Rational pathway design and adaptive evolution to develop an efficient cell factory for biofuel production from lignocellulose

Biofuels from plant cell walls offer potential to be sustainable and economically attractive alternatives to petroleum-based products.  Fuels from cellulosic biomass are particularly promising, but would benefit from lower processing costs. Clostridium thermocellum is a thermophilic bacterium which can grow at 60°C. In addition, C. thermocellum can rapidly solubilize and ferment cellulosic biomass.  These two features make it a good candidate microorganism for consolidated bioprocessing for biofuel production.   Here we started with a strain of C. thermocellum which had several gene deletions, including pfl, ldh, pta-ack and hydG.  This engineered strain reached 80% of theoretical ethanol yield when consuming 5 g/l cellobiose, however the yield decreased when substrate levels were increased.  Furthermore, the maximum titer was limited to 14 g/l although other strains of C. thermocellum have been adapted to grow in the presence of 50 g/l ethanol.  We report improvement of these performance parameters using a combination of rational strain design and adaptive evolution.