess the carbon flow via pathways relevant to H₂ production in vivo.  Work is ongoing to continue optimizing the strain for improved H₂ yield.