1-32: Redirection of carbon flux in Clostridium thermocellum for achieving high ethanol yield

Tuesday, May 1, 2012
Napoleon Ballroom C-D, 3rd fl (Sheraton New Orleans)
Yu Deng1, Daniel Olson1, Christopher D. Herring2, A. Joe Shaw3 and Lee Lynd1, (1)Thayer School of Engineering, Dartmouth College, Hanover, NH, (2)Mascoma Corporation, Lebanon, NH, (3)Mascoma Corp., Lebanon, NH
Clostridium thermocellum is a thermophilic anaerobic bacterium with the ability to rapidly ferment cellulose to ethanol and organic acids. The yield of ethanol in C. thermocellum is often only 25% of theoretical yield. In order to increase the ethanol yield, the flow of carbon and electrons was studied and redirected. Pyruvate kinase, catalyzing conversion of phosphoenolpyruvate to pyruvate, is absent from the genome and its activity was verified to be absent in C. thermocellum by enzymatic assay.  An alternate route exists for conversion of PEP to pyruvate through oxaloacte and malate, and the implications of this “malate shunt” were investigated using flux balance analysis. In order to bypass this shunt, the lactate dehydrogenase gene was replaced with the pyruvate kinase gene from Thermoanaerobacterium saccharolyticum, creating strain DS8. Expression of phosphoenolpyruvate carboxykinase was reduced in DS8 by directed mutation. The ethanol yield of the resulting strain was 3.3-fold higher than the wild-type strain. In another derivative of strain DS8, the gene for malic enzyme and part of malate dehydrogenase were deleted, producing a strain with an ethanol yield 3.1-fold higher than the wild-type strain. Amino acids analysis revealed that amino acid secretion was markedly lower in both strains.
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