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Consolidated bioprocessing (CBP) is believed to be one of the preferred strategies to produce biofuels directly from cellulose, which can greatly reduce the costs.Clostridium cellulolyticum H10, a typical mesophilic, gram-positive anaerobe, is one of the prominent candidates for CBP production of cellulosic ethanol.Here we presented the metabolic engineering of C.cellulolyticum H10 for higher ethanol production.We firstly improved the transformation efficiency of C.cellulolyticum H10 from 102 to 103 transformants per ug DNA, and developed a targeted gene inactivation method based on the targetron technology.Mutant H 10△mspI was thereafter constructed by disrupting the gene ccel2866 which encodes a type Ⅱ restriction endonuclease MspI.This mutant exhibited no MspI activity, and could be genetically manipulated with unmethylated DNA efficiently.Thus, with the well constructed genetic platform, we constructed three engineered strains to enhance the production of ethanol.H 10△ack was an acetate kinase (Ccel2136) inactivated mutant, no significant changes in fermentation pattern was observed compared with the wild type, which indicated other enzymes or pathways responsible for acetate production in C.cellulolyticum H10.Two genes, ccel2485 and ccel0137, were annotated as lactate dehydrogenase in H10.Mutant H10△ldh1 constructed by disrupting gene ccel2485 showed over 85% decrease of lactate production, and 36% and 26% increase of ethanol and acetate production, respectively.Double mutant H10△ldh1△ldh2 was successfully constructed by disrupting gene ccel0137 in H1 0△ldh1, and the stability of double mutation was confirmed after 100th generation.The production of lactate and ethanol of H10△ldh1△ldh2 was similar to that of H10△ldh1.This indicated that ccel0137 may not be responsible for lactate synthesis as lactate dehydrogenase in H10.In summary, our results firstly demonstrated the targeted gene inactivation in C.cellulolyticium H10, and suggested that the biofuel (e.g., ethanol) production of C.cellulolyticum could be greatly improved by metabolic engineering.