Engineered strain of Clostridium thermocellum for enhanced ethanol synthesis
Ranjita Biswas and Adam M. Guss
Biosciences Division, Oak Ridge National Laboratory, Oak Ridge TN
BioEnergy Science Center, Oak Ridge National Laboratory, Oak Ridge TN
Abstract
One major focus of biofuel research is to engineer a microorganism which can efficiently solubilize and ferment the plant sugars to ethanol. The cellulolytic system of Clostridium thermocellum, a gram positive thermophilic anaerobe, can solubilize crystalline cellulose and ferment it for production of ethanol, but it also produces other byproducts such as acetate, lactate, formate and H2. Deletion of the pathways leading to these byproducts can potentially redirect carbon flux towards ethanol pathway. To block H2 production, a hydrogenase-related gene (hydG) was deleted, which is involved in converting Fe-Fe hydrogenase apoenzymes into holoenzymes. This functionally inactivated all the Fe-Fe hydrogenases simultaneously, as they were unable to make active enzymes. In this DhydG background, various combinations of deletion knockout mutants were engineered. The deletion of pyruvate-formate lyase gene (pfl) blocked synthesis of formate from pyruvate with concomitant increase in ethanol formation. Similarly, deletion of lactate dehydrogenase (ldh) and phosphotransacetylase-acetate kinase genes (pta-ack) block synthesis of lactate and acetate from pyruvate, respectively. One such deletion mutant showed decreased production of acetate, lactate, formate and H2 concomitantly with a 3-fold increase in ethanol production on cellulose. Continued metabolic engineering of C. thermocellum could result in a viable candidate for consolidated bioprocessing of biofuels on an industrial scale.